text:
Rational design of a trispecific antibody targeting the HIV-1 Env with elevated anti-viral activity

HIV-1 broadly neutralizing antibodies (bNAbs) are being explored as passively administered therapeutic and preventative agents. However, the extensively diversified HIV-1 envelope glycoproteins (Env) rapidly acquire mutations to evade individual bNAbs in monotherapy regimens. The use of a “single” agent to simultaneously target distinct Env epitopes is desirable to overcome viral diversity. Here, we report the use of tandem single-chain variable fragment (ScFv) domains of two bNAbs, specific for the CD4-binding site and V3 glycan patch, to form anti-HIV-1 bispecific ScFvs (Bi-ScFvs). The optimal Bi-ScFv crosslinks adjacent protomers within one HIV-1 Env spike and has greater neutralization breadth than its parental bNAbs. Furthermore, the combination of this Bi-ScFv with a third bNAb recognizing the Env membrane proximal external region (MPER) results in a trispecific bNAb, which has nearly pan-isolate neutralization breadth and high potency. Thus, multispecific antibodies combining functional moieties of bNAbs could achieve outstanding neutralization capacity with augmented avidity.

Specific comments: 1. Given the weaknesses of this study outlined above, the current title is an overstatement and should be modified to reflect the limitation of the rational design strategy. The potency is certainly far from exceptional. 2. As stated already, when comparing the neutralizing breadth and potency of bispecific moieties, the data from the combination of two parental antibodies should be used. This is to provide meaningful comparisons. 3. On line 177, authors stated "SEC with negligible aggregation forms (Fig. S1B)". However, the SEC data presented suggests a significant portion of small shoulder peak was presented. Since the detailed SEC method was not presented, it is very difficult to discern if this small shoulder peak fraction (which seems to have multiple small peaks) was an aggregation. Detailed SEC method should be provided. A control molecule (monoclonal antibody that provide a single peak) should also be provided. 4. Reduced SDS-PAGE analysis is not an appropriate methodology to measure the homogeneity of the antibody. Non-Reduced SDS-PAGE analysis and data should be included. 5. SEC data for trispecific antibody should be presented to demonstrate the developability of this molecule. 6. Data presented in figure 3 C and D were interesting. It would be more informative if, for three dual resistant viruses (6631, CAP210 and 3817), the neutralization curve for bispecific antibodies, parental bNAbs and combination parental bNAbs against individual virus were presented.
; 7. With respect to future development, in addition to the positive aspects of the bi-and trispecific antibodies, the authors should also discuss the limitations of such irregular antibody-like structures, for example, potential immunogenicity, developability concerns, etc. The manuscript could be more useful to the field if they could show in vivo activity of their constructs, say in a humanized mouse model of HIV infection.

Reviewer #2 (Remarks to the Author):
This is a straightforward but interesting bit of protein engineering, in which two scFvs are strung together with an Fc by (G4S)n linkers and then paired with the heavy chain of a thrid antibody. As expected, when all three variable regions recognize epitopes that are targets of bnAbs, the potency and breadth are both extremely high. While the work does not break any new conceptual ground, it is probably interesting enough as a concrete application of previously validated ideas to merit, after suitable editing, publication in a journal like Nature Communications.
Suitable editing. The overall result is expected, if the engineering is OK, and the data speak for themselves, although it is, of course, nice to see that it works (and useful also). So the first step is to remove the entire Discussion, which is pure repetition, keeping only the last paragraph (which might be called Conclusions).
Since the MS suffers a bit from "template writing" and some hints of writing by a non-native speaker, the next steps are outlined below. line 29 We describe here joining single-chain .... [You didn't do the joining "here" --you are only desribing the joining "here". You did the joining in the lab.] line 33 has greater neutralization breadth than its parental bnAbs [use the 3-letter "has" rather than the more preteniious 'demonstrates"; "superior" is a value judgment, and scientific prose should be objective; the correct comparative in English is "greater ... than", not "over" or "compared to" or anything else] line 35 which has nearly pan-isolate neutralization breadth and high potency [the sentence as written is simply ungrammatical English] line 65 ... viruses, but these viruses have much lower genetic diversity than do circulating HIV-1 isolates. [see remark about correct English comparative above] line 69 The HIV rapidly developes resistance under pressure from a single bnAb, however, suggesting that passive treatment .... [fixing awkward syntax] lines 80-82 Preliminary data on bnAb cocktails suggest substantial advantages over either cART or single bnAb treatments for the management of HIV-1 infection. [you have an advantage "over", not "compared to" --also word order needs fixing, as shown] line 87 Their neutralization breadth could be further extended, however, and truly .... ["improved" is a value judgment; use an objective term like "extended"; avoid "however" at the beginning of a sentence, unless you mean "in whatever way" --as "However nasty and pedantic this reviewer might be, I still find his critique instructive."] line 100 delete "however" (not needed) The field of therapeutic antibody formats is quickly developing especially for infectious diseases as so much good science has shown the need to target the pathogen at multiple epitopes in order to weaken the pathogen's ability to adapt by mutation. Furthermore, the demonstration that passive immunization, provided by an antibody format, provokes additional mechanisms of ultimate protection such as strengthening the adaptive and endogenous humoral responses, makes the exploration of optimal configurations essential for success. Steinhardt and colleagues have taken a rationale design approach to the problem. Their study starts with a simple construct and builds to a higher and higher avidity through more complicated antibody-based structures leading to increasingly significant capacity to neutralize across a very broad panel of viral strains. The explanations are well thought through and presented with the following comments: The rationale for choices of target epitopes are validated with figure 1 providing a clear explanation of this as well as for the design of the constructs. The demonstration of the cobinding is convincing, with beautiful visual support provided by the electron micrographs of the binding interfaces using the subsaturating concentration group. Generally, as I am not an expert on the details of the viral neutralization assays, the data looks convincing. However, I challenge the helpfulness of providing a GEO mean in figure 3A and B as, for example in B, the BiSc-Fv has several red lines while the Bi-Nab format has only one red line, yet they have equivalent Geo Means (0.306 vs 0.297). The way the data is presented in figure 4 B is a better way to communicate the message. Staying on figure 3, the data presented in that figure are used by the authors to state in the results and conclusion that the linker length is relevant, i.e., '…molecules with shorter linker lengths of 4x or 3x displayed lower neutralization breadth…' This is not true as the breadth data presented in fig 3A show 80/85/80 or 85/85/80 % viruses neutralized for biScFv vs Bi-Nab for 5x/4x/3x formats, respectively. In other words, 4x is actually always the best. So either the data is wrong in the table or I am not able to read the table correctly. So please address perhaps by adding the numbers for breadth in the text of the results or changing this statement. The main concern that I have with this manuscript is the lack of better presentation of the analytics to prove/convince the reader of the homogeneity of the biAb or TriAb batches. The reason to know more accurately the level of aggregates is that in this biological system, having multimers may falsely increase the neutralization capacity, even if the level of aggregates is 'relatively' low. We know this from our own experience. Thus, it would be necessary to provide information on the size range of the column used. Also, please provide light scattering info to confirm the mass of the peaks as I don't agree that what is marked as the aggregate peak in fig S1 is actually that. There are peaks closer to the main peaks that should be where the aggregate peaks are. Furthermore, the trispecific structure is by the nature of putting all these Ab fragments together, a manufacturing challenge. Thus, having more analytical info than what is provided in fig S4 is essential to be able to validate the declaration of low aggregates/homogeneity to support the biological results that is the structure of a bi or tri Ab and not the aggregates. One minor comment is to fix the sentence in the Intro on line 79 as there seems not to be needed 'as well as' . This study and publication of the manuscript are very timely and will be of genuine interest to the field. Just this week, Science has published two articles in this domain, highlighting the importance to medicine: 'Why is the flu vaccine so mediocre?' J Cohen Science 22 Sep 2017. 'There's an increasing call now to improve the vaccine and organize the research community to more collaboratively try to develop a "universal" flu shot that works against many strains and lasts for many years, if not a lifetime.' Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques Ling Xu1 et al.,Science 20 Sep 2017: The development of an effective AIDS vaccine has been challenging due to viral genetic diversity and the difficulty in generating broadly neutralizing antibodies (bnAbs). Here, we engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: 1) the CD4 binding site, 2) the membrane proximal external region (MPER) and 3) the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to human bnAbs, and conferred complete immunity against a mixture of SHIVs in non-human primates (NHP) in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and could be applicable for diverse diseases, including infections, cancer and autoimmunity. I would suggest that as the authors see that sometimes the biAb or TriAb do not do as well as the parental mAb for certain viral strains, they might comment in the discussion on how yet a future approach could do even better (e.g., mixtures). But that suggestion is optional. Thus, post addressing the above comments, my opinion would be favorable for publication of this manuscript in Nature Communications.

Point-by-Point Response to Reviewer's Comments
We appreciate the overall positive and constructive comments from the reviewers. After carefully revised our manuscript, with the edited text highlighted, we are providing point-by-point response to their comments as below.

Comments
This study addresses the interesting question whether a single engineered antibody-like agent is capable of simultaneously targeting distinct HIV Env epitopes resulting in improved HIV neutralizing potency and breadth. Based on structural analysis of the interactions of HIV Env trimer with bNAbs, the authors generated a panel of Bi-ScFvs. Using negative stain EM, they showed that optimal Bi-ScFv is capable of crosslinking adjacent protomers within one HIV Env spike. However, when testing viral neutralizing activity in vitro, optimal Bi-ScFv improves only the neutralizing breadth, but not the potency (to any significance), over its parental bNAbs. In addition, the authors combined this bispecific antibody with a third bNAb 10E8 moiety and showed that a tri-specific antibody-like molecule further improves neutralizing breadth and potency in vitro. Based on these results, authors concluded that a combination of multi-functional moieties of individual bNAbs can elevate antibody binding avidity, hence improving HIV neutralizing potency and breadth.
There are several important limitations that raise questions as to whether the data provide insight into antibody bispecific binding, i.e., bivalent binding, binding avidity and neutralizing potency. Previously, Asokan and Mascola showed that bispecific antibodies (generated via CrossMab technology) capable of targeting two epitopes on Env improved neutralizing breadth when compared to their respective single parental bNAbs, but the overall breadth and potency was similar to the combination of two parental bNAbs. The bispecific antibodies' failure to improve neutralizing potency is likely due to the limited reach between two Fabs. Using the "molecular ruler" strategy, Galimidi and Bjorkman nicely showed that intraspike crosslinking can indeed enhance the binding avidity and importantly improve the potency of bNAbs. Furthermore, Bournazos and Ravetch showed that compared to the combination of two parental antibodies, a bispecific antibody design based on a longer IgG3 hinge can not only significantly improve the neutralizing potency and breadth, but also enhance anti-HIV activity in humanized mouse model. These studies showed consistently that engineered bispecific antibodies can improve neutralizing breadth due to the contribution from each Fab arm. However, improving avidity requires crosslinking two epitopes, and the binding avidity is expected to correlate with enhanced neutralizing potency. The strength of the current study is that authors utilized structure-based antibody rational design to optimize the multiepitope engagement of Env trimer with Bi-ScFv antibodies. But the optimally designed bispecific antibodies with dual engagement of Env epitope, validated by negative stain EM, can only improve breadth but failed to enhance virus neutralization potency to any significant degree. Scientifically, the reason for this discrepancy is not well addressed. Avoiding such discussion present a major weakness of the manuscript.
Response: We appreciate the reviewer's insight to the mechanism regarding the contrast effect of bispecific antibody on the improvement of neutralization breadth and potency: great gain of breadth but moderate improvement in potency. We believe that the gain of breadth is mainly caused by the combination of two parental bNAb moieties in the intra-spike protomer crosslinking mode used by the bispecific antibody.
The similarity of potency between that of the bispecific antibody and the parental bNAbs may be caused by a number of factors: 1) steric hindrance imposed by the Env structural elements from diverse HIV isolates which are not reflected by the current available structural information; and 2) the parental antibodies, VRC01 and PGT121 displaying remarkably low dissociation rates (slower than 10^-7 1/s) (provided as new data in Fig. S4 D & E) to the Env trimers such as BG505 SOSIP.664, which is virtually identical to the bispecific scFv. This observation is consistent with an elegant study by Galimidi et al. (2015, Cell 160, 433-446) revealing that for off-rates slower than 10^-5 1/s, avidity enhancement by multivalence binding is not substantial. Therefore, in this case, we do not see substantial enhancement of potency possessed by our bispecific antibody with many virus isolates. We addressed this point Perhaps a combination of other parental antibodies with higher off-rate for trimer will lead to more prominent gain of potency.
However, when tested with dual resistant viruses, the bispecific antibodies display greater potency than the individual parental antibodies (Fig. 3C & D) as well as a cocktail of the parental antibodies ( Fig. S3C &  D). In these cases, both parental Abs likely have poor affinity (possibly a fast off-rate as well) for the resistant viral Env spikes and fail to neutralize the viruses, whereas the bispecific antibody could overcome the low affinity by bivalent binding to the Env spike and mediate neutralization.

Comments
The authors should have compared the neutralization results of their bispecific and trispecific antibodies with the neutralization profiles of the particular double and triple combinations of the parental antibodies. If there is any simultaneous binding of two or three epitopes, the potency of the multispecific antibody should be superior. This is yet another major deficiency of the study.

Response
We had long-standing neutralization assay experience with antibody combinations in the lab (Mascola lab). We had tested more than 200 antibodies and antibody derivatives and compared their anti-viral capacities. The accumulated data suggest that, overall, the improvement of neutralization breadth by antibody combination was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes ( Kong et al., J Virol. 2015 Mar 1; 89(5): 2659-2671). Thus, the parental antibody neutralization profile as a side-by-side control used in this study is sufficient to show the effect of physical combination of bNAbs on neutralization breadth and potency.
Nevertheless, we performed a confirmatory neutralization assay in which the bispecific antibodies, parental antibodies, and a cocktail of parental antibodies were included to compare the breadth and potency of the antibodies against a VRC01-resistant virus panel (Fig. S3C & D). The outcome of this examination is highly consistent with our previous data with large virus panel (208-virus panel) (Fig. 3C &  D). Therefore, we are confident with our large virus panel neutralization data presented in this manuscript.

Comments
Furthermore, the authors do not directly compare their results to those generated by Pace et al, Sun et al, Gardner et al, Bournazos et al and Huang et al. Based on an "eyeball" examination, the constructs being reported here are significantly inferior in neutralization potency. Therefore, as potential HIV prevention products, they face superior competition.

Response
It is a good point to have a comparison of these anti-viral agents. However, due to the difference between the design rationale (different target) and the various virus neutralization assay panels used by these different studies, it is practically difficult to compare them fairly.
For instance, as stated in the introduction section of the manuscript, the studies by Pace et al, Sun et al, and Huang et al targeted host factor and one Env epitope, while the anti-viral agent in the study of Gardner et al is based on receptor CD4 (host factor). In contrast, our trispecific antibody targets three epitopes on viral Env. We believe that our unique multi-epitope targeting trispecific antibody is a good addition to these innovative agents. Indeed, it will be interesting to combine these approaches to generate novel antiviral agents in future studies.
The Bournazos et al study created a bispecific antibody targeting CD4bs and V3 glycan with 93% virus coverage (IC50 GMT 0.04 ug/ml), while our bispecific and trispecific antibody displays 95% (IC50 GMT = 0.3 ug/ml) and 99.5% virus coverage (IC50 GMT= 0.07 ug/ml), respectively. Again, it is hard to compare them, as the assay virus panel is different between these studies. Nevertheless, our approach generated antibodies with "comparable" antiviral potency and greater breadth. In addition, our trispecific antibody targets three Env epitopes, which has greater potential to combat viral resistance than bispecific antibodies. Furthermore, the cross-spike binding mode conveyed by rational design based tandem scFVs in this study will be of general interest for the field to engineer novel antibodies.

Comments
Lastly, if there is no significant new science being revealed, then the constructs must have practical application as HIV preventive agents. However, there is little evidence that these constructs could be developed into real products. Given the described antibody structures differ significantly from a normal antibody structure, there is real concerns regarding their stability, PK properties, and potential immunogenicity, especially given the linker sequences inserted. More in vitro analyses would be helpful, and preliminary in vivo determination of PK and anti-HIV activity would be informative. But they are completely missing.

Response
We appreciate the reviewer's suggestion for investigations toward practical applications including the in vivo PK, immunogenicity, and anti-HIV activity, although they are beyond the scope of our current study.
We are planning to perform such experiments in the future after we publish our work here and secure more fund to support the suggested studies.
Specific comments: 1. Given the weaknesses of this study outlined above, the current title is an overstatement and should be modified to reflect the limitation of the rational design strategy. The potency is certainly far from exceptional.
Response: There are two critical components of antiviral capacity: breadth and potency. Our Tri-NAb displays 99.5% virus coverage, which is exceptional among all the solely Env-targeting antibodies.
Regarding the potency, our Tri-NAb achieves IC50 GMT around 0.07 ug/ml, quite comparable to the other solely Env-targeting antibodies.
We revised the title of our manuscript to "Rational Design of a Trispecific Antibody Targeting the HIV-1 Env with Outstanding Anti-viral Activity" 2. As stated already, when comparing the neutralizing breadth and potency of bispecific moieties, the data from the combination of two parental antibodies should be used. This is to provide meaningful comparisons. Response As stated previously, we performed a confirmatory neutralization assay in which the bispecific antibodies, parental antibodies, and a cocktail of parental antibodies were included to compare the breadth and potency of the antibodies against a VRC01-resistant virus panel (Fig. S3C & D). The outcome of this examination is highly consistent with our previous data with large virus panel (208virus panel) (Fig. 3C & D). The effect of antibody combination on neutralization breadth and potency was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Therefore, we are confident with our large virus panel neutralization data presented in this manuscript without testing all the 208 viruses with the combination of two parental antibodies. The data of parental antibodies as control is sufficient to serve as control.

On line 177, authors stated "SEC with negligible aggregation forms (Fig. S1B)". However, the SEC data presented suggests a significant portion of small shoulder peak was presented. Since the detailed SEC method was not presented, it is very difficult to discern if this small shoulder peak fraction (which seems to have multiple small peaks) was an aggregation. Detailed SEC method should be provided. A control molecule (monoclonal antibody that provide a single peak) should also be provided. Response
We appreciate that the reviewer pointed out the interpretation of the SEC data. We recognized the "shoulder peak" in the SEC and revised the text accordingly. We provided the details of the SEC in the methods section, and added VRC01 as a control in the SEC (Fig. S4A). In addition, we performed DLS ( Fig. S4A-C) and showed the details of the molecular sizes of the bispecific antibodies (Lines 240-246).

Response
We added the Non-Reduced SDS-PAGE analysis data in FIG. S1 and S5B.

SEC data for trispecific antibody should be presented to demonstrate the developability of this molecule. Response
We added the SEC data for trispecific antibody (Fig. S5F-H, Lines 298-301).

Data presented in figure 3 C and D were interesting. It would be more informative if, for three dual resistant viruses (6631, CAP210 and 3817), the neutralization curve for bispecific antibodies, parental bNAbs and combination parental bNAbs against individual virus were presented.
Response We added the data in Fig. 3D, and added an independent confirmatory data set in Fig. S3C & D.

With respect to future development, in addition to the positive aspects of the bi-and tri-specific antibodies, the authors should also discuss the limitations of such irregular antibody-like structures, for example, potential immunogenicity, developability concerns, etc. The manuscript could be more useful to the field if they could show in vivo activity of their constructs, say in a humanized mouse model of HIV infection. Response
We revised our manuscript and addressed these points (Lines 414-419). As discussed previously, we are planning to perform such in vivo efficacy experiments in the future after we publish our work here and secure more fund to support the suggested studies.

Reviewer #2
Comments This is a straightforward but interesting bit of protein engineering, in which two scFvs are strung together with an Fc by (G4S)n linkers and then paired with the heavy chain of a thrid antibody. As expected, when all three variable regions recognize epitopes that are targets of bnAbs, the potency and breadth are both extremely high. While the work does not break any new conceptual ground, it is probably interesting enough as a concrete application of previously validated ideas to merit, after suitable editing, publication in a journal like Nature Communications.

Response
We appreciate the encouragement of the reviewer and we are very thankful for the truly constructive and instructive suggestions to improve the manuscript. We made most of the revision accordingly. Response: All the points listed above were corrected.
Comment lines 163 and 165 wrong use of "topology" --"sequence" is fine in both places and in a later place also Response: We thank the reviewer for making the suggestion. We think that "topology" describes the configuration of the antibody molecule well, however. The other reviewers are fine with this term and use. Therefore, we will remain using "topology" to refer to the sequence order of the antibody functional moieties. line 387 the Tri-nAb is an excellent candidate for the next ....
Response: All the points listed above were corrected.

Comment
Why not use Fig. S2 instead of Fig 2C --show real data, not some hand-picked single image Response: We think that the images in Fig. 2C makes the point well that the bispecific scFv crosslinks the adjacent protomers within one trimer. For more details, the reader can look at more images in Fig. S2. Indeed, the images in Fig. 2C represent those in Fig. S2 well.

Reviewer #3 Comments
The field of therapeutic antibody formats is quickly developing especially for infectious diseases as so much good science has shown the need to target the pathogen at multiple epitopes in order to weaken the pathogen's ability to adapt by mutation. Furthermore, the demonstration that passive immunization, provided by an antibody format, provokes additional mechanisms of ultimate protection such as strengthening the adaptive and endogenous humoral responses, makes the exploration of optimal configurations essential for success. Steinhardt and colleagues have taken a rationale design approach to the problem. Their study starts with a simple construct and builds to a higher and higher avidity through more complicated antibody-based structures leading to increasingly significant capacity to neutralize across a very broad panel of viral strains. The explanations are well thought through and presented with the following comments: The rationale for choices of target epitopes are validated with figure 1 providing a clear explanation of this as well as for the design of the constructs. The demonstration of the co-binding is convincing, with beautiful visual support provided by the electron micrographs of the binding interfaces using the subsaturating concentration group. Response: We appreciate the enthusiasm of the reviewer.

Comments
Generally, as I am not an expert on the details of the viral neutralization assays, the data looks convincing. However, I challenge the helpfulness of providing a GEO mean in figure 3A and B as, for example in B, the BiSc-Fv has several red lines while the Bi-Nab format has only one red line, yet they have equivalent Geo Means (0.306 vs 0.297). The way the data is presented in figure 4 B is a better way to communicate the message.
Response: We thank the reviewer for looking at the data carefully and pointed that we should have done a better job to clarify the data. The original IC50 Geo mean presented in Fig. 3A and B was calculated using IC 50 values only against SENSATIVE viruses. Therefore, PGT121, with many "blue" strips (resistant, therefore not counted), displays a much lower IC50 Geo mean than the other antibodies with better breadth.
To clarify this point, in the current Fig. 3A and B, we renamed the original IC50 Geo mean as "IC50 GMT"(Geometric titer), which was derived from SENSATIVE virus IC50 titers. The field uses IC50 Geomean to describe the potency against SENSATIVE viruses, which is the "working" concentration of antibodies.
In addition, we used another term, "Total C50 GMT" to describe IC 50 when all IC 50 values including those for the resistant viruses. In the case of resistant viruses, an IC 50 value of 50 ug/ml (the highest antibody concentration in the neutralization assay) was assigned to the corresponding antibody. In another word, IC50 GMT describes the potency against the Sensitive viruses, which reflects the effective working concentration of particular antibody; whereas Total IC50 GMT describes the overall potency of a given antibody with the consideration of breadth (since IC50 values against resistant viruses are included). For example, PGT121 has 63% breadth with many resistant viruses also has very low IC50 GMT but high Total IC50 GMT. For antibodies such as the Bi-NAb with higher breadth than PGT121, the IC50 GMT and Total IC50 GMT is very close. We think that the two forms of IC50 GMT values would help to better describe the neutralization breadth and potency of a given antibody. Response: We thank the reviewer for spotting this inconsistency in the text. We corrected the text in lines 234-236, reads as " We also noted that the five-or four-tandem G4S linker length (5X or 4X) was slightly better than the shorter variants (3X) (Fig. 3A, S3A Response: We provided additional SEC and DLS data to address the homogeneity of the biAb or TriAb batches in Fig. S4A-C, and Fig. S5 F-H, respectively, and described in text (lines 240-247, 298-301) accordingly. Briefly, the Bi-ScFv is highly homogenous in size, while Bi-NAb displays a minor aggregate peak in size distribution by intensity in DLS, which is virtually undetectable in size distribution by volume plot in DLS (account for 0.1% of the total particle population), indicating practically negligible level of aggregation. The Tri-NAb shows a single peak in DLS with no aggregate form, similar to the parental antibody VRC01.

Comments
One minor comment is to fix the sentence in the Intro on line 79 as there seems not to be needed 'as well as' . Response: Corrected.

Comments
This study and publication of the manuscript are very timely and will be of genuine interest to the field. Just this week, Science has published two articles in this domain, highlighting the importance to medicine: 'Why is the flu vaccine so mediocre?' J Cohen Science 22 Sep 2017. 'There's an increasing call now to improve the vaccine and organize the research community to more collaboratively try to develop a "universal" flu shot that works against many strains and lasts for many years, if not a lifetime.' Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques Ling Xu1 et al.,Science 20 Sep 2017: The development of an effective AIDS vaccine has been challenging due to viral genetic diversity and the difficulty in generating broadly neutralizing antibodies (bnAbs). Here, we engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: 1) the CD4 binding site, 2) the membrane proximal external region (MPER) and 3) the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to human bnAbs, and conferred complete immunity against a mixture of SHIVs in non-human primates (NHP) in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and could be applicable for diverse diseases, including infections, cancer and autoimmunity.
I would suggest that as the authors see that sometimes the biAb or TriAb do not do as well as the parental mAb for certain viral strains, they might comment in the discussion on how yet a future approach could do even better (e.g., mixtures). But that suggestion is optional.
Response: we took this notion and added the text in lines 410-414, reads, "In the neutralization assay using a virus panel consisting 208 virus isolates, the Tri-NAb missed one highly resistant virus strain, 6471.V1.C16 (Fig. S7), which can be neutralized by one of the parental bNAbs, 10E8. This suggests that a combination of multi-specific antibody with selected complimentary monoclonal antibody may achieve superior efficacy to conventional antibody cocktail or multi-specific antibody alone." Comment Thus, post addressing the above comments, my opinion would be favorable for publication of this manuscript in Nature Communications. Response: We thank the reviewer for the supportive comments.
The authors have addressed all my concerns satisfactorily. Thank you as much easier to evaluate and form an opinion with the data presented in this new manner. I have no further items to communicate. I find the data strong and their interpretation accurate. The study and it's data will add significant value to the field and hopefully bring an effective therapeutic strategy to patients.

Reviewer #4 (Remarks to the Author):
This is an interesting study by Steinhardt et al. describing a new tri-specific reagent capable of neutralizing HIV. While it cannot be considered groundbreaking given the publication of numerous other multi-valent proteins against HIV, some with greater clinical potential than the one described here, the work is well executed and does add enough to what is known as to warrant publication. With respect to the comments by Reviewer #1, they raise valid criticisms and questions regarding the lack of improved potency for the tri-specific reagent. These questions are largely answered by the responses by the authors to the critique. It is conceivable that the specific choice of linker designs could limit any possible gains in potency that might be observed by other published configurations and the field should be made aware of this. The reviewer's point regarding the comparison to neutralization assays with combinations of single agents is a reasonable one to raise, but the I agree with the authors that it has been shown fairly conclusively that combinations of single agents display additive (rather than synergistic) properties. For good measure, the authors have performed the requested assays and confirmed this for the combination of two antibodies. The last point made by the reviewer regarding the inferiority of this particular tri-specific reagent to others is accurate based on the available data, but I don't feel should preclude publication given that this could be another tool in the arsenal of reagents that have been developed against HIV. In my opinion, it is important for there to be second and third-line reagents in the pipeline in case the primary candidates fail during development.


s2fieldsofstudy: Biology Psychology

text:
Apraxia profiles—A single cognitive marker to discriminate all variants of frontotemporal lobar degeneration and Alzheimer's disease

Introduction Apraxia is common in neurodegenerative dementias but underrepresented in clinical workup for differential diagnoses. Methods Praxis-profiles were assessed with the Dementia Apraxia Test in 93 patients with early stages of biologically supported Alzheimer's disease or frontotemporal lobar degeneration: semantic primary-progressive aphasia, nonfluent primary-progressive aphasia, and behavioral variant frontotemporal dementia. Associations with core cognitive deficits of the dementia subtypes (i.e., visuospatial, sociocognitive, and semantic-linguistic) were explored. Results Patients showed significant apraxia compared with healthy controls but also disease-specific praxis-profiles. Using only the Dementia Apraxia Test, all four dementia subtypes could be correctly discriminated in 64.4% of cases, and in 78.2% when only distinguishing Alzheimer's disease versus frontotemporal lobar degeneration. Praxis-profiles showed consistent associations with core cognitive impairments of the different dementia subtypes. Discussion The Dementia Apraxia Test is a valid, time-efficient and versatile cognitive marker to delineate variants of frontotemporal lobar degeneration and Alzheimer's disease in clinical routine, facilitating differential diagnoses of dementia subtypes in early disease stages.

Background
Apraxia relates to a neuropsychological deficit regarding imitation or pantomiming of limb or face postures despite intact sensorimotor skills and task comprehension [1,2]. Impairments in praxis may occur early in a range of neurocognitive disorders and can be used as a cognitive marker for early neurodegenerative dementias [3][4][5]. Despite its recent inclusion as a basic cognitive domain in the Diagnostic and Statistical Manual of Mental Disorders (5th Edition), praxis assessment is underrepresented in both routine neuropsychological workup and current diagnostic criteria for neurocognitive disorders [6,7].
Disease progression, rate of functional decline, caregiver burden, and therapeutic approaches differ profoundly between neurodegenerative dementia subtypes [8,9]. A correct differential diagnosis in early disease stages is thus essential for patients, caregivers, and health practitioners.
In the absence of ready-to-use biomarkers for frontotemporal lobar degeneration (FTLD), clinical criteria for the behavioral variant frontotemporal dementia (bvFTD) and the two language variants, nonfluent primary progressive aphasia (nfPPA) and semantic variant primary progressive aphasia (svPPA), have been consecutively refined, improving their diagnostic accuracy [10,11]. Nevertheless, neither unambiguous operationalizations of diagnostic core features (e.g., "loss of empathy" for bvFTD; "semantic memory dysfunction" for svPPA) nor straightforward and brief clinical tests with sufficient differential value to discriminate between these dementia subtypes simultaneously are available. The correct application of the clinical criteria and a clinical differentiation of the FTLD variants from each other and from Alzheimer's disease (AD) thus remains challenging, particularly in nonexpert settings and for patients in early disease stages [12][13][14].
Although standardized neuropsychological testing is recommended to differentiate between the different dementia types in early disease stages, a range of former neuropsychological principles have recently been questioned or shown to be invalid for a reliable differentiation between FTLD and early stage AD: Impairments in verbal memory tests may occur to similar degrees in patients with bvFTD and AD either due to confounding executive influences or due to AD-like hippocampal atrophy in subsamples of patients with bvFTD [15,16]. Similarly, cognitive domains that place high demands on language (including verbal memory tests) are frequently confounded with deficits in task comprehension and/or semantic memory in patient with PPA [17,18]. Taken together, a considerable overlap regarding performance in standard neuropsychological domains exists between patients with different underlying neurodegenerative etiologies, particularly when only timeefficient screening tests are available [9,19,20].
Standardized assessment of praxis-profiles is time efficient, reliable, and places comparatively little demands on potentially confounding cognitive influences such as working memory or language comprehension. It may thus serve as a versatile neuropsychological tool to differentiate clinically heterogeneous dementia subtypes in early disease stages. However, although mentioned as a basic cognitive domain, apraxia is largely neglected in clinical or neuropsychological routine examinations. Here, we explored patterns of praxis disturbances and tested the clinical feasibility of a single praxis screening for the differential diagnosis between early stages of AD and the three most frequent clinical variants of FTLD (bvFTD, svPPA, and nfPPA). We hypothesized that specific praxis-profiles (operationalized by divergent performance in different praxis domains) are associated with core cognitive deficits of the different dementia syndromes (i.e., visuospatial deficits in AD, linguistic-semantic deficits in PPA, and social cognitive impairment in bvFTD).

Participants
A total of 93 patients with early (,3 years after the first symptom-onset) neurodegenerative diseases were enrolled in the memory disorder unit at the Department of Neurology at the University Hospital M€ unster, Germany. The initial diagnostic workup was conducted at our inpatient clinic and included neurological examination, history taking with patients and caregivers, consultation of medical records, comprehensive neuropsychological testing as presented in detail elsewhere [19], motor and speech assessment, and analysis of cerebrospinal fluid (CSF) for dementia biomarkers. Structural T1-and fluid-attenuated inversion recovery-magnetic resonance imaging images of the brain were available from all patients. In addition, 18-Fluorodeoxyglucose Positron Emission Tomography scans were available in 70% (46/66) of patients with suspected FTLD. All recruited patients matched the current criteria for probable AD, probable bvFTD, or imagingsupported PPA evaluated by a multidisciplinary team of senior neurologists and neuropsychologists [10,11,21]. Briefly, patients with probable AD (N 5 27) presented with memory decline objectified in episodic memory tests and had high or at least intermediate evidence for the pathophysiological process of AD based on neuroimaging and biomarker constellation [21]. Patients with probable bvFTD (N 5 31) presented with symptom constellations of social conduct decline, apathy, loss of empathy, and/or executive dysfunction in neuropsychological assessment as well as a consistent frontal and/or anterior temporal atrophy or hypometabolism [10]. Patients with svPPA (N 5 21) showed prominent naming and fluency deficits but circumlocutory speech as initial symptoms, whereas patients with nfPPA (N 5 14) initially presented with effortful, halting speech either with or without agrammatism. All patients with PPA showed signs of either brain atrophy or hypometabolism consistent with clinical diagnosis (imaging supported). Patients with mixed PPA or logopenic variant were not included due to their high clinical and pathologic heterogeneity [22]. Dementia biomarker constellation was not indicative of AD in patients with PPA or bvFTD diagnosis. Exclusionary criteria for all patients were as follows: history of stroke, brain tumor, traumatic brain injury, major psychiatric disorders, severe vascular lesion load (Fazekas score 2), and inflammatory CSF or motor impairment (including parkinsonism).
Healthy age-matched control subjects (HC; N 5 34) were relatives of patients, members of the hospital staff, or community-dwelling elderly screened for neurological and psychiatric disorders. HCs with a Mini-Mental State Examination (MMSE [23]) , 28 were excluded. The study was approved by the local ethics committee (2012-365-f-S), and participants gave written informed consent.

Praxis assessment
We used the Dementia Apraxia Test (DATE) as a screening for praxis impairment as previously described [24]. The test was recently recommended by the European Neurodegenerative Diseases Working Group, and it provides good psychometric properties, standardized stimulus material, and instructions as well as a reliable clinical rating system [6]. It provides a total apraxia score (DATE total) composed of five subscales (two subscales for limb apraxia and three for buccofacial apraxia): for limb apraxia (DATE 1), the subscales imitation of meaningless hand postures (limb imitation) and pantomiming of common objects (object pantomime) are tested. For buccofacial apraxia (DATE 2), the test assesses imitation of face postures (face imitation), emblematic buccofacial postures (e.g., "show me how you clear your throat"; buccofacial emblems), and repetition of pseudowords to test for apraxic speech. Each subscale of the DATE can be described with regard to two dimensions: demands on stimulus-specific semantic knowledge (e.g., knowledge about a common tool vs. novel stimuli) and the involved body part (face vs. limbs). For example, whereas imitation of meaningless hand postures and repetition of pseudowords represent novel stimuli and place relatively little demands on semantic memory, pantomiming of common objects or emblematic buccofacial postures crucially depend on semantic knowledge about the respective stimuli and/or gesture.

Neuropsychological background testing
To evaluate the overall cognitive status and frontalexecutive impairment, we used the MMSE and the Frontal Assessment Battery, respectively [25]. To screen for specific neuropsychological dysfunctions, we used the Language Aphasia Screening Test [26] for semantic-linguistic abilities, the subtest "number location" from the Visual Object and Space Perception Battery [27] for visuospatial impair-ment, and the Emotion Recognition subtest of the Mini-Social Emotional Assessment [28] to explore sociocognitive deficits. Moreover, a standardized caregiver questionnaire for abnormal behavior, also tapping into social cognition, the Frontal Behavioral Inventory [29] was employed. All test procedures were administered and scored by neuropsychologists in accordance with the respective manual.

Statistics
Statistical analyses were performed using SPSS, version 25 (IBM). Group differences in demographic data, disease severity scores, and neuropsychological background tests were analyzed using analysis of variances. Group differences in praxis-profiles (DATE) were explored using a multivariate analysis of variance. Normality of distribution was confirmed by visual inspection and Kolmogorov-Smirnov tests. Multivariate discriminant function analysis was performed to determine how well dementia subtypes can be distinguished based on the DATE. Before this, the variance-covariance matrix was checked for strong inhomogeneity. To explore associations between neuropsychological background tests (see Section 2.3) and praxis performance, we computed Kendalls-Tau-b rank correlations for ordinal data using the whole patient sample. Table 1 summarizes demographics, disease severity scores, and clinical characteristics of the patients and HC. Significant between-group differences were found for age (F 5 2.705, df 5 4, P 5 .03) and sex (c 2 5 23.306, df 5 4, P , .000). Patients with AD were significantly older than patients with bvFTD, and there was a significantly higher proportion of males among bvFTD compared with the other groups, reflecting typical demographic differences between AD and bvFTD. Groups did not differ regarding years of education or disease duration. Comparison of CSF biomarker profile revealed lower total amyloid-b and CSF levels in the AD group compared with all other dementia groups. Total tau was significantly higher in the AD group than bvFTD. Performance in MMSE and Frontal Assessment Battery was significantly lower in all dementia subtypes compared with HC. Between dementia subtypes, MMSE scores were significantly lower in svPPA versus bvFTD patients, and scores in the Frontal Assessment Battery were lower in AD versus nfPPA. The average Frontal Behavioral Inventory score was significantly higher in patients with bvFTD than in patients with AD and nfPPA, whereas no difference between svPPA and bvFTD was seen. As expected, both PPA groups had significantly lower scores than all other dementia patients in the Language Aphasia Screening Test, but no differences emerged between nfPPA and svPPA. Patients with AD reached significantly better results than all other dementia subtypes in the Mini-Social Emotional Assessment emotion recognition score. For the Visual Object and Space Perception Battery "number location" subtest, no significant group differences were found. Taken together, although significant differences between specific groups on specific neuropsychological background tests were found, no single screening test was able to reliably differentiate between all four dementia subtypes. Table 2 summarizes results of the DATE for dementia subtypes and HC. DATE total score was significantly lower in all dementia subgroups compared with HC. The scale DATE 1-limb apraxia differed significantly between HC and patients with AD or PPA diagnosis. On the scale DATE 2-face apraxia, all dementia subtypes performed significantly lower compared with HC and patients with bvFTD and nfPPA performed lower than patients with AD. Fig. 1 indicates DATE subscale performance for each group. Significant differences between dementia types were found on all five subscales. Patients with AD showed significantly lower performance in limb imitation than patients with bvFTD. On the subscale object pantomime, results differed significantly between patients with svPPA and bvFTD. Patients with nfPPA were most impaired in face imitation, and bvFTD also showed a statistical trend toward lower performance in face imitation compared with AD patients. On the subscale buccofacial emblems, patients with svPPA and nfPPA scored significantly lower compared with AD. Patients with nfPPA were significantly more impaired on the subscale pseudowords compared with all other dementia subtypes. Taken together, we found group-specific praxis-profiles showing major deficits in limb imitation for AD, major deficits in buccofacial praxis domains for both bvFTD and nfPPA, major impairment in subscales demanding semantic memory for svPPA, and a differential deficit for nfPPA on the subscale measuring apraxic speech. To test how well these between-group differences in the different subscales (i.e., praxis-profiles) could discriminate between groups, we next used a discriminant function analysis on the data.

Differentiation of dementia subtypes based on praxis-profiles
Discriminant function analysis, including the dementia subtypes as grouping variable and the five subscales of the DATE as discriminating variables, identified three significant discriminant functions ( Fig. 2A). Function 1 (Wilks'l 5 0.443, c 2 5 66.438, P , .000) discriminated between AD versus bvFTD and AD versus nfPPA. The function showed positive correlations with all buccofacial praxis subscales: pseudowords (r 5 .697), face imitation   Fig. 2A displays means of the discriminant scores for all three functions grouped by dementia subtype. Regarding the efficiency for differential diagnosis using only the DATE, a total of 64.4% of cases could be correctly classified to the correct dementia subtype. Fig. 2B represents the percentage of classified cases for each dementia subtype to the different groups. Inspection of Fig. 2B shows that some groups were easier to classify than others: In patients with svPPA, a relatively large percentage of cases (20%) was falsely classified as having AD, whereas patients with AD were rarely classified as having FTLD (,15%). In fact, when only FTLD (bvFTD, nfPPA and svPPA merged) versus AD patients were discriminated, the classification accuracy of the DATE raised to 78.2%.

Associations between DATE subscales and cognitive screening tests
To confirm our hypothesis that praxis performance taps into specific cognitive abilities and that the disease-specific praxis-profiles reflect cognitive core symptoms of the different dementia subtypes, we computed rank correlations between the DATE subscales and neuropsychological background tests across the whole patient sample. Significant correlations were found between the VOSP "number location" subtest performance and subscales measuring limb apraxia: limb imitation (r 5 .258) and object pantomime (r 5 .240), indicating associations between visuospatial processing and these subscales. The language screening test LAST showed significant correlations with subscales that are highly   showing negative correlations with subscales depending on buccofacial abilities (i.e. face imitation, buccofacial emblems, and pseudowords), indicating that more behavioral dysfunction may be associated with less accurate face imitation. Table 3 summarizes results of these correlational analyses.

Discussion
Praxis impairments have extensively been studied in stroke, and its assessment in early stages of neurodegenerative diseases has only recently been a subject of systematic research [2,3,30]. Previous studies suggested that apraxia is promising as a cognitive marker for AD versus bvFTD [6,7]. We showed here that different neurodegenerative dementia subtypes display characteristic patterns of praxis impairment in early stages when directly contrasted using a single apraxia screening test. These praxis-profiles were effective differential diagnostic markers and showed close and coherent associations with already established and typical core cognitive deficits of AD, bvFTD, svPPA, and nfPPA.

The DATE as a versatile tool for clinical dementia subtype classification
Neuropsychological tests are designed to tap into dysfunction of specific neuroanatomical structures (e.g., episodic memory tests primarily quantify medial temporal lobe function) [31]. Clinicians, however, are often confronted with a large overlap between dementia subgroups regarding neuropsychological test performance [19,20]. In light of diffuse neuronal loss common to neurodegenerative diseases irrespective of the underlying etiology, overlapping cognitive symptoms are the rule rather than the exception in clinical routine. "Profiles" of deficits reflecting foci of relative neuronal loss in certain brain regions (e.g., frontal lobe vs. parietal lobe) or in large-scale functional networks may better account for cognitive and clinical heterogeneity in dementia syndromes than performance in single cognitive domains [32,33]. A detailed functional assessment of remote neuroanatomical structures is however time-consuming, and some tests are  not applicable in certain patient groups (e.g., due to comprehension deficits in aphasic patients). Standardized brief tests that display characteristic patterns in different etiologies are sparse. In line with this, we found that no standard neuropsychological background screening test used here was able to reliably differentiate between all four diagnostic groups and HC simultaneously. In contrast, patient's performance across the DATE subscales varied systematically depending on the diagnosis. While there was overlap between dementia subtypes in single praxis domains, variance between groups extracted by multivariate discriminant function analysis revealed disease-specific patterns. Based on this between-group variance, a correct classification of 64.4% of patients into the correct diagnostic category (according to current clinical guidelines) was achieved, a proportion that is remarkable given that chance-level is at 25% with four diagnostic categories. For the differentiation between all clinical variants of FTLD combined versus AD, an even better diagnostic value of 78.2% correct classifications was reached. We interpret these findings as a proof-of-principle that disease-specific profiles in praxis performances can be effectively used for differential diagnosis across a range of early neurodegenerative diseases. Given that the DATE can be administered in less than 15 minutes, our results argue to include praxis assessment into the diagnostic workup for early neurodegenerative diseases.

Specific profiles of praxis impairments in each dementia syndrome
Previous studies have shown that praxis can be impaired early in AD and bvFTD with evidence for more severe impairment in limb praxis for AD and in buccofacial praxis for bvFTD [5,34]. Only two studies have specifically explored apraxic deficits in patients with PPA, showing higher degrees of praxis deficits across all domains in patients with nfPPA compared with other subtypes [4,35]. Because definitions of apraxia and methods of assessment differed profoundly between studies, results cannot be directly compared, and the relevance of such findings for differential diagnosis is unclear. To our knowledge, this is the first study to directly contrast results in a single standardized test for praxis abilities in clinically well-defined samples of the most frequent young-onset neurodegenerative dementia syndromes, AD, bvFTD, nfPPA, and svPPA.
In line with our previous work on the degree and profile of praxis impairment, patients with AD were impaired in limb imitation and to a lesser degree in object pantomime, whereas performance in buccofacial praxis domains was mostly unimpaired. Current evidence from structural neuroimaging points to a crucial dependence on left and medial parietal lobe integrity for limb imitation and to more ventrally temporal neural substrates for object pantomime [30,36,37]. Such results form the basis of the proposed dual-route model for gesture production [38]. Consistent with this, parietal and temporal cortices are early targets of neurodegeneration in AD and have been previously associated with visuospatial processing and object semantics, respectively, thus reflecting core features of cognitive and neural dysfunctioning in AD. In line with this, limb apraxia subscales as measured by the DATE were significantly correlated with performance in the VOSP 7, a cognitive test for visuospatial processing.
In contrast, patients with bvFTD showed major impairments across the buccofacial praxis subscales. Although the neural basis of buccofacial praxis abilities is far less clear, functional neuroimaging studies in healthy participants point to a crucial role of frontal, subcortical, and anterior temporal areas (including the insular) for decoding information from visually presented faces [39]. As both faces and speech convey information relevant for social conduct, deficits in buccofacial praxis may be related to core symptoms of bvFTD such as loss of empathy, thus reflecting core symptoms of this dementia subtype [10]. Although more evidence is needed here, our result of significant correlations between buccofacial praxis subscales and measures for social cognition (i.e., emotion recognition and behavioral abnormalities) support this hypothesis.
We also revealed disease-specific profiles of praxis impairment using the DATE in patients with the two language variants of FTLD. Similar to patients with bvFTD, patients with svPPA showed relatively little impairment in limb imitation and pseudoword repetition, domains that do not place high demands on semantic memory and foreknowledge. Instead, the praxis-profile of svPPA was characterized by pronounced and unique impairment regarding buccofacial emblems (e.g., "show me how you clear your throat") and to a lesser degree regarding object pantomime. Correlations between these scales and the Language Aphasia Screening Test suggest that these impairments are mediated by deficits regarding stimulus-specific semantic memory. This interpretation also fits well with the neuroanatomical evidence that semantic knowledge is predominantly subserved by areas in the anterior (ventral) temporal lobe, the key target of neurodegeneration in svPPA [40].
Compatible with previous studies, patients with nfPPA showed the most pronounced praxis impairment from all dementia groups across all subscales but with a unique feature of apraxia of speech (pseudoword repetition) [4,35]. Clinically, nfPPA is the group with the most severe and global language production and comprehension deficits, similar to the prototypic Broca's aphasia following left media infarction. Neuroanatomically, nfPPA is characterized by a large-scale damage to the left temporal and posterior frontal lobe [40]. Within the framework of the aforementioned dual-route model for gesture production, atrophy in these areas may ultimately impair both routes, the ventral "what" route, associated with the semantic components of gestures (e.g., object and manipulation knowledge) and the dorsal "how" route, crucial for online-processing during imitation and spatial decoding of novel stimuli [38].

Conclusion
Brief cognitive tests for standard neuropsychological domains (e.g., memory, language, and executive functions) often yield inconclusive results regarding clear differential diagnoses of clinical variants of FTLD and AD. In the case of PPA, a wide range of neuropsychological tests is invalid as they heavily rely on comprehension and language skills. The present study showed that distinctive praxis impairments can efficiently and validly be assessed across clinically heterogeneous neurodegenerative dementia syndromes, supporting clinicians in the differential diagnosis of AD and FTLD subtypes in early disease stages. Results also showed that standardized assessment of praxis domains effectively taps into core cognitive deficits of these dementia syndromes, validating previous hypotheses regarding the cognitive basis of differential praxis-profiles in dementia (i.e., limb apraxia associated with visuospatial impairment, buccofacial imitation associated with sociocognitive abilities, and pantomime/communicative gestures associated with semantic memory and language). The current results thus extend the evidence that apraxia is a multifaceted rather than a homogeneous cognitive disorder. Future studies should directly explore the structural and functional neural bases for praxis domains beyond the known brainbehavior links for limb imitation and object pantomime in early neurodegenerative diseases.


extfieldsofstudy: Chemistry Medicine

text:
Transforming Growth Factor-β and Interleukin-10 Synergistically Regulate Humoral Immunity via Modulating Metabolic Signals

Inhibitory cytokines, such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), are humoral factors involved in the suppressive function of regulatory T cells and play critical roles in maintaining immune homeostasis. However, TGF-β and IL-10 also have pleiotropic effects and induce humoral immune responses depending on conditions, and thus their therapeutic application to autoimmune diseases remains limited. Here, we show that a combination of TGF-β and IL-10, but not single cytokine, is required to suppress B cell activation induced by toll-like receptor (TLR) stimulation. In in vivo analyses, the simultaneous presence of TGF-β and IL-10 effectively suppressed TLR-mediated antigen-specific immune responses and ameliorated pathologies in imiquimod (TLR7 agonist)-induced lupus model and lupus-prone MRL/lpr mice. Intriguingly, TGF-β and IL-10 synergistically modulated transcriptional programs and suppressed cellular energetics of both glycolysis and oxidative phosphorylation via inhibition of the mammalian target of rapamycin complex 1 (mTORC1)/S6 kinase 1 (S6K1) pathway in TLR-stimulated B cells. On the other hand, enhancement of mTOR signaling and mitochondrial biosynthesis in TLR-stimulated B cells counteracted the synergistic inhibitory effects. The inhibitory cytokine synergy of TGF-β and IL-10 via suppression of energy metabolism was also observed in human TLR-stimulated B cells. There is increasing evidence supporting the importance of adequate metabolic signals in various immune cells to exert their immune function. In this study, we have shown that a previously unrecognized synergy of inhibitory cytokines regulates systemic humoral immune responses via modulating immunometabolism in B cells. Our findings indicate that inhibition of B cell metabolism mediated by two synergistic cytokines contributes to the induction of immune tolerance and could be a new therapeutic strategy for autoimmune diseases such as systemic lupus erythematosus.

Inhibitory cytokines, such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), are humoral factors involved in the suppressive function of regulatory T cells and play critical roles in maintaining immune homeostasis. However, TGF-β and IL-10 also have pleiotropic effects and induce humoral immune responses depending on conditions, and thus their therapeutic application to autoimmune diseases remains limited. Here, we show that a combination of TGF-β and IL-10, but not single cytokine, is required to suppress B cell activation induced by toll-like receptor (TLR) stimulation. In in vivo analyses, the simultaneous presence of TGF-β and IL-10 effectively suppressed TLRmediated antigen-specific immune responses and ameliorated pathologies in imiquimod (TLR7 agonist)-induced lupus model and lupus-prone MRL/lpr mice. Intriguingly, TGF-β and IL-10 synergistically modulated transcriptional programs and suppressed cellular energetics of both glycolysis and oxidative phosphorylation via inhibition of the mammalian target of rapamycin complex 1 (mTORC1)/S6 kinase 1 (S6K1) pathway in TLR-stimulated B cells. On the other hand, enhancement of mTOR signaling and mitochondrial biosynthesis in TLR-stimulated B cells counteracted the synergistic inhibitory effects. The inhibitory cytokine synergy of TGF-β and IL-10 via suppression of energy metabolism was also observed in human TLR-stimulated B cells. There is increasing evidence supporting the importance of adequate metabolic signals in various immune cells to exert their immune function. In this study, we have shown that a previously unrecog nized synergy of inhibitory cytokines regulates systemic humoral immune responses via modulating immunometabolism in B cells. Our findings indicate that inhibition of B cell metabolism mediated by two synergistic cytokines contributes to the induction of immune tolerance and could be a new therapeutic strategy for autoimmune diseases such as systemic lupus erythematosus. Immune Regulation by Cytokine Synergy Frontiers in Immunology | www.frontiersin.org June 2018 | Volume 9 | Article 1364 inTrODUcTiOn The balance of cytokines controls immune responses and is implicated in the pathogenesis of autoimmune diseases (1). Inhibitory cytokines like transforming growth factor-β (TGF-β) (2)(3)(4) and interleukin-10 (IL-10) (5,6) are key regulators of immune homeostasis, as mice deficient for either of these regulatory cytokines develop severe inflammatory diseases (7,8).
Blockade of pro-inflammatory cytokines offers effective treatment of inflammatory autoimmune diseases such as rheumatoid arthritis (9,10). However, therapeutic effects of blockade of proinflammatory cytokines for other systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are limited (11). Regulatory T cells (Tregs) are a subpopulation of T cells, which play an essential role in maintaining immunological self-tolerance, and their immunoregulatory mechanisms have been vigorously investigated and considered for treatment application in recent years (2). Inhibitory cytokines are one of the major contributors to immune tolerance by Tregs but therapeutic application of inhibitory cytokines remains limited; difficulties in use are due to their pleiotropic and context-dependent effects (12,13). The pathogenesis of SLE is supposed to be a sustained loss of self-tolerance and autoantibody production contributed by genetic and environmental factors (14). Among immune cells, B cells are centrally involved in the immunopathogenesis of SLE (15). Only belimumab, an anti-B cell activating factor (BAFF), is a targeted biologic agent for SLE approved by the US Food and Drug Administration (14,15). B cells are one of the primary targets for regulating autoimmune diseases because high-affinity antibodies are produced through interaction of germinal center B (GCB) cells with follicular helper T (TFH) cells (16,17). Also, extrafollicular B cell activation by sensing nucleic acid via innate receptors, including toll-like receptors (TLRs), contributes to the production of autoantibodies and the pathogenesis of SLE (18,19).
The effects of representative inhibitory cytokines, TGF-β, and IL-10, on B cells have been examined. Physiological importance of signaling from TGF-β family cytokines in T cells has been elucidated using Tgfbr2 fl/fl CD4-Cre + mice which show TFH cell accumulation and self-reactive B cell activation (20). We recently revealed that TGF-β3 produced by CD4 + CD25 − LAG3 + Tregs (LAG3 + Tregs) is required for the adequate control of humoral immunity (3,21,22), indicating immunological roles of TGF-β3 in immune tolerance. On the other hand, the direct effects of IL-10 on B cells are reported to be stimulatory (12), although IL-10 exerts a suppressive function on TFH cells (23). Cytokines display diverse functions in combination with each other, and the combined response of multiple cytokines, which has greater effects than the sum of its parts, is termed cytokine synergy (24). However, synergistic effects of TGF-β and IL-10 in humoral immunity have not yet been investigated.
In this study, we have investigated the roles of TGF-β and IL-10 on humoral immune responses. We examined the effects of these cytokines on B cells from mice or humans under TLR stimulation in vitro. Further, therapeutic potentials of these inhibitory cytokines on a mouse model of SLE are also verified. Our findings of synergistic regulation of humoral immunity by TGF-β and IL-10 via regulating cellular metabolism provide novel insights for clinical applications of inhibitory cytokines on autoimmune diseases.

construction and injection of the Plasmid Vector
Full-length fragments of murine Il10, Tgfb1, and Tgfb3 were subcloned into the pCAGGS vector, which has the CAG (cytomegalovirus immediately early enhancer/chicken β-actin hybrid) promoter, kindly provided by Junichi Miyazaki (Osaka University Medical School, Japan). The recombinant plasmids were transformed into Escherichia coli JM109 (Toyobo, Osaka, Japan) and isolated in large scale using the EndFree plasmid Maxi kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Purified plasmid DNA (100 µg) of pCAGGS-Mock, pCAGGS-Tgfb1, and pCAGGS-Tgfb3 in 100-200 µl Ringer's solution was administered i.v. Purified plasmid DNA (50 µg) of pCAGGS-Il10 in 1 ml Ringer's solution was administered i.v. Serum cytokine protein levels after each vector administration were measured using a mouse latent TGF-β1 ELISA kit (BioLegend) and a mouse latent TGF-β3 ELISA kit (MyBioSource, San Diego, CA, USA), according to the manufacturer's protocol.
injection of Plasmid Dna into Mrl/lpr Mice MRL/lpr mice were administered 100 µg pCAGGS-Mock or pCAGGS-Tgfb3 plasmid vectors i.v. every 4 weeks and were analyzed at the age of 21 weeks. Spleen weights were also measured then. Proteinuria was semi-quantitatively assessed using dipsticks (Albustix; Bayer, West Haven, CT, USA). Anti-dsDNA antibody levels in the sera of 21-week-old mice were measured using a mouse anti-dsDNA ELISA Kit (Shibayagi, Gunma, Japan), according to the manufacturer's protocol. Mice were randomly assigned to specific treatment groups, and to prevent research outcomes from being influenced by observer bias, proteinuria progressions were evaluated by an examiner blind to the experimental conditions.

Quantification of nP-specific antibody responses
Anti-NP IgG, IgG2b, IgM, or IgA levels were quantified by ELISA on plates coated with NP8-bovine serum albumin (BSA) (Biosearch Technologies) for the capture antigen. Serially diluted pooled sera from NP-KLH or NP-LPS immunized C57BL/6 mice were utilized as a standard. Following the incubation with sample and control sera, HRP-conjugated goat anti-mouse IgG, IgG2b, IgM, or IgA antibody (Bethyl Laboratories) and further TMP substrate (KPL, Gaithersburg, MD, USA) were added to the plates.

Fluorescence Microscopy and Detection of gFP-lcii
Splenic B cells from GFP-LC3 mice were cultured for 3 days with the indicated cytokines. Cells were washed with 0.05% saponin for 3 min and fixed for 10 min at room temperature with 4% paraformaldehyde. Images were captured using a fluorescence microscopy instrument (IX83, Olympus, Tokyo, Japan). GFPpositive puncta were counted manually in a blinded manner. For FCM assessment of GFP-LCII, cultured cells were treated with 100 µM chloroquine the last 4 h, washed with 0.05% saponin for 3 min, and analyzed.

Quantitative real-Time Pcr
Total RNAs were extracted with RNeasy Micro Kit (Qiagen) and were reverse-transcribed to cDNA with random primers (Invitrogen) and SuperScript III (Invitrogen), as

rna-sequencing
Splenic B cells (3.3 × 10 6 ) in 24-well plates were stimulated by 10 µg/ml LPS in the presence of 2 ng/ml TGF-β3 and/or 50 ng/ml IL-10 for 3 days. Total RNA of flow cytometrically purified B220 + 7-AAD − B cells from the cultured cells was extracted with RNeasy Mini kit (Qiagen) and libraries were prepared using TruSeq Stranded mRNA HT Sample Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. Sequencing was performed using HiSeq 2500 instrument (Illumina). Sequence reads were mapped to the mouse genome GRCm38/mm10 by TopHat2, and read counts were obtained using HTSeq. The detection of differentially expressed genes (DEGs) measured by the informative/non-informative (I/NI) value more than 0.2 as well as the normalization were performed using the R package Dexus. Hierarchical clustering and heatmap visualization of the DEGs were further performed using the R package Heatplot. DEGs were clustered into 16 groups and pathways in each group were investigated using Inguinal Pathway Analysis software (Qiagen). Fisher's exact test was used for the calculation of P-value determining whether the pathways were enriched with genes of interest.

extracellular Flux analysis
An XF96 Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used for the quantification of oxygen consumption rate (OCR) and ECAR. OCR was measured in XF media (Agilent Technologies, Santa Clara, CA, USA) supplemented with 1 mM sodium pyruvate and 10 mM glucose and To address the similarities and differences among inhibitory cytokines affecting humoral immunity, we first analyzed the effects of TGF-βs on B cells in vitro. Consistent with our previous report that TGF-β3 inhibited B cell proliferation under anti-IgM stimulation (3), TGF-β1 and TGF-β3 inhibited proliferation of B cells in the presence of anti-CD40 antibody-mediated co-stimulation without affecting cell death ( Figure 1A; Figure S1A in Supplementary Material). Total IgG production under stimulation with anti-CD40 and IL-4 was also suppressed by TGF-β1 or TGF-β3 ( Figure 1B). Further, either TGF-β1 or TGF-β3 suppressed the differentiation of B220 + CD138 + plasmablasts in anti-CD40-and anti-IgMstimu lated B cells (Figure 1C). To examine their function in vivo, we constructed a plasmid pCAGGS vector (27,28) that expressed Tgfb1 or Tgfb3, thereby inducing high serum levels of these cytokines (Figures S1B in Supplementary Material) (3,21,27). Administration of pCAGGS-Tgfb3 inhibited the development of B220 + GL-7 + Fas + GCB cells and suppressed antigen-specific anti body production in C57BL/6 mice immunized with a T celldependent antigen (Figures 1D,E; Figure S1C in Supplementary Material). In contrast, pCAGGS-Tgfb3 could not suppress CD4 + CD25 − CXCR5 + PD-1 + TFH cells with statistically significance ( Figure S1D in Supplementary Material). On the other hand, pCAGGS-Tgfb1 could neither suppress NP-specific antibody production nor GCB cells (Figures 1D,E). These findings indicate that TGF-β3 could regulate T cell-dependent humoral immune responses in vivo by inhibiting B cells.
synergistic inhibitory effect of TgF-β and il-10 on Tlr-related humoral immunity Innate and adaptive immune systems cooperate in the pathogenesis of autoimmunity (29), and the characteristic expression of TLRs on B cells makes cell-intrinsic connections of innate signals to adaptive immunity (30,31). Thus, we next examined whether TGF-βs could regulate TLR-stimulated B cell activation by using TLR stimulants which have pathogenic roles in SLE (14,32).
Notably, either TGF-β1 or TGF-β3 failed to inhibit LPS-stimulated B cell proliferation and instead enhanced the antibody production (Figures 2A,B; Figure S2A in Supplementary Material), consistent with previous observations (12,33). It is well known that Tregs produce not only TGF-βs but also IL-10 (2-4, 6, 22). Then, we hypothesized that the concomitant existence of TGF-βs and IL-10 would be important for the regulation of humoral immune homeostasis. As expected, contrary to the single addition of TGF-β or IL-10, simultaneous addition of TGF-β and IL-10 inhibited the proliferation and antibody production of LPS-stimulated B cells (Figures 2A,B; Figure S2A in Supplementary Material). Similar results were observed in B cells stimulated with R848, which is a selective ligand for TLR7 ( Figure 2C). We next verified whether TGF-β and cytokines other than IL-10 could show synergistic inhibitory effects on B cells. Although TGF-βs plus IL-6 provoke pro-inflammatory responses with inducing T helper 17 (Th17) cells as a cytokine synergy (34,35), TGF-β plus IL-6 show a synergistic enhancement, but not a synergistic inhibition, in antibody production of B cells ( Figure 2D). Also, a combination of TGF-β plus IL-17, a pro-inflammatory cytokine produced by Th17 cells (36), did not have synergistic inhibitory effects in antibody production of B cells ( Figure 2D).
To assess immunological roles of TGF-β and IL-10 on TLRrelated systemic immune responses in vivo, we investigated antigen-specific immune responses with blocking antibodies and pCAGGS plasmid vectors. Since both IL-10 and TGF-β have proinflammatory and inhibitory effects in vivo (12,13), we firstly evaluated the effects of endogenously produced IL-10 and TGF-βs in T cell-independent TLR stimulation. As a result, the administration of anti-pan TGF-β and anti-IL-10 neutralizing antibodies enhanced the T-cell-independent immune responses ( Figure 2E; Further, antigen-specific antibody production after the immunization with TLR-stimulating adjuvant (37) was suppressed with co-administration of pCAGGS-Tgfb1 or pCAGGS-Tgfb3 and pCAGGS-Il10, but not single pCAGGS plasmid vectors ( Figure 2F). These observations suggest that the synergistic effects between IL-10 and either TGF-β1 or TGF-β3 are comparable. Although the therapeutic effects of TGF-β1 on SLE produced by CD4 + CD25 + Foxp3 + Tregs, which also produce IL-10 (2), have not been verified, IL-10-producing LAG3 + Tregs exist in the steady state and TGF-β3 produced by LAG3 + Tregs showed therapeutic effects on lupus pathologies (3,6). Therefore, we focused on the cytokine synergy between TGF-β3 and IL-10, both of which are highly produced by LAG3 + Tregs (3,6). To evaluate the synergistic inhibitory effects of TGF-β3 and IL-10 directly in mice models with innate immune signals, we analyzed 4-hydroxy-3-nitrophenylacetyl (NP)-LPS immunized mice treated with pCAGGS-Tgfb3 and/or pCAGGS-Il10. Intriguingly, simultaneous treatment with both pCAGGS-Tgfb3 and pCAGGS-Il10, but not with pCAGGS-Tgfb3 or pCAGGS-Il10 alone, effectively suppressed NP-LPS-induced antibody production ( Figure 2G).
Next, we injected each pCAGGS plasmid vectors to murine models of lupus in order to assess the therapeutic effects of these cytokines. Importantly, treatment with both pCAGGS-Tgfb3 and pCAGGS-Il10 suppressed autoantibody productions in a murine lupus model induced with TLR7 agonist, imiquimod (26) (Figure 3A; Figure S3A in Supplementary Material). TLR signaling is also implicated in the pathogenesis of lupus-prone MRL-Fas lpr/lpr (MRL/lpr) mice (38)(39)(40). On the other hand, the pathogenic role of IL-10 in systemic autoimmunity has been suggested previously. For example, elevated levels of serum IL-10 correlate with disease activity of SLE (41) and anti-IL-10 antibody exhibits therapeutic effects in SLE (42). Consistent with the previous report (43), we also confirmed elevated levels of serum IL-10 in MRL/lpr mice ( Figure 3B). As expected, administration of pCAGGS-Tgfb3 alone showed therapeutic effects against anti-dsDNA antibody production, splenomegaly, and proteinuria progression in MRL/lpr mice (Figures 3C-E; Figure  S3B in Supplementary Material) and suppressed the development of TFH cells and B220 + CD138 + plasmablasts (Figures 3F,G). On the contrary, the MRL/lpr mice treated with pCAGGS-Tgfb3 did not exhibit a decrease in the number of CD4 + T cells and B220 + (44)(45)(46). These results indicate that TGF-β and IL-10 synergistically inhibit TLR-related humoral immune responses, and TGF-β3 has therapeutic potentials for the lupus-prone mice.

suppression of Mammalian Target of rapamycin complex 1 (mTOrc1)-Directed signaling in B cells by TgF-β3 and il-10
To identify the mechanisms underlying the inhibitory cytokine synergy, we conducted RNA-sequencing of LPS-stimulated B cells cultured in the presence of TGF-β and/or IL-10. Gene clustering of DEGs categorized the genes into 16 clusters; genes in cluster 6 showed a characteristic gene expression pattern that was enhanced by either TGF-β3 or IL-10 alone and suppressed by TGF-β3 and IL-10 in combination ( Figure 4A). Quantitative PCR analyses confirmed that genes related to cell proliferation, plasma cell differentiation, and endoplasmic reticulum-stress responses, such as Myc, Prdm1, and Xbp1, were suppressed, although transcriptional repressors such as Bcl6 and Bach2 were upregulated, in LPS-stimulated B cells treated with TGF-β3 and IL-10 (Figures 4B,C; Figure S4A in Supplementary Material). Intriguingly, simultaneous phosphorylation of STAT3 and Smad3 was observed in LPS-stimulated B cells with IL-10 and TGF-β3 This finding might suggest the importance of a cross talk with STAT3 and Smad3 for the cooperative action of the two cytokines. We next examined specific transcriptional regulation observed in TGF-β3-and IL-10-treated B cells. Pathway analyses of genes in the cluster 6 in Figure 4A suggested involvement of energy metabolism in TGF-β3and IL-10-treated B cells (Figure 5A). Potential upstream regulators predicted by Ingenuity pathway analysis (IPA) software showed that TGF-β3 and IL-10 downregulated mTORC1 signaling, which plays a central role in cellular metabolism (47,48) ( Figure 5B). mTOR signaling is important in enhancement of humoral immune responses (49), and an mTORC1 inhibitor ameliorates lupus pathologies (50). Thus, we evaluated the phosphorylation of downstream targets of mTORC1. Although the  Figure S4D in Supplementary Material). Expression of hypoxia inducible factor-1α (HIF1α) which regulates glycolysis (51,52) tended to decrease under the combination of TGF-β3 and IL-10. As expected, mTORC1 activator MHY1485 (53) counteracted the synergistic inhibitory effect of TGF-β3 and IL-10 on antibody production ( Figure 5E). Further, we administered anti-IL-10 and anti-TGF-β neutralizing antibody to NP-LPS-immunized mice and analyzed intracellular phosphorylation level of S6 in splenic B cells. Blockade of IL-10 and TGF-β induce phosphorylation of S6 in B cells (Figure 5F; Figure S4E in Supplementary Material).
These findings indicate that TGF-β3 and IL-10 synergistically regulate genetic programs through modulating mTORC1 signaling in B cells activated by innate immunity signals.
suppression of TgF-β3-induced Upregulation of autophagy by il-10 in lPs-stimulated B cells We went on to examine whether autophagic activities could be modulated through the suppression of mTORC1 signaling by the combination of TGF-β3 and IL-10. Autophagy is a conserved self-degradative process (54) essential for plasma cell development (55). Although mTORC1 signaling negatively regulates autophagy (56), enhanced mTORC1 signaling in LPS-stimulated B cells is essential for cell proliferation (57) and also high autophagic activities are observed in those cells (55). To determine autophagic activities in vivo, we utilized mice expressing transgenic GFP fused to microtubule-associated protein 1A/1B-light chain 3 (LC3) (25). As expected, LPS stimulation on B cells increased formation of GFP-LC3 + dots and GFP fluorescence, and TGF-β3 further enhanced the GFP-LC3 + dots and GFP fluorescence ( Figure 6A; Figures S5A,B in Supplementary Material). TGF-β3 prominently enhanced the GFP-LC3 + dots in LPSstimulated B cells (Figure 6A; Figure S5A in Sup plementary Material), and also enhanced GFP fluorescence ana lyzed by flow cytometry ( Figure S5B in Supplementary Material). Although IL-10 alone did not affect the autophagic activity in LPS-stimulated B cells, IL-10 counteracted the enhancement of autophagosome formation mediated by TGF-β3 ( Figures S5A,B in Supplementary Material). Next, we assessed whether the increased autophagic activities with TGF-β3 could affect antibody production of LPSstimulated B cells by using 3-methylademine (3-MA), an autophagy inhibitor. As expected, the enhanced antibody production by TGF-β3 in LPS-stimulated B cells was suppressed with 3-MA ( Figure S5C in Supplementary Material), autophagic activities might be partially involved in TGF-β3-induced enhancement of antibody production. Taken together, the simultaneous presence

TgF-β3 and il-10-Mediated inhibition of B cell Metabolic capacity
There is growing interest in bioenergetic homeostasis as a target of immunotherapy (50,52,58). It was reported that activated B cells utilize both glycolysis and oxidative phosphorylation (59). To evaluate the direct effects of metabolic modulators, we utilized pyruvate, which is a substrate of mitochondrial respiration in plasma cells (60) and a combination of mitochondrial fusion promoter M1 and fission inhibitor Mdivi1, which en hances mitochondrial respiration (61). As expected, pyruvate or a combination of M1 and Mdivi1 upregulated Prdm1, a key transcriptional regulator for the generation of antibodysecreting cells (62), in LPS-stimulated B cells. In contrast, glycolysis inhibitor, 2-deoxyglucose (2-DG), or a combination of mitochondrial inhibitors, rotenone and antimycin-A (Rot/AA), suppressed Prdm1 expression ( Figure 6B). In accordance with these observations, TGF-β3 and IL-10 synergistically regulated genetic programs related to energy metabolism, such as Cox5a, Cox5b, Ndufs7, and Atp5d expression ( Figure S6A in Supplementary Material). Then we hypothesized that B cell energy metabolism could be suppressed by TGF-β3 and IL-10 with relevance to the inhibition of mTORC1 signaling, and  we used a combination of membrane potential independent stain MitoTracker Green and membrane potential dependent stain MitoTracker DeepRed to assess respiring mitochondria (63,64). As expected, the combination of TGF-β3 and IL-10, but not single addition of TGF-β3 or IL-10, suppressed mitochondrial membrane potential in LPS-stimulated B cells ( Figure 6C).
Further, extracellular flux analyses revealed that both glycolysis and oxidative phosphorylation, which were enhanced by LPS stimulation, showed significant reduction in the presence of both TGF-β3 and IL-10 ( Figures 6D,E). Importantly, M1/ Mdivi1 counteracted the inhibitory effect of TGF-β3 and IL-10 through enhancing maximal OCR (Figures 6F,G). Further, in vivo combination of anti-TGF-β and anti-IL-10 neutralizing antibodies enhanced the mitochondrial membrane potential in B cells from NP-LPS-immunized mice ( Figure 6H). These results indicate that the synergistic inhibition by TGF-β3 and IL-10 on energy metabolism determines B cell immune function.
To further investigate whether these findings observed in mice could hold true in humans, we analyzed the effects of TGF-β3 and IL-10 on TLR-stimulated B cells isolated from healthy humans. We recently reported that high concentrations of TGF-β3 (1 ng/ml) alone could effectively suppress CpG-oligodeoxynucleotides (ODN)-stimulated human B cell antibody production with inhibition of BLIMP-1 expression (65). Nonetheless, determining the role of TGF-β3 at physiological concentration is of interest for therapeutic purposes in humans. In healthy humans, the median serum concentrations of TGF-β3 is at the 11.8 pg/ml (66). As expected, neither IL-10 nor 10 pg/ml TGF-β3 alone inhibited metabolic signals, but addition of IL-10 with 10 pg/ml TGF-β3 effectively suppressed CpG-ODN-stimulated human B cell antibody production with reduced oxidative phosphorylation and glycolysis (Figures 6I,J; Figures S6B,C in Supplementary Material). These results collectively indicated that the inhibitory cytokine synergy of TGF-β3 and IL-10 on TLR-stimulated B cells occurs via suppression of energy metabolism in a physiological condition and is a key mechanism for induction of humoral immune tolerance.

DiscUssiOn
TGF-βs and IL-10, potent inhibitory cytokines, have been investigated for their therapeutic potential in systemic inflammatory diseases (12,13). However, the immunostimulatory effects of IL-10 on B cells and humoral immunity including lupus pathologies (12), and pleiotropic immunological roles of TGF-βs (13) make application of these potent inhibitory cytokines elusive. Herein, we proposed an inhibitory cytokine synergy with TGFβ and IL-10 that control TLR-related humoral responses via controlling glycolysis and oxidative phosphorylation of B cells. Although recent evidence indicates a role for IL-10 in SLE pathogenesis (41,42), it was also reported that lupus like phenotypes of TGF-β3-sufficient MRL/lpr mice are exacerbated by genetic deletion of IL-10 (67). These findings suggest that physiological expression levels of IL-10 might be necessary for the control of lupus pathology. Although the mechanisms of action of these cytokines in immune cells remained poorly elucidated, latest reports uncovered that TGF-β signaling inhibited natural killer cell activation via suppressing mTOR signaling (68) and IL-10 inhibited LPS-induced macrophage activation via suppressing glycolysis (63). There is growing evidence that metabolic reprogramming affects immune function in macrophages, and LPSstimulated pro-inflammatory M1 macrophages mainly rely on aerobic glycolysis, while IL-4-mediated anti-inflammatory M2 macrophages enhance oxidative phosphorylation and fatty acid oxidation (69). In B cells, mTORC1 activity is recognized to be important for the differentiation and antibody production, and metabolic reprogramming upon various stimulation determines the fate of B cells (70). Controlling mTORC1 and glycolysis by inhibitory cytokines is predicted to regulate humoral immunity (70) but cytokine-mediated metabolic inhibition in B cells has not been reported. Our results showed that a combination of TGF-β and IL-10 inhibited TLR-stimulated immune reaction with suppressing bioenergetics of B cells, and an mTORC1 activator or a reagent enhancing mitochondrial respiration counteracted the synergistic inhibitory effects. TLR stimulation is supposed to be essential in the activation of autoreactive B cells (31). Accumulating evidences has highlighted the central role of TLR signaling in the induction of immune responses in SLE (19). Thus, the inhibitory cytokine synergy that regulate the TLR signaling-mediated metabolic signals in B cells have a potential to be a novel therapeutic strategy for autoantibody-related autoimmune diseases.
Patterns of cytokine combination determine its distinct synergistic effects on the target cells (24). In T cells, TGF-β is required signal for Treg differentiation, but co-existence of TGF-β plus IL-6 and IL-23 induce Th17 differentiation (35). Although a combination of TGF-β and pro-inflammatory cytokines, such as IL-12 and IL-23, could induce a specific transcriptional factors and downstream signaling (24,71), synergistic inhibition by a combination of inhibitory cytokines has not been previously elucidated. Here, we uncovered TGF-β3 and IL-10-mediated synergistic inhibition of TLR-stimulated B cells via modulating energy metabolisms. We showed cooperative transcriptional modulation including suppressing mTOR signaling by a combination of TGF-β and IL-10 in TLR-stimulated B cells through RNA-sequencing. Although the molecular mechanisms of cytokine synergy have not fully elucidated, synergy through cooperative action is supposed to occur at the transcriptional level (24). Previous reports indicate that that Smad-STAT signaling networks are cell-type specific and context-dependent. For example, a direct cross talk between Smad3 and STAT3 is bridged by p300 in hepatoma cells (72), a synergistic action of LIF-induced-Smad1 and BMP2induced-STAT3 is bridged by p300 in neural cells (73), and Smad2/3 and STAT3 cooperatively interact in Th17 cell differentiation (74). In this study, we observed simultaneous activation of STAT3 and Smad3 in LPS-stimulated B cells with IL-10 and TGF-β3. Further studies should be conducted to verify the cross talk with STAT3 and Smad3 mediated by the inhibitory cytokine synergy in LPS-stimulated B cells.
B cell-specific mTOR deficiencies are associated with decreased high-affinity antibody production (49), and enforced mTOR signaling promotes plasma cell differentiation and immunoglobulin production (75). Enhanced glycolytic states in activated B cells (50,59) and requirement of mitochondrial pyruvate for long-lived plasma cells (60) imply the importance of inhibition of mTORC1 signaling, glycolysis, and mitochondrial respiration in B cell lineage to regulate excessive humoral immune responses. Biological processes in immune cells are supported by energy metabolism, and immune-metabolism relationships could be a basis for future therapy (52,76). Indeed therapeutic effects of metabolic intervention were observed in models of SLE and transplantation, and rapamycin, an mTORC1 inhibitor, and pioglitazone, a selective peroxisome proliferator-activated receptor-γ agonist, is currently being tested in a clinical trial for SLE (50,58,76). The fact that CD4 + CD25 + Tregs produce both TGF-β1 and IL-10 (2, 4), and CD4 + CD25 − LAG3 + Tregs produce both TGF-β3 and IL-10 (3,6,22) imply the existence of a mechanism for maintaining humoral immune tolerance by the co-production of these cytokines. In this study, although both of TGF-β1 and TGF-β3 effectively suppressed B cell activation in vitro, only TGF-β3 effectively regu lated the humoral immune responses in vivo. It is well known that extracellular activation of latent TGF-β are mediated by proteases, oxygen radicals, thrombospondin type I, integrins, glycoprotein A repetitions predominant, etc. (77). The difference of therapeutic effects between pCAGGS-Tgfb3 and pCAGGS-Tgfb1 in vivo might be due to their complexed physiological activation mechanisms including multiple factors of extracellular matrix components. Further, we showed that TGF-β3 ameliorated lupus pathologies with suppressing TFH cells and antibody-producing cells while maintaining total CD4 + T cells and B220 + B cell count in IL-10-sufficient MRL/lpr mice. The cell-type selective effects of TGF-β3 for targeting GC responses could have an advantage in adverse effects such as opportunistic infection typically caused by other immunosuppressive therapies. Potent anti-fibrotic properties of TGF-β3 (78), while TGF-β1 has strong pro-fibrotic effects (13) further ensure the safety for the therapeutic application.
In summary, our findings indicate that the combined presence of TGF-β3 and IL-10 in a local environment could target humoral immune responses. Regulation mediated by inhibitory cytokine synergy could be a future therapeutic option for autoimmune diseases, including SLE.

eThics sTaTeMenT
This study was carried out in accordance with the guidelines of the ethics committee of the University of Tokyo Institutional Animal Care and Use Committee. The protocol was approved by the ethics committee of the University of Tokyo Institutional Animal Care and Use Committee (approval number: G10095). Also, this study was carried out in accordance with the guidelines of the ethics committee of the University of Tokyo. The protocol was approved by the ethics committee of the University of Tokyo (approval number: G3582). All subjects gave written informed consent in accordance with the Declaration of Helsinki.

aUThOr cOnTriBUTiOns
All authors extensively contributed to the work presented in this paper. TK and MI carried out all experiments and contributed equally to this work. TK, MI, TO, KM, YI, SS, HS, KY, and KF conceived, designed, and analyzed the experiments and contributed to writing the manuscript.