extfieldsofstudy: Biology Medicine

text:
Constitutive expression of calreticulin in osteoblasts inhibits mineralization.

Recent studies have shown that the multifunctional protein calreticulin can localize to the cell nucleus and regulate gene transcription via its ability to bind a protein motif in the DNA-binding domain of nuclear hormone receptors. A number of known modulators of bone cell function, including vitamin D, act through this receptor family, suggesting that calreticulin may regulate their action in bone cells. We have used a gain-of-function strategy to examine this putative role of calreticulin in MC3T3-E1 osteoblastic cells. Purified calreticulin inhibited the binding of the vitamin D receptor to characterized vitamin D response elements in gel retardation assays. This inhibition was due to direct protein-protein interactions between the vitamin D receptor and calreticulin. Expression of calreticulin transcripts declined during MC3T3-E1 osteoblastic differentiation. MC3T3-E1 cells were transfected with calreticulin expression vectors; stably transfected cell lines overexpressing recombinant calreticulin were established and assayed for vitamin D-induced gene expression and the capacity to mineralize. Constitutive calreticulin expression inhibited basal and vitamin D-induced expression of the osteocalcin gene, whereas osteopontin gene expression was unaffected. This pattern mimicked the gene expression pattern observed in parental cells before down-regulation of endogenous calreticulin expression. In long-term cultures of parental or vector-transfected cells, 1 alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3) induced a two- to threefold stimulation of 45Ca accumulation into the matrix layer. Constitutive expression of calreticulin inhibited the 1,25(OH)2D3-induced 45Ca accumulation. This result correlated with the complete absence of mineralization nodules in long-term cultures of calreticulin-transfected cells. These data suggest that calreticulin can regulate bone cell function by interacting with specific nuclear hormone receptor-mediated pathways.

reticulin were established and assayed for vitamin D-induced gene expression and the capacity to mineralize. Constitutive calreticulin expression inhibited basal and vitamin D-induced expression of the osteocalcin gene, whereas osteopontin gene expression was unaffected. This pattern mimicked the gene expression pattern observed in parental cells before down-regulation of endogenous calreticulin expression. In longterm cultures of parental or vector-transfected cells, let,25-dihydroxyvitamin D 3 (1,251OH]2D3) induced a two-to threefold stimulation of 45Ca accumulation into the matrix layer. Constitutive expression of calreticulin inhibited the 1,25(OH)2D3-induced 45Ca accumulation. This result correlated with the complete absence of mineralization nodules in long-term cultures of calreticulin-transfected cells. These data suggest that calreticulin can regulate bone cell function by interacting with specific nuclear hormone receptor-mediated pathways.
~"~ALREXICUIAN2+ was initially identified as the major I[ Ca storage protein in the sarcoplasmic reticulum of skeletal muscle (Ostwald and MacLennan, 1974). Subsequent work has revealed that the protein can also be detected in the endoplasmic reticulum of nonmuscle tissues (Fliegel et al., 1989;Opas et al., 1991). Calreticulin possesses many diverse functional domains such as high affinity, low capacity-and low affinity, high capacity-CaZ+-binding sites, a COOH-terminal KDEL endoplasmic reticulum retention signal, and a nuclear localization signal (for review see Michalak et al., 1992).
Recent studies have shown that calreticulin can indeed be detected in the nucleus (Opas et al., 1991;Dedhar, 1994;Dedhar et al., 1994). Although the precise molecular mechanisms allowing the escape of calreticulin from the endoplasmic reticulum and regulating its translocation to the nucleus are currently unknown, two elegant series of experiments have elucidated the function of nuclear calreticulin (Burns et al., 1994;Dedhar et al., 1994). The protein recognizes a conserved amino acid motif, KXFFK/RR (where X is either G, A, or V), common to the DNA-binding domain of all known members of the nuclear hormone receptor family (Burns et al., 1994;Dedhar et al., 1994). These receptors act as ligand-inducible transcriptional regulators (Fuller, 1991). By interacting with the conserved motif located between the DNA-binding zinc fingers (for review see Glass, 1994) of the nuclear receptors, calreticulin inhibits their capacity to bind DNA in vitro and to activate gene expression in vivo (Burns et al., 1994;Dedhar et al., 1994). The ability of calreticulin to inhibit nuclear hormone receptor activity has been directly demonstrated for the glucocorticoid (Burns et al., 1994), the androgen , and the retinoic acid receptors (Dedhar et al., 1994). The functional importance of this inhibition has been demonstrated by the observation that calreticulin overexpression can influence the retinoic acid-dependent dif-ferentiation of multipotential embryonal carcinoma cells . Interestingly, a similar KLGFFKR amino acid motif is also found in the cytoplasmic domains of all integrin tx subunits (Rojiani et al., 1991), and calreticulin has also been shown to interact with a-integrins through this sequence . These results suggest the existence of a calreticulin-dependent signal transduction pathway linking the binding of extracellular matrix components by the integrin family of cell-surface receptors to the coordinate regulation of gene expression in the nucleus (Dedhar, 1994).
The active form of vitamin D, la,25-dihydroxyvitamin D 3 (l,25-[OH]2D3,1 also named calcitriol), is a known modulator of bone cell growth and function (for review see Narbaitz, 1992). Vitamin D mainly exerts its pleiotropic effects after binding to its specific receptor, which is itself a member of the steroid hormone receptor superfamily (Fuller, 1991). The ligand-bound vitamin D receptor (VDR) then interacts with its cognate-binding site, termed vitamin D response element (VDRE), to affect the transcription of target genes (for review see Ozono et al., 1991). In osteoblastic cells, vitamin D can stimulate the activity of alkaline phosphatase (Kurihara et al., 1986), and the expression of type I collagen (Kurihara et al., 1986), osteocalcin (Yoon et al., 1988;Demay et al., 1989), and osteopontin (Noda et al., 1990). Functional VDREs have been identified and characterized for the promoters of the osteocalcin (Kerner et al., 1989;Demay et al., 1990) and osteopontin (Noda et al., 1990) genes.
In addition to the binding of the VDR to certain VDREs as a homodimer (Carlberg et al., 1993), the VDR can form heterodimers with retinoid X receptors (RXRs) (Yu et al., 1991;Kliewer et al., 1992). The RXRs are a family of nuclear receptors binding the retinoid 9-cis retinoic acid Levin et al., 1992;Mangelsdorf et al., 1992). Both the VDR and RXRs possess the conserved KGFFK/RR amino acid motif within their respective zinc finger DNA-binding domains Burns et al., 1994), suggesting that VDR-mediated transcriptional activation in bone cells may be inhibited by calreticulin and thus that calreticulin may be involved in the regulation of osteoblastic differentiation and function.
We have used the osteoblastic MC3T3-E1 cell line (Sudo et al., 1983) to investigate this putative regulatory function of calreticulin. We report that purified calreticulin inhibited the binding of VDR homodimers and VDR-RXR heterodimers to the previously characterized osteopontin and osteocalcin VDREs. Direct protein-protein interactions between the VDR and calreticulin were demonstrated. The expression of the endogenous calreticulin gene was shown to be down-regulated during osteoblastic differentiation of MC3T3-E1 cells. Constitutive expression of calreticulin achieved by transfection of MC3T3-E1 cells with calreticulin expression vectors inhibited basal and vitamin D-induced stimulation of osteocalcin expression. However, the stimulation of the expression of osteopontin by vitamin D was unaffected. Mineralization was also inhibited in the calreticulin-expressing clones, as assessed by 1. Abbreviations used in this paper: 1,25(OH)2D3, 1ct,25-dihydroxyvitamin D3; RXR, retinoid X receptor; VDR, vitamin D receptor; VDRE, vitamin D response element. the reduction in calcium incorporation into the extracellular matrix and the complete absence of mineralization nodules. These results support a role for calreticulin in the regulation of osteogenesis through interaction with specific nuclear hormone receptor-mediated pathways.

Gel Retardation Assays
The VDR and RXR-13 cDNAs were kind gifts from Dr. J. W. Pike (Ligand Pharmaceuticals, San Diego, CA) and Dr. V. Gigu~re (McGill University, Montr6al, Qu6bec), respectively, and were in vitro transcribed and translated using the TNT-coupled wheat germ lysate system following the instructions of the manufacturer (Promega Corp., Madison, WI). Purified recombinant VDR and RXR-c~ expressed in insect cells using baculovirus expression vectors (MacDonald et al., 1991) were generously provided by Dr. M. Haussler (University of Arizona, Tucson, AZ). For gel-retardation assays, 5 I~l from the TNT reactions or 40 ng of the baculovirus-expressed receptors were incubated with or without purified calreticulin (Rojiani et al., 1991) together with 4 x 103 cpm of probe in gel retention mix (10 mM Tris-HC1, pH 7.3, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 2 mM MgCle, 0.2% NP-40, 12% glycerol, 1.3 mg/ml BSA, 10 -7 M 1,25-[OH]2D3) in a final vol of 25 I~1. Nonspecific competitor DNA consisted of 8 Ixg poly (dI-dC) and 0.2 Ixg salmon sperm DNA or 500 ng of poly (dI-dC) for the in vitro-translated and baculovirus-expressed receptors, respectively. The receptor and calreticulin proteins were preincubated for 30 min on ice before addition of the probe DNA. The probes used were synthetic oligonucleotides corresponding to the murine osteopontin VDRE (Noda et al., 1990) or the murine osteocalcin VDRE (Rahman et al., 1993). Probes were labeled by Klenow fill-in or kinasing (St-Arnaud and Moir, 1993). After incubation for 20 min at room temperature, bound probe was separated from free oligonucleotide on a 7% 30:1 acrylamidePoisacrylamide nondenaturing gel in lx TBE (89 mM Tris-borate [pH 8.3], 2 mM Na 2 EDTA). Samples were migrated at 150 V for 3 h with recirculating lx TBE. Gels were then dried and autoradiographed.

Immunoprecipitation
The VDR was labeled with [35S]methionine using the in vitro express translation kit as instructed by the manufacturer (Stratagene Corp., La Jolla, CA). The labeled receptor was incubated for 60 min on ice with or without purified calreticulin (Rojiani et al., 1991) in RIPA buffer (10 mM Tris-HC1, pH 8.0, 150 mM NaCI, 1% Triton X-100, 1% sodium deoxycholate) before addition of 10-30 ixl of anti-calreticulin antibody (Lieu et al., 1988). To ascertain that VDR and calreticulin interacted through the KXFFK/RR motif, an additional immunoprecipitation reaction included 100 ixg of the synthetic peptide KLGFFKR . Immunoprecipitation reactions were incubated for 4 d at 4°C and antigen-antibody complexes were collected by the addition of protein A-Sepharose (Pharmacia Canada, Baie d'Urf6, Qu6bec). The immunocomplexes were washed four times in RIPA buffer before analysis by SDS-PAGE on a 7.5% gel. The fixed, dried gel was subsequently autoradiographed.

RNA Extraction and Analysis
Cells were cultured for 14 d in medium supplemented for mineralization and then treated with 10 8 M 1,25-(OH)2D3 in ethanol or vehicle alone for 24 h. Total RNA was isolated by the lithium chloride-urea technique of Auffray and Rougeon (1980). Northern blot hybridization was performed as described elsewhere (St-Arnaud et al., 1988). The probes used included the 1.9-kb SacI fragment from the human calreticulin cDNA (McCauliffe et al., 1992); a 470-bp EcoRI-PstI fragment from the murine osteocalcin cDNA (Celeste et al., 1986); the full-length murine osteopontin cDNA (Smith and Denhardt, 1987); and the MAT1.1 probe for mouse a-tubulin (Lemishka et al., 1981).

Expression Vectors and Transfections
The SV-40 promoter-driven pECE-Cal expression vector was constructed by subcloning the 1.9-kb HindIII-calreticulin cDNA fragment from Cal-2  into the corresponding site of the pECE vector (Ellis et al., 1986) in the sense orientation. 20 ~g of pECE-Cal together with 0.3 ~.g of the pGEM7(KJ1)R selection plasmid (Rudnicki et al., 1992) were transfected into MC3T3-E1 cells by electroporation (2 × 106 cells; 300 V; 1000 ixF; 0.2 cm electrode gap). To establish control clones, MC3T3-E1 cells were transfected with 2 Ixg of pGEM7(KJ1)R selection plasmid alone. After electroporation, the cells were plated in 100-mm-diam dishes. Selection was initiated the next day by adding 1.2 mg/ml G-418 (Canadian Life Technologies, Burlington, Ontario) to the culture medium. Three control clones and four clones transfected with pECE-Cal were picked and established. The clones were maintained as the parental MC3T3-E1 cells except that the culture medium included 200 txg/ml of G-418. Expression of the recombinant calreticulin protein in transfected clones was assessed by Western blotting of whole-cell extracts with an anti-calreticulin antibody (Rokeach et al., 1991) as previously described .

Assay of 45Ca Accumulation
Accumulation of 45Ca into the matrix layer was measured as described by Matsumoto et al. (1991). Cells were cultured for 14 d before treatment with 10 8 M 1,25-(OH)2Ds or vehicle for 2 d.

Calreticulin Inhibition of DNA Binding by VDR
We used gel retardation assays with in vitro-translated or baculovirus-expressed VDR and RXR proteins to determine the effect of purified calreticulin on the binding of VDR homodimers and VDR/RXR heterodimers to characterized VDREs. Fig. 1 A shows that calreticulin inhibited the binding of the heterodimer to the murine osteopontin VDRE (Noda et al., 1990) in a dose-dependent manner (lanes 8-11). A similar result was obtained using the VDRE from the promoter region of the murine osteocalcin gene (Rahman et al., 1993) (Fig. 1 B, lanes 3~5). The amount of calreticulin required for the inhibition of VDR binding to each VDRE was not significantly different. The inhibition by calreticulin was also observed with VDR homodimers ( Fig. 1 A, lanes 4-7), demonstrating that calreticulin can interact with the VDR protein, and that the inhibition of DNA binding was not mediated solely through interaction of calreticulin with RXR. Unprogrammed wheat germ lysate ( Fig. 1 A, lane 2) or purified calreticulin alone ( Fig. 1 A, lane 3 and B, lane 2) did not bind to the VDRE probes.
Recent results have suggested that calreticulin interacts directly with nuclear hormone receptors through a conserved KXFFK/RR amino acid motif nested within the DNA-binding domain of the receptor proteins (Dedhar et al., 1994;Burns et al., 1994). We addressed the VDR-calreticulin interaction using immunoprecipitation reactions with labeled VDR, purified calreticulin, and anti-calreticulin antibodies. Antibodies directed against calreticulin did not immunoprecipitate the in vitro-labeled VDR (Fig. 1  C, lane 1). However, when purified unlabeled calreticulin was added to the reaction, the labeled VDR protein was coimmunoprecipitated by the anti-calreticulin antibodies ( Fig. 1 C, lane 2). This result demonstrates a direct interaction between VDR and calreticulin. This reaction was mediated through the KGFFRR motif of the VDR, as a synthetic KLGFFKR peptide added simultaneously with calreticulin competed for the interaction of the VDR with calreticulin and substantially decreased the amount of coimmunoprecipitated labeled receptor ( Fig. 1 C, lane 3).

Calreticulin Expression during Osteoblastic Differentiation
MC3T3-E1 cells exhibit a preosteoblastic phenotype during exponential growth. On reaching confluency, the cells begin to express osteoblastic phenotype markers such as alkaline phosphatase and type I collagen (Sudo et al., 1983). Long-term cultures form multilayers of cells that express late osteoblastic differentiation markers and form calcified bone tissue (nodules of mineralization) in vitro (Sudo et al., 1983). This pattern closely recapitulates the osteoblastic differentiation sequence (Stein et al., 1990;Zhou et al., 1994).
We extracted RNA from subconfluent, confluent, and mineralizing cultures of MC3T3-E1 cells to analyze the expression of the endogenous calreticulin gene. Fig. 2 B shows that the 1.9-kb calreticulin mRNA could be detected in early and confluent cultures (lanes 1 and 2, respectively). No calreticulin expression could be detected in mineralizing cultures (Fig. 2 B, lane 3). Fig. 2 A shows that comparable amounts of total RNA were loaded in each lane. This pattern of expression of the endogenous calreticulin gene was observed in two independent experiments (data not shown).
We also probed the RNA blot for expression of osteoblastic phenotype markers. Fig. 2 C shows that the osteopontin gene was expressed at all stages of culture. Thus osteopontin expression could be detected in cultures that did or did not express the endogenous calreticulin gene (compare Fig. 2, B and C). On the contrary, the osteocalcin mRNA was only detected in mineralizing cultures (Fig.  2 D). Thus osteocalcin gene expression appeared to coincide with the inhibition of endogenous calreticulin gene expression in the cultured cells (Fig. 2, B and D, lane 3).

Expression of Recombinant Calreticulin
We isolated four clones stably transfected with the pECE-Cal vector (clones SV-Cal 4, SV-Cal 5, SV-Cal 6, and SV-Cal 12). Three clones transfected with the selection vector alone served as controls. Fig. 3 shows the relative expression levels for the calreticulin protein in parental MC3T3-E1 cells and the control and transfected clones. Elevated calreticulin protein expression was achieved in all four clones transfected with the calreticulin expression vector. The increase in calreticulin expression was estimated to vary between two-and fivefold over parental levels. The expression of the recombinant calreticulin protein was constitutive and maintained in long-term cultures of the transfected clones (data not shown). In both parental MC3T3-E1 cells and transfected clones, calreticulin was detected in the nucleus and the perinuclear region using immunocytochemistry (not shown), as demonstrated for other cell types (Dedhar, 1994); the relative abundance of nuclear calreticulin was increased in the clones transfected with the calreticulin expression vector (data not shown).
Enhanced calreticulin expression affected the morphology of the cells. This change was more evident in early cultures Figure 1. Calreticulin interacts with the VDR protein to inhibit the binding of VDR homodimers and VDR-RXR heterodimers to characterized VDREs. (A) VDR and RXR-13 were in vitro translated and incubated with a labeled oligonucleotide corresponding to the murine osteopontin VDRE in the presence or absence of purified calreticulin (10 ng/p-1). Probe, unprogrammed lysate, and calreticulin alone were migrated as controls in lanes 1, 2, and 3, respectively. (B) Baculovirus-expressed VDR and RXR-a were incubated with a murine osteocalcin VDRE oligonucleotide probe. Increasing amounts of purified calreticulin (10 ng/p-1) were added as indicated above lanes 4-6. Probe and calreticulin were migrated in lanes I and 2. The binding reactions were analyzed using the gel retardation assay on a 7% nondenaturing gel. (C) SDS-PAGE analysis of immunoprecipitation reactions. 35S-labeled VDR was incubated with or without purified calreticulin and an anti-calreticulin antibody. In the presence of calreticulin, the labeled receptor was associated with the immunocomplexes (lanes 2 and 3). Addition of a synthetic peptide (KLGFFKR; 100 p.g) substantially reduced the amount of immunoprecipitated receptor (lane 3). that had just reached confluency. The clones overexpressing calreticulin had a spindle-shaped appearance compared with the characteristic cuboidal osteoblastic morphology of the parental cells and the control clones (Fig. 4). This morphological change became less apparent in long-term, multilayer cultures (data not shown).

Calreticulin Overexpression Selectively Inhibits Gene Expression
Long-term (14 d) cultures of control and calreticulinexpressing clones were treated with 1,25-(OH)2D3 for 24 h and total R N A was extracted to analyze the influence of calreticulin overexpression on vitamin D-stimulated gene expression in bone cells. Fig. 5 shows that constitutive calreticulin expression inhibited the induction of osteocalcin expression by vitamin D in all clones studied. Basal levels of osteocalcin m R N A were also drastically down-regulated in calreticulin-expressing clones (Fig. 5). These observations are consistent with the pattern of osteocalcin gene expression observed in parental cells, where osteocalcin m R N A could not be detected in subconfluent and confluent cultures of MC3T3-E1 cells that expressed endogenous ealreticulin m R N A (Fig. 2). Similarly, vitamin D treatment of subconfluent and confluent MC3T3-E1 cul-tures did not induce osteocalcin gene expression (data not shown). Interestingly, the vitamin D-induced stimulation of the expression of the osteopontin gene was unaffected by calreticulin (Fig. 5). This result is in accord with the data obtained in parental cell cultures, where osteopontin expression could readily be detected in early cultures of MC3T3-E1 cells that expressed significant levels of endogenous calreticulin m R N A (Fig. 2). Thus our observations suggest that calreticulin overexpression had specific effects on the modulation of gene expression by nuclear hormone receptors and did not inhibit gene expression in general. Indeed, the expression of a-tubulin, which was analyzed to monitor for equal loading of R N A for each sample, was unaffected by calreticulin overexpression (Fig. 5).

Constitutive Calreticulin Expression Inhibits Mineralization
Multilayer cultures of MC3T3-E1 cells deposit mineral and form calcified bone tissue after 14-21 d in culture (Sudo et al., 1983). This osteogenic process can be documented by counting the number of mineralization nodules and by measuring the incorporation of labeled calcium into the extracellular matrix. The calcium accumulation Figure 2. Expression pattern of calreticulin and osteobtast phenotype markers during MC3T3-E1 osteoblastic differentiation. Total RNA was isolated from subconfluent (day 2), confluent (day 4), and mineralizing (day 21) MC3T3-E1 cells and analyzed by Northern blot assay. (A) Ethidium bromidestained ribosomal RNA showing equivalent loading for all samples. The RNA was transferred onto a nylon membrane, cross-linked, and hybridized to specific probes for calreticulin (B), osteopontin (C), and osteocalcin (D). The major 1.5-kb osteopontin transcript is shown in C.
into the matrix has been shown to be stimulated by treatment of the cultures with 1,25-(OH)2D 3 (Matsumoto et al., 1991). We have used both assays to analyze the influence of constitutive calreticulin expression on mineralization in osteoblastic cells. Fig. 6 A shows a 14-d-old culture of the control clone G7KJ1-2 that was stained for mineralization nodules using the von Kossa method (Mallory, 1942). Long-term cultures of all three control clones yielded similar results that were representative of the mineralization observed in parental MC3T3-E1 cultures (not shown). Fig. 6 B illustrates the complete absence of mineralization nodules observed in calreticulin-overexpressing clones. The data from a typical mineralization experiment is summarized in Table I. The cultures of the control clone G7KJ1-2 contained 53 +_ 4 nodules (mean _+ SEM, n = 4), whereas the cultures from all the clones expressing calreticulin constitutively contained no nodules (0.6 _+ 0.3; mean + SEM, n = 2 for each of the four clones).
Vitamin D-induced stimulation of 45Ca incorporation  into the extracellular matrix was also inhibited in calreticulin-transfected clones. We observed the previously reported twofold stimulation of calcium incorporation into the matrix by vitamin D (Matsumoto et al., 1991) in cultures of MC3T3-E1 cells and in cultures of all three control clones (Fig. 7). Constitutive expression of calreticulin inhibited calcium incorporation in response to vitamin D treatment in all four clones (Fig. 7).

Discussion
We have shown that constitutive expression of calreticulin in osteoblastic cells inhibited mineralization. This inhibition was most likely mediated through specific modulation of gene expression by calreticulin, as we have demonstrated that calreticulin could interact with the vitamin D receptor to inhibit its binding to cognate response elements in vitro, and selectively inhibit certain vitamin D-mediated transcriptional responses in vivo. Our results support a role for calreticulin in the regulation of osteoblastic differentiation and function. The skeleton is the body's major repository of calcium in the form of hydroxyapatite crystals. Mineralization, i.e., the deposition of mineral along the fibrils of the collagen Figure 5. Calreticulin overexpression selectively inhibits the vitamin D-induced stimulation of osteocalcin expression without affecting vitamin D-stimulated osteopontin expression. Total RNA was isolated from mineralizing (day 14) cultures of one control clone (lanes 1 and 2) and the four calreticulin overexpressing clones (lanes 3-10) treated with (even-numbered lanes) or without (odd-numbered lanes) 10 -8 M 1,25-(OH)2D 3 for 24 h. The RNA was analyzed by Northern blot assay with specific probes for osteocalcin, osteopontin, and a-tubulin (to monitor for variations in sample loading). Observed transcript sizes are indicated on the right. Multiple transcripts for murine osteopontin have been described previously (Miyazaki et al., 1990). matrix of bone, is under the control of the osteoblast (Termine, 1993). But the role of the osteoblast is not to act as a calcium-storage compartment since a proportion of the skeletal content of calcium is freely exchangeable with the extracellular fluids and serves as the major storage pool of calcium (Broadus, 1993). Moreover, the function of the osteoblast is not dependent on the availability of a large intracellular store of calcium, in contrast, for example, to the function of skeletal myotubes which require rapid calcium release to initiate muscle contraction. Thus it is likely that the role of calreticulin in osteoblasts is not limited to a calcium storage function.
Indeed, we have shown that calreticulin can influence gene expression and mineralization. The inhibition of mineral deposition cannot be simply explained by the sequestration of calcium inside the cells by the recombinant calreticulin protein. First, the levels of expression that we achieved in the transfected clones were not disproportionately high. Second, under the conditions used, the extracellular fluid was supersaturated with regard to calcium and phosphate. In experiments measuring the stimulation of labeled calcium into the extracellular matrix by vitamin D (Fig. 7), the calreticulin-expressing clones had similar baseline amounts of 45Ca incorporated in the matrix (not shown).
The mechanisms responsible for the actual inhibition of calcium incorporation when the calreticulin-transfected clones were treated with vitamin D are unclear. Vitamin D treatment had no effect on the expression of the endogenous calreticulin gene in J.,unpublished observations).
In addition to vitamin D, a number of steroid hormones or morphogens binding to members of the nuclear hormone receptor superfamily can modulate gene expression in osteoblasts. In MC3T3-E1 cells, glucocorticoids have been shown to inhibit prostaglandin E2 synthesis (Hughes-Fulford et al., 1992) and increase the expression of betaglycan Figure 6. Constitutive calreticulin expression inhibits mineralization. Long-term (day 14) cultures, supplemented for mineralization, of control clone G7KJ1-2 (A) and calreticulin-expressing clone SV-Cal 12 (B) were stained for mineralization nodules using the von Kossa method. The nodules appear as dark spots in the bright-field photograph. (Nakayama et al., 1994). Estrogen receptors are expressed in osteoblasts (Eriksen et al., 1988) and 17-[3 estradiol down-regulates the transcriptional activity of the interleukin-6 gene promoter and inhibits the expression of the glycoprotein 130, a subunit of cytokine receptors (Manolagas, S. C. 1995. Bone. 16 [Suppl. 1]:91S.). Retinoic acid stimulates the expression of alkaline phosphatase and induces differentiation of preosteoblastic cell lines (Ng et al., 1988;Heath et al., 1989). All of these receptor-mediated responses represent putative targets for modulation by calreticulin. Our results, showing that calreticulin inhibited some but not all of the transcriptional responses mediated by the VDR, suggest that certain of these nuclear hormone receptor-dependent pathways will be left intact while others may be affected. It would be interesting to investigate transcriptional responses to various steroids in bone cells expressing calreticulin constitutively. The response to vitamin D was not completely abrogated in clones expressing the recombinant calreticulin protein (Fig. 5). This could suggest that the VDR was in stochiometric excess over calreticulin in the transfected clones, thus allowing the excess of ligand-bound VDR to escape inhibition by calreticulin and activate transcription. Indeed, vitamin D treatment has been shown to induce the expression of V D R mRNA and protein in bone cells (Mahonen et al., 1990). Alternatively, the residual vitamin D response observed in calreticulin-transfected clones could be due to the documented nongenomic effects of vitamin D (for review see Norman et al., 1992), which may not be affected by constitutive calreticulin expression.
Our results have demonstrated that calreticulin inhibits VDR binding to both the osteopontin and the osteocalcin V D R E (Fig. 1). However, the two genes responded dissimilarly when the calreticulin-expressing clones were challenged with vitamin D (Fig. 5). Despite differences between the sequences of the two VDREs (Kerner et al., 1989;Demay et al., 1990;Rahman et al., 1993) that could affect VDR binding affinity to each response element (Nishikawa et al., 1993), we were unable to detect any difference in the capacity of calreticulin to inhibit VDR binding to each VDRE. Thus the structure of the response element cannot seem to account for the variation in transcription. It is more likely that the expression of each gene is dictated by the structure of the entire promoter region, and not just a single element. The pattern observed in the parental cells supports this view. Indeed, the osteopontin m R N A was readily detected in early cultures of MC3T3-E1 cells that also expressed calreticulin (Fig. 2). On the contrary, osteocalcin transcripts were only observed in long-term cultures that had down-regulated calreticulin expression (Fig. 2). Thus it appears that calreticulin expression inhibits both steady-state and stimulated osteocalcin expression, whereas it does not affect the transcription of the osteopontin gene. In this respect, the pattern of expression of these two osteoblastic phenotype markers that we measured in the calreticulin-expressing clones closely mimicked what was observed in the parental MC3T3-E1 cells.
There was no correlation between the level of calreticulin overexpression achieved in independent clones and the inhibition of basal and stimulated osteocalcin transcription, as all clones exhibited an identical blunted osteocalcin response (see Figs. 3 and 5). Our results suggest that it was not the magnitude of the expression of the recombinant protein per se that affected the osteocalcin response, but the fact that calreticulin expression was constitutive and therefore not down-regulated in long-term cultures of the transfected clones. Again, this observation parallels the pattern of osteocalcin gene expression that we documented in nontransfected MC3T3-E1 cultures (Fig. 2).
Attachment to extracellular matrix components modulates gene expression and differentiation of bone cells. For example, differentiation of canalicular cell processes was observed after contact of MC3T3-E1 cells with laminin (Vukicevic et al., 1990), and gene expression is affected when preosteoblastic cells are plated on various extracellular matrix components (Traianedes et al., 1993). Osteoblasts express various forms of the integrin family of cell surface receptors for matrix proteins (Hughes et al., 1993;Saito et al., 1994), and inhibition of integrin function using subunit-specific antibodies has been shown to prevent cytokine-induced osteoblastic differentiation of osteosarcoma cells (Dedhar, 1989). Calreticulin binds to a-integrin subunits  and recent results suggest that this interaction can modulate the affinity state of integrins (Dedhar, 1994). Moreover, calreticulin can modulate nuclear hormone receptor-dependent gene expression Burns et al., 1994). Taken together, these observations support the existence of a calreticulin-modulated signal transduction pathway linking substratum attachment via integrin receptors to the control of gene expression. Our results further support an important role for this pathway in the regulation of osteoblastic differentiation and function.
We thank Dr. Mark Haussler for providing the baculovirus-expressed nuclear receptors, E. Wan for oligonucleotide synthesis, and M. Lepik and J. Wishart for preparing the figures. This work was supported by grants from the Shriners of North America (R. St-Arnaud) and the Medical Research Council of Canada (S. Dedhar). R. St-Arnaud is a Chercheur-Boursier from the Fonds de la Recherche en Sant6 du Qu6bec and S. Dedhar is a Terry Fox Cancer Scientist of the National Cancer Institute of Canada.
Received for publication 7 April 1995 and in revised form 15 August 1995.
Note Added in Proof. After completion of this work, a report was published confirming the inhibition of vitamin D signal transduction by calreticulin (Wheeler, D. G., J. Horsford, M. Michalak, J. H. White, and G. N. Hendy. 1995. Nucleic Acids Res. 23:3269-3274).


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A rare cryptic translation product is presented by Kb major histocompatibility complex class I molecule to alloreactive T cells.

The identity of allogeneic peptide/major histocompatibility complex (MHC) complexes that elicit vigorous T cell responses has remained an interesting problem for both practical and theoretical reasons. Although a few abundant MHC class I-bound peptides have been purified and sequenced, identifying the unique T cell-stimulating peptides from among the thousands of existing peptides is still a very difficult undertaking. In this report, we identified the antigenic peptide that is recognized by an alloreactive bm1 anti-B6 T cell clone using a novel genetic strategy that is based upon measurement of T cell receptor occupancy in single T cells. Using lacZ-inducible T cells as a probe, we screened a splenic cDNA library in transiently transfected antigen-presenting cells (APCs) and isolated a cDNA clone that allowed expression of the appropriate peptide/Kb MHC complex in APC. The antigenic octapeptide (SVVEFSSL) exactly matched the consensus Kb MHC motif, but was surprisingly encoded by a non-ATG defined translation reading frame. Furthermore, the abundance of the naturally processed analog in untransfected cells was estimated to be <10 copies per cell. These results illustrate a novel strategy for identifying T cell-stimulating antigens in general and directly show that alloreactive T cells can respond to rather rare peptide/MHC complexes. These results also suggest that the total pool of processed peptides expressed on the APC surface may include those generated by cryptic translation of normally expressed transcripts.

T he clonotypic up antigen receptor of T cells recognizes peptide/MHC class I or class II complexes. Normally, T cells respond to foreign peptides presented by self-MHC molecules. T cells, however, also respond to allogeneic cells with a vigor that far exceeds normal responses to conventional foreign antigens (see references 1-3 for reviews) . This response, referred to as the allogeneic response, involves T cell recognition ofnonself, allogeneic MHC molecules . Compared to foreign antigens that are recognized by -1 in 105 T cells, alloantigens are recognized by up to -1 in 102 T cells. The high frequency of T cells responding to allo-MHC antigens is the basis for rapid rejection of allogeneic transplants and for graft versus host disease . Understanding the molecular basis of alloreactivity is therefore important, not only for manipulating T cell responses in these clinically relevant cases, but also for understanding the nature and recognition ofligands by the TCR in general.
Defining the ligands recognized by allogeneic T cells is key to understanding alloreactivity . At the present time, it is uncertain whether the individual responding T cells recognize nonself MHC molecules with or without peptides, 'Abbreviations used in this paper: CPRG, chlorophenol red (3-galactoside; X-GAL, 5-bromo-4-chloro-3-indolyl (3-n-galactopyranoside . and whether the MHC-bound peptides represent a small set of highly abundant or rare sequences (3) . Of the three examples where the actual peptides recognized by alloreactive (4,5) and xenoreactive T cells (6) have been identified, the processed peptides were abundantly expressed by the APC . For two of these peptides presented by the Ld and the Qa-1 MHC molecules, the donor proteins were also quite abundant (5,7). No donor protein has yet been identified for the peptide presented by HLA-A2.1 MHC to xenogeneic murine T cells (6) . These results have therefore favored the argument that alloreactivity is caused by T cells that recognize abundant peptide/nonself MHC complexes (8) . By contrast, analysis of panels of alloreactive T cells specific for Kb or its Kbm mutant alleles has suggested that distinct peptides are presented by allogeneic MHC. By HPLC analysis of APC extracts, both the Rammensee and Sherman groups showed that different peaks contained alloreactive T cell-stimulating peptides (9,10) . Thus, alloreactivity could be attributed to recognition of many different peptides presented by MHC that stimulate different sets of T cells . This interpretation has, however, remained inconclusive because the T cell-stimulating peptide sequences or their relative abundance in APC is unknown .
Identifying MHC-bound peptides that actually stimulate T cells is a daunting task . Biochemical purification of the peptides is difficult, not only because MHC molecules display thousands of different peptides on the cell surface (11)(12)(13), but also because individual antigenic peptides can represent only a few copies per cell (14,15) . Nevertheless, with remarkable efforts and sophisticated instrumentation, several groups have succeeded in determining the sequence of some T cell-stimulating peptides (4,6,14) . These peptides, as well as those identified as simply MHC-bound peptides in APC, are related by MHC allele-specific consensus motifs and, where known, are derived from endogenously synthesized proteins (12,13,16) . The latter fact has allowed an alternative strategy for identifying antigenic peptides by isolating the gene that allows generation of the T cell-stimulating peptide/MHC complexes in the APC .
Boon and colleagues were the first to isolate T cell-defined tumor antigen genes by expression cloning (17,18) . The antigenic peptide can subsequently be identified by testing the activity of synthetic peptides that match the consensus motif (19) and/or by narrowing the antigenic activity in deletion constructs (20) . We recently showed that expression-cloning strategies can be further generalized and improved by (a) generating the peptide/MHC complexes by transient rather than stable transfections, and (b) by using single T cell assays as an exquisitely sensitive read-out of peptide/MHC complexes generated in the APC (21,22) . In this report, we applied this expression cloning strategy for identifying the T cell-stimulating antigens that are expressed by allogeneic cells . First, lacZ-inducible T cells specific for allogeneic peptide/Kb MHC complexes expressed by wild-type B6 mice were generated in the congenic bml (Kbmi) mice . By screening a spleen cDNA library in transfected APC with these T cells as a single-cell probe, we isolated a a-tubulin cDNA that allowed antigen/MHC-specific lacZ response in the T cells . Curiously, the antigenic octapeptide JAL8 (SWEFSSL) was not located within the open reading frame of the a-tubulin sequence, but it was encoded at the vector/cDNA insert junction . Most surprisingly, this antigenic peptide was a cryptic translation product of this cDNA because it was encoded in a non-ATGdefined translational reading frame . Transfecting APCs with constructs containing an in-frame ATG as the translation initiation codon, or the addition of exogenous synthetic peptide, allowed expression of high levels of the T cell-stimulating peptide/Kb complex . In contrast to the high abundance of all previously identified allogeneic T cell-stimulating peptides, the abundance of the naturally processed peptide in Kb + cell extracts was <10 copies per cell . These results demonstrate that rare peptide/MHC complexes can stimulate alloreactive T cells, and that these methods are applicable to the identification of unknown T cell antigens in general . , and their Kb-expressing derivatives were generated as described (21,23) . Kb' Hepa 1 cells were also stably transfected with JMAL9 or 44S1 constructs by electroporation (20) . B3Z T cell hybrid is specific for OVA peptide SL8 (OVA 257-264)/Kb complex and has been described previously (20,21) .

Materials and Methods
Generation of LacZ-inducible bm l Anti-B6 T Hybrrdomms .
C57BL/6 (B6) and its congenic strain B6 .C-H26-'/Byj (bml) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) . 2-4-mo-old bml mice were injected twice with 2 X 10' B6 spleen cells intraperitoneally 7 d apart . Spleens of immunized bml mice were removed 1 wk after the second injection, and the CTL were enriched by coculture with fresh irradiated B6 spleen cells and rat Con A supernatant (10) . CTL were fused with the lacZ-inducible fusion partner BWZ .36/CD8 as described earlier (24) . Hybridoma cells were screened for their responsiveness to B6 spleen cells . Anti-B6, lacZ-inducible T cell hybrids were cloned by limiting dilution, and stable subdones were used in subsequent experiments . Construction and Screening the cDNA Library. A unidirectional cDNA library using mRNA from Con A+LPS-activated B6 spleen cells was constructed in the BstXI/NotI sites of the mammalian expression vector pcDNAI (Invitrogen, San Diego, CA) . The cDNA inserts were ligated to the vector using the BstXI/EcoRI linker (5' GAATTCCACCACA 3') at the 5' end and primer/ linker oligodT/Notl (5' AACCCGGCTCGAGCGGCCGCT 18 3') at the 3' end. Recombinant plasmids were selected by transforming the MC1061/P3 competent bacteria in ampicillin and tetracycline in L broth/agar . The bacteria representing the cDNA library were divided into 15 different pools each comprising _105 CFU. For expression screens, aliquots of DNA (100 ng/ml) prepared from each bacterial pool were transiently transfected into 2 X 10 4 Kb-COS cells per well of 96-well plates. 2 d later, 3 X 104 bm1BZ19 .4 T cells were added to each well, and after overnight incubation, cultures were stained with the 5-bromo-4-chloro-3indolyl (3-D-galactopyranoside (X-GAL) substrate as described (21,24) . Positive pools were scored as an increase in average number of "blue" lacZ+ T cells over the vector alone background control, as shown in the photomicrographs in Fig. 2 . The single plasmid that was responsible for the positive signal was isolated by screening sequentially smaller pools of recombinant bacteria from the positive pool and repeating the transfection and T cell assays with DNA isolated from each bacterial pool . The number of CFU at each stage were estimated as, Iv2 (_5-10 X 104), Iv2 .4 (_2 X 103), Iv2 Restriction maps and sequence of the isolated Iv44 cDNA were obtained by standard methods . The Iv44 deletion constructs were prepared as follows : 44S1 was prepared by removing the 3' 0 .6-kb fragment from Iv44 plasmid between the internal and the flanking Sphl sites in the polylinker, and then religating the large fragment . Similarly, the internal Clal and the 3' flanking Xbal sites were used to remove the 0 .86-kb fragment from 1-kb 44S1, and the remaining 134-bp insert-vec-for fragment was filled in with klenow and religated. For 44SBS1, DNA from the 44SC1 construct was obtained by excising the 5' flanking BamHI site in the vector, and the internal 3' Smal site in the insert and was cloned into the BarnHI/Smal sites of the pEVRF1 vector (25) . For RF1, RF2, and RF3 constructs, the 44SBS1 plasmid was cut at the EcoRI site at the vector-insert junction and was filled in with Klenow . The insert fragment was then removed by making a second cut at the internal Smal site at the 3' end. This blunt fragment was then cloned into the Smal sites of pEVKF0, pEVRF1, andpEVKF2 vectors that allow translation in each reading frame, as described previously (20,25).
Transient DNA Transfections . Stable COS cell transfectants expressing Kb or Db were transiently transfected with CsCl purified large scale or with mini prep plasmid DNAs by the DEAE-dextran/chloroquin protocol, as described in detail previously (20) . Transfections were performed with 3 X 104 COS cells per well in 96-well flat bottom tissue culture plates. Two days later, transfected cells were tested for the expression of specific peptide/MHC complexes by overnight coculture with 3-10 X 104 T cells. LacZ response induced in activated T cells was measured as described below.
T Cell Activation Assays . Peptide/MHC ligand-specific T cell responses were measured by the lacZ activity induced in the T cells (24,26). 3-10 X 10 4 T cells were cocultured overnight with 2-5 X 104 appropriate APC, (normal spleen cells, cell lines, or transfected cells) with or without exogenous peptides (synthetic or extracts) in 96-well plates . The ligand-induced T cell response was determined using the lacZ substrate chlorophenol red (3-galactoside (CPRG), as described (20,24). In each well of the 96-well plate, the conversion of CPRG to chlorophenol red was measured at 595 and 655 ran as reference wavelength with a plate reader (model 3550 ; Bio Rad Laboratories, Hercules, CA). Data shows the mean absorbance of replicate cultures, and they are representative of at least three independent experiments .
Extraction of Processed Peptides . Total acid soluble peptide pool from EG7 or EL4 cells was extracted by TFA, as described (27,28) . Briefly, 107-10' cells were washed with PBS, lysed in 2 nil 0.1% TFA in water, and homogenized by ultrasonication using a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) . The homogenate was spun at 12,000 g for 30 min. The supernatant was passed through a 10-kD filter (Ultra Free-MC; Millipore Corp ., Bedford, MA). The filtrate was dried in a vacuum centrifuge, resuspended in PBS, and analyzed for processed peptides in T cell activation assays described above.

Results
Generation of Peptide/Kb-specific, LacZ-inducible T Cells.
Alloreactive T cells specific for the Kb MHC class I molecule were generated by immunizing the congenic bml mutant mice with the spleen cells of wild-type B6 mice . These strains were chosen because the bml mutant mice differ from the wild-type B6 mice in three amino acid substitutions within the Kb-1 MHC u chain alone, thus constraining the alloreactive bml anti-B6 T cell response to only the wild-type Kb MHC (29) . After in vitro enrichment, the alloreactive T cells were fused with the BWZ.36/CD8 fusion partner to derive lacZ-inducible, T cell hybrids (24) . Fig. 1 shows three representative T hybridoma clones responding to either the immunogen (B6 spleen) or to Kb+ cell lines. None of the T cells recognized T2-Kb cells that express predominantly empty MHC class I molecules (30) . Hybridoma bm1BZ3 recognized an antigen shared by the B6 spleen and Kb-COS cells, but absent on Kb L cells, while hybrids bm1BZ18 and bmlBZ19 recognized antigens shared by B6 spleen cells and Kb L cells, which were absent in Kb-COS cells. Further evidence that Kb MHC determined the responsiveness of these T cells was obtained by demonstrating that only anti-Kb, but not anti-Db antibodies, inhibited their response (Fig . 1 B) . This was observed when either EL-4 (Kb, Db) or Kb-L (Kb, H-2k) were used as APCs . Hybridoma bmlBZ19.4 was chosen for use as a single T cell probe because of its low reactivity towards Kb-COS cells that were used as recipient APCs to screen for its cognate antigenic cDNA .
Isolation of the bmlBZ19.4 T Cell-stimulating Antigen Gene.
To identify the gene that could generate the T cell-stimulating peptide/Kb complex, we screened a cDNA library prepared from B6 spleen cells. Kb-COS cells were transfected with pools of cDNAs clones in 96-well plates . 2 d later, the transfected cells were assayed for expression of the peptide/Kb MHC complex that stimulated lacZ response in individual bmlBZ19 .4 T cells. Photomicrographs of representative cultures in Fig. 2 show that compared to the vector alone background, a few lacZ + blue T cells were present in one of the cultures, and that their number increased dramatically upon transfecting Kb-COS cells with yet smaller cDNA pools. By sequential sib-selection of cDNA pools of decreasing size, a single plasmid DNA (Iv2 .4 .52.44) that allowed stimulation of the bmlBZ19.4 T cells was identified . This cDNA clone (referred to as lv44) specifically stimulated the T cells only upon transfection into Kb-COS, but not when it was expressed in Db-COS or in parental COS cells (see below and data not shown) . We conclude that APC Antibody Dilution expressing the Iv44 plasmid DNA generated the peptide/ Kb complex, and that this was most likely caused by the antigenic peptide encoded within the cDNA insert. Location of the Antigenic Peptide in the Iv44 cDNA. The cDNA insert in Iv44 plasmid was 1 .6 kb in length. Partial sequence of^300 nucleotides at both the 5' and 3' ends ofthe insert showed that Iv44 was identical to the mouse a-tubulin mRNA sequence (MUSTUBA6M, GenBank/EMBL accession no. M13441) . Iv44 cDNA, however, lacked the first 131 nucleotides of the full-length clone that included the natural ATG start codon. Nevertheless, a truncated tubulin polypeptide (414 versus 449 amino acids) could be generated from this cDNA using the second in-frame ATG codon as a translation initiation codon (31,32). Kb1-7 (44S1), Kbl (44SC1) alone or with no Kb motifs at all (44SBS1) . These constructs were transfected into Kb-or Db-COS cells that were tested for their ability to stimulate bm1BZ19 .4 T cell response (Fig. 3 B-D). The T cell response clearly required both the cDNA constructs and the Kb MHC molecule because no T cell response occurred when the same constructs were expressed in Db-COS cells.
The smallest active construct that contained a Kb1 motif was 44SC1, but surprisingly, the construct 44SBS1, lacking all but two residues of the Kb1 motif, was as active as the parental clone Iv44. This result indicated that none of the nine Kb-binding motifs represented the bmlBZ19 .4 T cellstimulating peptide . To directly test the activity of the Kbl motif that was present in 44SC1, but was disrupted in 44SBS1, we prepared a mini-gene construct (Fig. 3 A, Kbl) that encoded only the peptide PRAVFVDL_and the translational initiation methionine codon.-This construct was clearly inactive (Fig. 3 C), and it ruled out the possibility that the two Kb1 residues shared between 44SC1 and ), yet it has been described as capable of being presented by Kb MHC and eliciting tumor-specific CTL re-sponses (33) . Second, the antigenic peptide could be encoded by one of the other non-ATG-defined translational open reading frames in the construct. This unconventional mechanism has been observed to yield T cell-stimulating peptide/ MHC complexes in several independent model systems (17,20,34) . Finally, the peptide presented by Kb need not have been encoded by the constructs at all, but instead, could have resulted from induction of yet another cellular gene in transfected APC. In this case, the minimal 44SBS1 construct would contain the transcription/translation-inducing activity for the induced antigen gene .
All attempts to localize the antigenic activity within the open reading frame of the ot-tubulin insert in the 44SBS1 construct failed . First, no T cell response was detected with A  (Fig. 4 A, frame a) in exogenous presentation assays, even at supraoptimal 100-11,M concentration (data not shown) . Second, the minigene constructs Kb10 and K611, encoding partial (xxxx-[F]xxx) motifs that bind Kb MHC in vitro with nonconsensus COOH-terminal anchor residues (35), (Fig. 4 A, Kb10 and K'11) were inactive . Third, the RF1, RF2, and RF3 constructs designed to allow translation of each ofthe three reading frames of the insert sequence alone were also completely inactive (Fig. 4 C). Taken together, these results strongly suggested that the antigenic activity of the 44SBSI construct was not caused by the insert sequence alone, but that it required both the insert and the vector sequences . Examination of the sequence of the entire transcript of 44SBS1 in each of the three translational reading frames revealed a peptide sequence at the vector/cDNA insert june- tion that matched the consensus Kb motif (Fig. 4 A). This sequence was indeed the bm1BZ19 .4 T cell-stimulating antigenic peptide . The mini-gene construct (JMAL9) encoding only these residues (ASVVEFSSL) with a methionine codon added for translation initiation was at least 100-fold more active than either the Iv44 or 44SBS1 constructs (Fig.  4 D). This dramatic difference in endogenous presentation activity is consistent with the known differences in the translational efficiencies of ATG versus non-ATG codons (20) . Based on our recent analysis ofnon-ATG codons that allow generation of peptide/MHC complexes (36), it is quite likely that translation of the antigenic precursor is initiated at the upstream TGG and/or GAT codons that are in-frame with the antigenic peptide (Fig. 4 A, arrows) . Furthermore, the generation of the T cell-stimulating peptide via cryptic translation was not limited to transiently transfected DNAs, but was also observed in stably transfected Hepal cells ( Conclusive proof that the "out-of-frame" JAL9 nucleotides did encode the antigenic peptide was obtained with synthetic peptides . By contrast to the synthetic peptides representing the ATG-defined open reading frame of the insert (Fig. 4 A, frame a), which were completely inactive, both the nona (ASVVEFSSL) and the octa (SVVEFSSL) peptides of translational frame c (Fig. 4 A), stimulated bm1BZ19 .4 T cells strongly (Fig. 5 B) . Note that the dose-response curves show that these peptides were active at even picomolar concentrations, and that the 8 mer was 3-10-fold more active than the 9 mer. These results are entirely consistent with previous analyses of naturally processed peptides presented by the Kb MHC (12,28,37,38) . With the sole exception  Insert sequence (uppercase letters) and its flanking pEVRF0 vector DNA (lowercase letters) are shown with each of three translational reading frames (a-c) in single-letter amino acid code . Linker nucleotides for ligating the cDNA to the vector are underlined . The 5' flanking vector sequence provided an in-frame ATG initiation codon that is in-frame with the a-tubulin open reading frame in frame a. The asterisks indicate termination codons, and vertical arrows show the presence of alternate non-ATG initiation codons that could be used for translating the peptide shown in frame c (36) . The residues encoded by the oligo constructs JMAL9, Kb10, and Kbll are indicated within parentheses and by dashed lines . An in-frame ATG codon (M) was added to each minigene construct to allow translation . (B-D) bmIBZ19 .4 T cellstimulating activity of 44SBS1 is encoded in a non-ATG-defined translational reading frame . Antigenic activity of the indicated cDNA constructs was tested by transient transfection into Kb-COS cells, as described in legend to Fig. 3 . Labeled curves show T cell responses obtained with the constructs shown in Fig. 4 A . The RF1, RF2, and RF3 constructs were identical to 44SBS1, except that the insert sequence alone, without the junction sequence, was placed downstream of an in-frame ATG codon (20,25) . Because o£ the rather high activity of the JMAL9 relative to .01 0 .1 1 10 100 the Iv44 or 44SBS1 constructs, the cultures were incubated with the lacZ substrate for only 1 .5 h rather than the usual 4 h .
of the tumor peptide FEQNTAQP (33), these peptides conform to the consensus motif xxxx[F,Y]xx[l,L,M], and exhibit T cell-stimulating activity at extremely low-concentrations . Other Kb-restricted T cells that recognize conventional (B3Z, anti-OVA257-264 (SL8)/Kb) or allogeneic Kb MHC (bm1BZ18 .5) did not recognize the JAL8 or JAL9 peptides (Fig. 5, C and D) . Similarly, none of eight other bml anti-B6 alloreactive T cells tested recognized either JAL8 or JAL9 peptides (data not shown) . We conclude that the JAL8 octapeptide is a cryptic translation product presented by Kb MHC, and that it serves as a unique ligand for bmlBZ19 .4 T cells .
Relative Abundance of the Naturally Processed Peptide/K' Complex in Normal APC . What is the abundance of the naturally processed counterpart of JAL8 peptide? While the DNA constructs and the synthetic peptides allowed genera- tion of the bm1BZ19.4-stimulating complex, a similar complex was naturally expressed by normal B6 spleen cells as well as by other Kb+ cell lines (Fig. 1, A and B) . To analyze this naturally processed peptide, we extracted the total pool of processed peptides from EL-4 (H-2 6 ) and its OVAexpressing derivative, EG-7 (39) . Because EG-7 cells express the OVA-derived SL8/Kb complex, the presence and amount of naturally processed SL8 peptide in the extracts provides a critical internal control. While both B3Z and bmlBZ19.4 T cells were capable of responding to similarly low concentrations (-1-3 pM) of the synthetic SL8 and JAL8 peptides, respectively (Fig. 6 A), only the naturally processed SL8 peptide was detected in extracts of EG-7 cells (Fig. 6 C) . No bm1BZ19.4-stimulating activity was detected in either EL-4 or EG-7 extracts (Fig. 6 B) or in extracts of five B6 spleens (data not shown) . From the sensitivity ofthe assay for detecting synthetic JAL8 peptide, we estimate that the abundance of the naturally processed JAL8 counterpart in EL-4 or in normal spleen cells is less than eight copies per cell. This estimate was validated by the fact that the same EG-7 extract yielded 90 SL8 copies per cell. Peptides Recognized by Alloreactive T Cells Remarkably, our result is virtually identical to the completely independent estimate of 88 SL8 copies/EG-7 cell by Rammensee's group (40) . Thus, the natural peptide/Kb complex recognized by bmlBZ19.4 T cells is expressed at a very low level in Kb-expressing cells.
What is the source of the naturally processed JAL8 analogue in untrans£ected Kb-APC? A match for the JAL8 peptide sequence was not found in either the protein (Swiss-Prot release 37 .0) or nucleic acid (GenBank/EMBL release 86.0) databases. Note that because peptides with amino acid substitutions can cross-react with T cells (41,42), and that the low abundance of the naturally processed peptide in cell extracts precluded comparison ofjAL8 peptide with its natural analogue by HPLC (28,43), it is uncertain at this time whether the naturally processed peptide is identical to JAL8, and whether it is derived from a normal cellular protein or via cryptic translation ofan alternate reading frame.

Discussion
The bml anti-B6 alloreactive bmlBZ19.4 T-cells recognize the octapeptide SVVEFSSL (JAL8)/Kb complex. The JAL8 peptide was identified by a novel genetic strategy for isolating cDNAs that allow expression of the T cellstimulating complex in transfected APCs . Surprisingly, the JAL8 peptide was a cryptic translation product of this cDNA clone and its natural counterpart was present at <I 0 copies per cell in APCs . These results show that as for normal T cell responses to foreign antigens, alloreactive T cells can respond to rare peptide/MHC complexes .
The explanation for why allogeneic MHC molecules elicit vigorous T cell responses has remained elusive for decades . Early hypotheses (44), proposing structural or functional differences among TCRs expressed by alloreactive versus conventional antigen-specific T cells are currently less attractive, and attention has focused instead on the MHC ligands for the TCR (3) . Strong evidence that peptides play a critical role in alloreactivity is now available with mutant cells that express empty MHC molecules on their surface, and are consequently not recognized by alloreactive T cells (e .g., Fig . 1) unless processed peptides from wild-type cells are added exogenously (9,10,45) . Interestingly, each of the two allogeneic and one xenogeneic peptide/MHC complexes that have been characterized so far appear to be expressed as thousands o£ copies on APC surface (4-6) . Based on the high level of expression ofthese peptides and the estimates of the frequency of T cells that recognize them, it has been argued that alloreactive T cells are primarily stimulated by abundant peptide/MHC complexes (8) .
Identification of JAL8 peptide allowed us to estimate the level of expression of its naturally processed counterpart in APCs . Similar to all naturally processed peptides characterized so far, synthetic JAL8 peptide was active in stimulating bm1BZ19 .4 T cells at low picomolar concentrations (Figs . 5 and 6) . In contrast to the B3Z-stimulating SL8 peptide that was expressed at^100 copies in EG-7 cells, the bm1BZ19 .4stimulating peptide was expressed below the detection threshold of 10 copies in the same extracts. This low level of expression is at the extreme end of the range recently established HeLa cells as APC and the T cells.
After overnight culture, lacZ response induced in T cells was measured as described in the legend to Fig. 1 .
for the minimum number of peptide/MHC complexes that are required for T cell stimulation (15) . Interestingly, similar low levels of peptide expression (<30 copies per cell) were also estimated for expression of the processed OVA257-264 (SL8) peptide when it was generated as a cryptic translation product from experimental constructs (36). Thus, barring the unlikely but formal possibility that the natural analogue of JAL8 peptide is exceptional in being active only at very high concentrations, APCs express rather low levels of the bm1BZ19 .4-stimulating peptide . Furthermore, the fact that of the eight bm1 anti-B6 hybrids that were tested, only bm1BZ19 .4 cells were stimulated by JAL8/Kb complex suggests that a diverse set of peptides are actually presented by allogeneic MHC molecules to a correspondingly diverse set of alloreactive T cells, fulfilling early predictions (1), and explaining the high frequency and vigor of alloreactive T cell responses . It is interesting to contrast the peptide purification and our expression-cloning approaches to account for conflicting conclusions on the abundance of peptides recognized by alloreactive T cells . Because success of peptide purification depends on isolatable amounts of processed peptides expressed in the target cells, a correlation between peptide abundance and ease ofits purification is expected. By contrast, the antigenic JAL8 peptide was identified using an expression cloning strategy that depends on the generation of MHC-bound peptides derived from the transfected donor gene in the APCs at a level sufficient to stimulate T cells. In addition to the differences in the mechanism (exogenous versus endogenous) for generation of the peptide/MHC complexes, we also used an exquisitely sensitive single T cell activation assay as a probe for ligand expression (21,26) . Thus, as demonstrated previously with model antigens (20,21,24), and here with the JAL8 peptide, rare peptide/ MHC complexes may be more accessible by the expression-cloning methods described here . Furthermore, it is possible that different immunization procedures may favor selection of T cells that respond to abundant or rare ligands . This is particularly relevant to xenogeneic immunizations where species-specific differences in MHC/CD8 coreceptor interactions markedly influence T cell responses (46). Future discoveries of other alloreactive T cell-stimulating antigens, particularly those presented by the same MHC molecule, are needed to clarify this issue.
The unexpected finding that JAL8 peptide was a cryptic translation product and was expressed in APC raises the issue of the source of peptides for display by MHC . Undoubtedly, MHC bind and present peptide fragments that are obtained from intracellular polypeptides (47) . It is not clear, however, whether these peptides are solely byproducts of cellular protein turn-over or are also generated via alternate mechanisms. Intriguing observations in several experimental systems have demonstrated that peptide/MHC complexes are generated from donor genes that should not have been expressed (17,20,34). Boon and his colleagues were the first to formulate the "pepton" hypothesis explaining how gene fragments lacking obvious transcriptional promoters or translational control sequences could yield peptide/MHC complexes (48) . Subsequent analysis from this laboratory showed that the generation of these peptides could be explained by cryptic translation initiated by non-ATG codons (20). By screening DNA constructs in APCs for their ability to generate T cell-stimulating peptide/MHC complexes, we identified the set of non-ATG translation initiation codons, and we determined that despite being below detection levels in cell extracts, the amount of these cryptic translation products in either transiently or stably transfected APC were sufficient to cause T cell activation (36). The fact that JAL8 peptide is a cryptic translation product of lv44 cDNA and that it was initially identified in a complex cDNA mixture (Fig. 2) provides strong independent support to the notion that peptide/MHC complexes in APCs can be generated by cryptic translation .
Presentation of cryptic translation products can have a significant impact on the diversity of peptides displayed by References MHC . With current technology, it is not possible to directly determine what fraction, if any, of the large pool of naturally processed peptides includes cryptic translation products of normally expressed genes . Nevertheless, two recent observations merit attention. First, Townsend and colleagues showed that frame shift mutations in colon tumors can generate novel polypeptides that are presented by MHC and are recognized by T cells (49) . Second and more intriguing, Nakayama's group identified a CTL-stimulating, tumor-specific peptide that was encoded within the 5' untranslated region ofthe c-akt gene (50) . How an "untranslated" sequence served as a peptide source was, however, not explained . Conceivable mutations, for example, those that can introduce an upstream ATG codon to allow translation of the 5' c-akt sequence, may exist in these cells. Alternatively, the identified peptide may be derived from the translated open reading frame ofsome other gene that is yet absent from the databases . We have noticed, however, that the upstream nucleotide sequence of c-akt gene (MMSTPK, GenBank accession no. X65687) contains three in-frame CTG codons that can serve as cryptic translation initiators and can account for generation of the peptide/MHC complex recognized by the CTL (36). Regardless of the yet unknown mechanisms that generate frame-shift mutations or allow cryptic translation, our findings reported here, together with other independent examples, can be conceptualized as maximization of RNA coding information for antigen presentation . Note that from the point o£ view of T cell surveillance of novel antigens, such as viral gene products in the target cells, it is irrelevant whether the processed peptides arise via degradation of full-length proteins or of cryptic translation products. In fact, maximizing the input of peptide precursors may be the simplest solution to overcoming the bottlenecks in peptide/MHC display imposed by sequence specific motifs in peptide/MHC interactions (51,52), as well as by sequence selectivity in peptide transport by TAP (53)(54)(55)(56) .
We acknowledge the gifts ofreagents from other investigators, Mr. P. Paz for generating the Kb-HeLa transfectants, and the excellent technical assistance ofMr. Vu Nguyen in DNA sequencing.