10.14639/0392-100X-N2023
Objectives. Detailed characterisation of CD45RA+ and CD45RO+ T cell subsets in both CD4+ and CD8+ cells in blood and tissue of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) is lacking. We aimed to investigate T cell differentiation levels in blood and tissue and correlate this with CT score and white blood cell count. Methods. This case-control study was conducted on 21 patients with CRSwNP and 20 healthy controls. Lund-Mackay score was used for radiologic staging of chronic rhinosinusitis. T cell subsets were characterised in blood and nasal polyp tissue using CD4, CD8, CCR7, CD45RA, CD45RO, CD27, and CD95 monoclonal antibodies. Results. Most variations in T cell subsets were detected in tissue rather than blood. A higher level of CD8+ TCM, CD4+ TSCMs and lower level of CD8+ TEM were detected in the blood of CRSwNP patients vs controls. In CRSwNP patients, substantial decrease of tissue CD8+ TNs, CD8+ TCMs, CD4+ TNs, and CD4+TSCMs with marked increase in the percentages of TEMs and TEMRAs were detected among CD4+ and CD8+ T cells compared with levels in blood. Conclusions. The reduction of TNs, TSCMs, and TCMs and accumulation of TEMs and TEMRAs subsets that function as effector cells in sinonasal mucosa, especially CD8+ T cells, might indicate the role of immunomodulation of memory/effector T-cells in the pathogenesis of CRSwNP.
text/xml
1827-675X
2023
en
Mohamed M. Osman
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
chronic rhinosinusitis
Delineation of T cell subsets in chronic rhinosinusitis with nasal polyps
Text.Serial.Journal
https://www.actaitalica.it/
42
Rhinology
2022-11-23
2022-01-27
2022-12-13
2022-12-13
441-449
1
Mohamed M. Osman
2022/11/23
10.14639/0392-100X-N2023
441
Department of Otorhinolaryngology & Head and Neck Surgery, Faculty of Medicine, Assiut University, Assiut, Egypt
1827-675X
chronic rhinosinusitis
en
449
Delineation of T cell subsets in chronic rhinosinusitis with nasal polyps
https://www.actaitalica.it/article/download/2023/870
42
ACTA OTORHINOLARYNGOLOGICA ITALICA 2022;42:441-449; doi: 10.14639/0392-100X-N2023




Rhinology


Delineation of T cell subsets in chronic rhinosinusitis
with nasal polyps
Definizione di sottopopolazioni di cellule T nella rinosinusite cronica con poliposi
nasale
Asmaa M. Zahran1, Omnia El-Badaway2, Ibrahim K.I. Elsayh3, Mohamed M. Osman4
1
 Department of Clinical Pathology, South Egypt Cancer Institute, Assiut University, Assiut, Egypt; 2 Department of Medical
Microbiology & Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt; 3 Third year Medical student, Faculty of Medicine,
Assiut University, Assiut, Egypt; 4 Department of Otorhinolaryngology & Head and Neck Surgery, Faculty of Medicine, Assiut
University, Assiut, Egypt


SUMMARY
Objectives. Detailed characterisation of CD45RA+ and CD45RO+ T cell subsets in both
CD4+ and CD8+ cells in blood and tissue of patients with chronic rhinosinusitis with nasal
polyps (CRSwNP) is lacking. We aimed to investigate T cell differentiation levels in blood
and tissue and correlate this with CT score and white blood cell count.
Methods. This case-control study was conducted on 21 patients with CRSwNP and 20
healthy controls. Lund-Mackay score was used for radiologic staging of chronic rhinosi-
nusitis. T cell subsets were characterised in blood and nasal polyp tissue using CD4, CD8,
CCR7, CD45RA, CD45RO, CD27, and CD95 monoclonal antibodies.
Results. Most variations in T cell subsets were detected in tissue rather than blood. A higher
level of CD8+ TCM, CD4+ TSCMs and lower level of CD8+ TEM were detected in the blood of
CRSwNP patients vs controls. In CRSwNP patients, substantial decrease of tissue CD8+
TNs, CD8+ TCMs, CD4+ TNs, and CD4+TSCMs with marked increase in the percentages of TEMs
and TEMRAs were detected among CD4+ and CD8+ T cells compared with levels in blood.
Conclusions. The reduction of TNs, TSCMs, and TCMs and accumulation of TEMs and TEMRAs
subsets that function as effector cells in sinonasal mucosa, especially CD8+ T cells, might
                                                                                                  Received: February 21, 2022
indicate the role of immunomodulation of memory/effector T-cells in the pathogenesis of
                                                                                                  Accepted: August 2, 2022
CRSwNP.
KEY WORDS: nasal polyps, T cell, chronic rhinosinusitis                                           Correspondence
                                                                                                  Omnia HB. EL-Badawy
                                                                                                  Department of Medical Microbiology and Immu-
RIASSUNTO                                                                                         nology. Faculty of Medicine, Assiut University,
Obiettivi. La caratterizzazione delle sottopopolazioni di cellule T CD45RA+ e CD45RO+             Assiut, Egypt
provenienti da sangue e tessuti di pazienti affetti da rinosinusite cronica con poliposi nasale   Tel. +2 010 05212176. Fax +2 088 2332278
(CRSwNP) è ad oggi incompleta. Scopo del lavoro è definire i livelli di differenziazione di       E-mail: omniaalbadawy@aun.edu.eg
cellule T in campioni ematici e tissutali e correlarli con CT score e conta linfocitaria.
Metodi. Lo studio caso-controllo è stato condotto su 21 pazienti affetti da CRSwNP e 20             How to cite this article: Zahran AM, El-Bada-
controlli. Le sottopopolazioni di cellule T sono state caratterizzate da campioni emati-            way O, Elsayh IKI, et al. Delineation of T cell
ci e tissutali di polipi nasali, utilizzando anticorpi monoclonali per CD4, CD8, CCR7,              subsets in chronic rhinosinusitis with nasal pol-
CD45RA, CD45RO, CD27, e CD95.                                                                       yps. Acta Otorhinolaryngol Ital 2022;42:441-449.
Risultati. La maggior parte delle variazioni delle sottopopolazioni di cellule T è stata ri-        https://doi.org/10.14639/0392-100X-N2023
scontrata nei campioni tissutali. Un livello superiore di CD8+ TCM, CD4+ TSCMs e un livello
inferiore di CD8+ TEM sono stati riscontrati nel sangue di pazienti affetti da CRSwNP. Nei        © Società Italiana di Otorinolaringoiatria
                                                                                                  e Chirurgia Cervico-Facciale
pazienti con CRSwNP sono stati rilevati una riduzione di CD8+ TNs, CD8+ TCMs, CD4+ TNs, e
CD4+TSCMs tissutali e un marcato incremento percentuale di TEMs e TEMRAs tra linfociti T CD4+
                                                                                                                 OPEN ACCESS
e CD8+ rispetto ai loro livelli ematici.
Conclusioni. La riduzione di sottopopolazioni TNs, TSCMs, and TCMs e l’accumulo di TEMs e         This is an open access article distributed in accordance with
                                                                                                  the CC-BY-NC-ND (Creative Commons Attribution-Non-
TEMRAs con funzione di cellule effettrici all’interno della mucosa nasale potrebbe suggerire      Commercial-NoDerivatives 4.0 International) license. The
il ruolo immunomodulante delle cellule T di memoria ed effettrici nella patogenesi della          article can be used by giving appropriate credit and mentio-
CRSwNP.                                                                                           ning the license, but only for non-commercial purposes and
                                                                                                  only in the original version. For further information: https://
PAROLE CHIAVE: poliposi nasale, cellule T, rinosinusite cronica                                   creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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Introduction                                                      the least differentiated memory T cell subset and display a
                                                                  naïve-like phenotype (CD45RO-CD45RA+CCR7+) and can
Nasal polyps are believed to arise in the nasal mucosa
                                                                  be distinguished from TNs by their distinctive expression of
because of chronic inflammation. Chronic rhinosinusitis           CD95 (Fas) 7.
with nasal polyps (CRSwNP) is mostly characterised by a           Detailed characterisation of CD45RA+ and CD45RO+ T cell
moderate to severe T helper type 2 (Th2)-mediated inflam-         subsets among both CD4+ and CD8+ cells in blood and tis-
mation with hypereosinophilia and increased IgE concen-           sue of patients with CRSwNP is lacking. Hence, we aimed
trations 1. CRSwNP is associated with higher colonisation         to delineate T cell differentiation level in blood and tissue
rates with bacteria such as Staphylococcus aureus and fun-        and correlate it with CT score and white blood cell count.
gi like Aspergillus fumigatus and Candida albicans 2. Up-
per airway colonisation with bacteria may be accompanied
by biofilm formation and increasing inflammatory cells in         Materials and methods
tissue that contribute to nasal tissue damage leading to a        This case-control study was conducted on 21 patients with
recalcitrant disease 3.                                           CRSwNP admitted to the Otorhinolaryngology Depart-
Numerous studies have underlined the role of activated            ment, Faculty of Medicine, Assiut University. Twenty age
memory CD4+ T cells as the primary producer of Th2 cy-            and sex-matched healthy blood donors were enrolled as a
tokines in asthma and other atopic diseases. Th2 cytokines        control group.
such as IL-4 and IL-13 contribute to many of the patho-           Non-contrast computerised tomography scans were done
physiological features of asthma, including airway inflam-        for all patients, including coronal, axial and sagittal cuts
mation, mucus secretion and airway hyper-responsiveness.          (3 mm thickness) in bone and soft tissue window set-
The production of Th2 cytokines was initially ascribed to         tings. Lund-Mackay’s radiologic scoring system was used
CD4+ T cells. In addition, several studies have provided ev-      to stage chronic rhinosinusitis 11. Each sinus was given a
idence that CD8+ T cells are able to secrete Th2 cytokines        score of 0, 1, or 2 if it was totally patent, partially opaci-
and are essential for allergic inflammation and airway sen-       fied, or completely opacified, respectively. The ostiomeatal
sitivity 4.                                                       complex (OMC) was allocated a score of 0 if not occluded
The functional state of T cells is dependent on their degree      or 2 if occluded. The maximum obtainable score for each
of differentiation. When naive CD4+ and CD8+ T lympho-            side is 12, with a total combined score of 24. Lund Mackay
cytes are challenged with a cognate antigen, they prolifer-       scores of 4 or higher largely support a clinical diagnosis
ate and differentiate, and acquire specialised migratory and      of chronic rhinosinusitis, whereas scores less than four are
effector capabilities 5. Expression of the lymph node–hom-        unspecified 12.
ing molecules L-selectin (CD62L) and chemokine receptor
7 (CCR7), the activation marker (CD27), and the RA/RO             Flow cytometry
isoforms of the leukocyte common Ag (CD45) distinguish-           Two ml of peripheral blood were collected from each par-
es memory T cells from naïve T cells (TNs) and classifies         ticipant and polyp biopsy specimens were collected during
antigen-experienced T cells into at least four differentia-       endoscopic sinus surgery. The polyps were minced finely
tion subsets: stem cell-like memory T cells (TSCMs), central      and passed through a cell strainer in phosphate-buffered sa-
memory T cells (TCMs), effector memory T cells (TEMs) and         line (PBS) solution. The filtrate was centrifuged, then treat-
terminally differentiated effector T cells (TEMRAs) 6,7.          ed with red blood cell (RBC) lysis buffer and washed twice
Naïve T cells, which have not yet encountered an antigen,         with PBS. Processed tissue and 100 µl of the whole blood
express CD45RA, CCR7 and CD62L in addition to CD27                sample were each incubated with 10 µl of fluoroisothiocy-
and preferentially migrate to secondary lymphoid organs           anate (FITC)-conjugated anti-CD27, phycoerythrin (PE)-
like lymph nodes and spleen. After interaction with its li-       conjugated anti-CD8, PE-cyanine 7 (PE-CY7)-conjugated
gand (CD70), CD27 delivers the costimulatory signals              anti-CD45RO, allophycocyanin (APC)-conjugated anti-
needed for expansion of primed T cells and differentiation        CD45RA, APC-H7-conjugated anti-CD4, PerCP-Cy5.5-
to effector cells with a fast antigen recall response 8. TCMs     conjugated anti-CCR7 and V500-conjugated anti-CD95 for
lose CD45RA and gain expression of CD45RO during the              15 minutes at 4°C in the dark. All monoclonal antibodies
transition from naive to memory T cells 9. Upon antigenic         were purchased from Becton Dickinson (BD, CA, USA).
restimulation, TCMs down-regulate CCR7 and CD27 and dif-          RBCs lysis and washing with PBS were done. An isotype-
ferentiate into TEms 8,9 and lastly into TEMRAs, which are con-   matched negative control was used to detect background
sidered to be terminally differentiated. TEMRAs lack CCR7         staining signals in each sample.
and CD45RO but re-express CD45RA 10. TSCMs represent              Data analysis was done by FACSCanto flow cytometer us-



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ing FACS DIVA 7.0 software (BD, USA). Approximately             Table I. Clinical and laboratory features of patients.
100,000 events were acquired for each sample. Lympho-            Variables                                                    Patients
cytes were selected based on their light scatter properties.                                                                  N = 21
CD4+ and CD8+ T cells were then gated, and five distinct         Age (years)                                                  36.6 ± 4
subsets were characterised in each of CD4+ and CD8+ T cells      Sex
according to CD45RA and CD45RO expression, followed                Male                                                      15 (71.4%)
                                                                   Female                                                    6 (28.6%)
by gating based on CD27, CCR7, and CD95. These sub-
                                                                 Haematological findings
sets include TNs; CD45RO−CD45RA+CCR7+CD27+CD95−,                   TLC (x109/L)                                               7.5 ± 0.5
TSCMs;     CD45RO−CD45RA+CCR7+CD27+CD95+, TCMs;                    Neutrophil percent/count (x109/L)                       52 ± 2/3.8±0.4
CD45RO CD45RA-CCR7+, TEMs; CD45RO+CD45RA-
          +                                                        eosinophil percent/count (x109/L)                     3.7 ± 0.6/0.3 ± 0.04
CCR7− and TEMRAs; CD45RO−CD45RA+CCR7−CD27−.                      Lund-Mackay total score                   8                   2 (9.5%)
                                                                                                          13                   1 (4.8%)
                                                                                                          15                   1 (4.8%)
Statistical analysis                                                                                      16                   4 (19%)
IBM Statistical Package for the Social Sciences, version 25                                               19                   2 (9.5%)
(IBM SPSS statistics, USA) was used for statistical analy-                                                21                   1 (4.8%)
sis of data. Continuous variables were expressed as mean                                                  22                   2 (9.5%)
                                                                                                          24                   8 (38%)
± standard error (SE), whereas categorical data were pre-       TLC: total leukocyte count.
sented as numbers (percentages). The Shapiro-Wilk test
was employed to determine the normality of data distribu-
tion. Independent sample t-test and Mann-Whitney U test         blood CD45RA+ T cells or CD45RO+ T cells between pa-
were used to compare variables among the different para-        tients and controls. Higher levels of CD8+ TCM and lower
metric or non-parametric groups, respectively. A Wilcoxon       levels of CD8+ TEM were detected in the blood of CRSwNP
signed-rank test was performed to evaluate the differences      patients vs controls (p = 0.01 & p < 0.0001, respectively).
in cell frequencies between biopsy and blood samples. As-       CD4+ TSCMs was the only CD4+ T cell subset showing in-
sociations between variables were explored using Kend-          creased frequency in the blood of CRSwNP patients com-
all’s tau and Spearman correlation coefficient. A p-value       pared with controls (p = 0.01).
was considered significant when less than 0.05.                 CD4+ T cells were lower in polyp tissue than blood of
                                                                CRSwNP patients (p < 0.0001), whereas tissue and blood
Results                                                         levels of CD8+ T cells did not show a significant difference.
Clinical and laboratory features of patients                    Also, no significant difference was observed between CD4+
As presented in Table I, the average age of patients with       and CD8+ T cells in tissue from CRSwNP patients. How-
CRSwNP was 36.6 ± 4 years and 71% were males. The mean          ever, the CD4/CD8 ratio was substantially lower in tissue
total leukocyte count (TLC) was 7.5 ± 0.5. The neutrophil       than in blood of CRSwNP patients (1 ± 0.09 vs 1.5 ± 0.08,
mean percentage was 52 ± 2, while the eosinophil mean per-      p = 0.004), and 12 had a CD4/CD8 ratio < 1 in tissues.
centage was 3.7 ± 0.6. More than half of patients had a total   In comparison with controls, CRSwNP patients showed a
CT score ≥ 19 and nearly 38% of patients scored 24 by CT.       significant decrease in the total percentage of CD45RA+
                                                                T cells and a significant increase in the total frequency
Changes in CD8+ and CD4+ T cell subsets in CRSwNP patients      of CD45RO+ T cells among tissue CD8+ (p = 0.004 &
Changes in CD8+ and CD4+ T cell subsets in blood and            p = 0.02, respectively) and CD4+ (p < 0.0001 & p = 0.007,
polyp tissue are shown in Figures 1 and 2, respectively. No     respectively) T cells. Moreover, tissue CD45RO was higher
significant changes were detected in the levels of CD4+ and     than CD45RA expression by both CD8+ and CD4+ T cells
CD8+ T cells in the blood of CRSwNP patients compared           of CRSwNP patients (p < 0.0001, p = 0.001, respectively).
with controls. The percentage of CD4+ T cells in blood was      Tissue CD8+ cells showed a substantial decrease of the
significantly higher than that of CD8+ T cells in CRSwNP        TNs and TCMs cells (p = 0.001, p = 0.02, respectively), and
patients (p < 0.0001). All but two patients had a CD4/CD8       a marked increase in TEM and TEMRA cells (p < 0.0001,
ratio > 1 in blood.                                             p = 0.001, respectively) in CRSwNP patients compared
Similar to controls, blood CD45RA+ T cells were higher          with controls. Likewise, CD4+ T cells displayed a signifi-
than CD45RO+ T cells among both CD8+ and CD4+ T cells           cant decrease of TNs and TSCMs cells (p = 0.001, p = 0.005,
of CRSwNP patients (p = 0.03, p < 0.0001, respectively).        respectively), and a marked increase in TEMs and TEMRAs cells
In addition, no noticeable changes were detected in either      (p < 0.0001, p = 0.02, respectively).



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Figure 1. (A, B, C) plots showing levels of CD4+ and CD8+ cells in patients’ tissue and blood and control blood, respectively; (D ,E, F) percentages of CD45RA+CD8+
and CD45RO+CD8+ cells in patients’ tissue and blood and control blood, respectively; (G, H, I) frequencies of CD8+ TNs and CD8+ TSCMs in patients’ tissue and blood
and control blood, respectively; (J, K, L) levels of CD8+ TCMs and CD8+ TEMs in patients’ tissue and blood and control blood, respectively; (M, N, O) levels of CD8+
TEMRAs in patients’ tissue and blood and control blood, respectively; (P) comparison of percentages of CD8+ T cell subsets between patients and control blood and
(Q) between patients’ blood and tissue. *p < 0.05, **p < 0.01; ***p < 0.0001.


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Figure 2. (A, B, C) percentages of CD45RA+CD4+ and CD45RO+CD4+ cells in patients’ tissue and blood and control blood, respectively; (D,E,F) frequencies of
CD4+ TNs and CD4+ TSCMs in patients’ tissue and blood and control blood, respectively; (G, H, I) levels of CD4+ TCMs and CD4+ TEMs in patients’ tissue and blood and
control blood, respectively; (J, K, L) levels of CD4+ TEMRAs in patients’ tissue and blood and control blood, respectively; (M) comparison of percentages of CD4+ T cell
subsets between patients and control blood and (N) between patients’ blood and tissue. *p < 0.05; ** p < 0.01; ***p < 0.0001.



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Generally, the percentages of TEMs and TEMRAs were high-                          Discussion
er among CD8+ than CD4+ T cells in blood (p = 0.047,
                                                                                  The abnormal immune system activation in sinonasal mu-
< 0.0001, respectively) and tissue (p = 0.001, p< 0.0001,
                                                                                  cosa is a key player in the aetiology and pathophysiology of
respectively). Conversely, the percentages of TNs and TC-                         CRS. The recruitment of T cells and elevated T cell inflam-
Ms
   were higher among CD4+ than CD8+ T cells in blood                              matory cytokines, particularly type 2 in the mucosa of CRS
(p = 0.005, p = 0.07, respectively) and tissue (p < 0.0001,                       patients, emphasises that T cells significantly contribute to
p = 0.004, respectively).                                                         the pathogenesis of CRS 1. To better understand the patho-
                                                                                  physiologic mechanisms of CRS, characterisation of the
Correlations of T cell subsets with Lund-Mackay CT score                          immune cells that infiltrate nasal and sinus tissues is criti-
and blood cell count                                                              cal. Therefore, in this study, we analysed the T cell subset
Blood CD8+ TCMs had a positive correlation with neutrophil                        distribution in blood and tissue and assessed their relations
percentage (r = 0.3, p = 0.03), whereas blood CD4+ TEMRAs                         with CT score and blood cell count.
had a negative correlation with neutrophil count (r = -0.3,                       Comparable with controls, the percentage of CD4+ T cells
p = 0.04). Only blood CD4+ TEMs have shown positive corre-                        in blood was significantly higher than CD8+ T cells in
                                                                                  CRSwNP patients. CD4+ T cells were decreased in polyp
lation with the total CT score (r = 0.3, p = 0.03). Tissue CD8+
                                                                                  tissue compared to blood of CRSwNP patients. Even though
T cells were directly related to the neutrophil count (r = 0.3,
                                                                                  no noticeable differences were detected in CD8+ T cells in
p = 0.04), and tissue CD8+ TEMs were positively correlated                        tissue and blood, all but two patients had a CD4/CD8 ratio
with eosinophil percentage and absolute count (r = 0.3,                           > 1 in blood, and 12 patients had a CD4/CD8 ratio < 1 in
p = 0.03, and r = 0.3, p = 0.04, respectively). The relations                     tissues. Also, the CD4/CD8 ratio was substantially lower
among demographic data, blood cell count, CT scores and                           in tissue than in the blood of CRSwNP patients, which is
levels of different T cell subsets in blood and polyp tissue                      largely attributable to the decrease in CD4+ T cells.
specimens of all patients are presented in Figure 3.                              The distribution of CD4+ and CD8+ T cells in nasal polyps




Figure 3. A heat map showing the demographic data, CT scores and laboratory data [total leukocyte count (TLC), neutrophil (neut) count and percentage (%),
eosinophil (eosin) count and percentage (%), total CD4+ and CD8+ cells, percentages of TNs, TSCMs, TCMs, TEMs and TEMRAs among CD4+ and CD8+ cells in blood and
polyp tissue] of patients. The figure shows a decrease of the tissue CD8+ TNs, CD8+ TCMs, CD4+ TNs, and CD4+TSCMs and an increase in the percentages of TEMs and
TEMRAs among CD4+ and CD8+ T cells compared with their levels in blood.



446
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has been broadly discussed in previous studies. Immuno-          cells to the inflamed nasal mucosa where reactivation oc-
histochemical analysis of sinus mucosal tissue showed el-        curs. Pants et al. found that the majority of CD8+ T cells
evated levels of CD8+ T cell subset in the nasal mucosa of       in nasal polyp tissue of CRSwNP patients were TEMRAs.
CRSwNP than controls 13. Several authors also described          However, they did not detect significant changes in ef-
a predominance of mucosal CD8+ T cells than CD4+ T               fector memory CD4+ T cells in those patients, indicating
cells in patients with nasal polyps 14-16 and an inverse me-     that CD8+ T cells are probably the main cells involved in
dian of CD4/CD8 T cell ratio compared with controls 14.          inflammation in nasal polyps. Moreover, they did not de-
However, Pants and coauthors also noted that the mucosa          tect any differences in CD4 or CD8 T cell subset ratios in
of some patients with chronic inflammation had an excess         nasal mucosa in relation to sinus microbiology or coexist-
of CD4+ T cells with a relative decrease of CD8+ T cells 15.     ing allergy. In addition, they did not find any alterations in
In addition, Derycke and colleagues found a rise in CD4/         CD8+ and CD4+ T cell subsets between peripheral blood of
CD8 T cell ratio in nasal polyps 17. Recently, Bartemes et al.   patients and healthy controls 15.
found lower levels of both CD8+ and CD4+ T cells in nasal        To date, this is the first study to analyse TSCMs in the na-
polyp tissue than in peripheral blood 18. On the other hand,     sal polyps microenvironment. TSCMs are considered the
Morinaka and Nakamura reported no significant differences        least differentiated subset of memory T cells. Staphy-
in the percentages of CD4+ and CD8+ T lymphocyte subsets         lococci have been shown to trigger type 2 cytokine re-
and CD4/CD8 ratio between nasal turbinate mucosa and             lease from innate lymphoid cells (ILCs) and T cells 24.
nasal polyps 19. Immunochemical analysis in another study        Gattinoni and coauthors demonstrated the rapid respon-
showed higher levels of CD8+ and CD4+ T cells in nasal tur-      siveness of CD95+ naïve-like CD8+ and CD4+ TSCMs fol-
binate mucosa in chronic sinusitis patients vs controls. Ad-     lowing exposure to staphylococcal enterotoxin B, ac-
ditionally CD4+ T cells clustered in lamina propria, whereas     companied by the production of IFN-γ, TNF-α and IL-2,
CD8+ T cells were scattered in the lamina propria and ap-        while TNs stayed relatively quiescent. TSCMs have the stem
peared in the epithelium next to seromucous glands 20.           cell-like capabilities of self-renewal and are multipotent
In line with previous findings 16,17,21, among both CD8+ and     progenitors that can generate all memory/effector T-cell
CD4+ T cells, there was a shift from naïve phenotype in          subsets in vitro 25.
blood to an effector/memory phenotype in nasal polyp tis-        A multicentre study carried out in Europe, Asia and Oce-
sues of our CRSwNP patients. However, detailed charac-           ania revealed that tissues from CRSwNP patients in West-
terisation of CD45RA+ and CD45RO+ T cell subsets among           ern countries were Th2 skewed, while those from Beijing
both CD4+ and CD8+ cells in blood and tissue of CRSwNP           showed mixed Th1/Th2/Th17 pattern 26. Eosinophils are
patients is lacking. Most of the variations in T cell sub-       inflammatory cells with a significant role in the pathogen-
sets detected in the current study were in tissue rather than    esis of CRSwNP. In addition, levels of peripheral blood
blood. Our results showed a higher level of CD8+ TCM and         eosinophils have been suggested to be a factor of disease
CD4+ TSCMs with a lower level of CD8+ TEM in the blood of        severity 27. Moreover, according to recent literature, de-
CRSwNP patients compared with controls. In contrast, a           fining not only the eosinophilic but also the neutrophilic
substantial decrease of tissue CD8+ TNs, CD8+ TCMs, CD4+         inflammation in CRS patients could lead to prediction of
TNs, and CD4+TSCMs with a marked increase in the percent-        prognosis 28. Eosinophils are involved in the regulation
ages of TEMs and TEMRAs was detected among CD4+ and CD8+         of T cell function with respect to Th1/Th2 polarisation.
T cells in CRSwNP patients.                                      An earlier study reported a positive correlation of CD8+ T
Generally, the percentages of TEMs and TEMRAs were high-         cells with the eosinophil absolute count and percentage 29.
er among CD8+ than CD4+ T cells in blood and tissue.             Ma et al. evaluated the CD8+ T cell subset distribution in
Conversely, the percentages of TNs and TCMs were higher          CRSwNP patients and described that the percentage of
among CD4+ than CD8+ T cells in the blood and tissue of          IL-4+ type-2 cytotoxic T cell (Tc2) subset was directly
our CRSwNP patients. Tissue accumulation of TEMRAs is            correlated with absolute eosinophil count, while the fre-
enhanced by the presence of persistent infections 22. Alto-      quencies of IFN-γ+ Tc1 and IL-17A+ Tc17 subsets were
gether these findings indicate that the nasal polyp micro-       directly correlated with absolute neutrophil count 30.
environment might drive T cell differentiation, especially       Along with the previous findings, tissue CD8+ T cells
CD8+ T cells signifying increased activation by long-term        showed significant correlations with eosinophil and neu-
exposure to antigens which reflects their likely role in         trophil counts and percentages in our study, proposing
defense against microbes and or suppression of allergic          that, comparable to CD4+ T cell subsets, accumulation of
responses.                                                       CD8+ T cells in the tissue of nasal polyps associates with
Previous studies 15,23 suggested recruitment of memory T         the degree of inflammation in CRS.



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Altogether, TNs, TSCMs, and TCMs probably differentiated into                    ers. J Allergy Clin Immunol 2016;137:1449-1456. e4. https://doi.
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effector TEMs and TEMRAs subsets in sinonasal mucosa, es-                   2
                                                                                 Bachert C, Zhang N, Patou J, et al. Role of staphylococcal superan-
pecially CD8+ T cells. These changes in memory/effector                          tigens in upper airway disease. Curr Opin Allergy Clin Immunol
T-cells may reflect a constant exposure to microbial an-                         2008;8:34-38. https://doi.org/10.1097/ACI.0b013e3282f4178f
tigens or allergens. However, our study has some limita-                    3
                                                                                 Singh P, Mehta R, Agarwal S, et al. Bacterial biofilm on the sinus mu-
tions. It was not possible to get healthy tissue from controls                   cosa of healthy subjects and patients with chronic rhinosinusitis (with
                                                                                 or without nasal polyposis). J Laryngol Otol 2015;129:46-49. https://
which would probably have given wider insight on the al-                         doi.org/10.1017/s002221511400303x
terations in memory/effector T cell subsets in patients with                4
                                                                                 Larché M, Robinson DS, Kay AB. The role of T lymphocytes in the
CRSwNP. Also, histopathological examination might yield                          pathogenesis of asthma. J Allergy Clin Immunol 2003;111:450-463.
data on the impact of T cell activity on sinonasal mucosa.                       https://doi.org/10.1067/mai.2003.169
                                                                            5
                                                                                 Seder RA, Ahmed R. Similarities and differences in CD4+ and CD8+
It would be of interest to know if changes in T cell subsets                     effector and memory T cell generation. Nat Immunol 2003;4:835-
and cytokine secretion were present following exposure to                        842. https://doi.org/10.1038/ni969
microbial antigens.                                                         6
                                                                                 Sallusto F, Lenig D, Förster R, et al. Two subsets of memory T lym-
                                                                                 phocytes with distinct homing potentials and effector functions. Na-
                                                                                 ture 1999;401:708-712. https://doi.org/10.1038/44385
Conclusions                                                                 7
                                                                                 Schmueck-Henneresse M, Sharaf R, Vogt K, et al. Peripheral blood-
                                                                                 derived virus-specific memory stem T cells mature to functional
The reduction of TNs, TSCMs, and TCMs and accumulation of                        effector memory subsets with self-renewal potency. J Immunol
TEMs and TEMRAs subsets that function as effector cells in                       2015;194:5559-5567. https://doi.org/10.4049/jimmunol.1402090
sinonasal mucosa, especially CD8+ T cells, might indicate                   8
                                                                                 Hintzen RQ, de Jong R, Lens SM, et al. Regulation of CD27 expres-
the role of immunomodulation of memory/effector T-cells                          sion on subsets of mature T-lymphocytes. J Immunol (Baltimore, Md:
                                                                                 1950) 1993;151:2426-2435.
in the pathogenesis of CRSwNP.                                              9
                                                                                 Berger C, Jensen MC, Lansdorp PM, et al. Adoptive transfer of ef-
                                                                                 fector CD8+ T cells derived from central memory cells establishes
Conflict of interest statement                                                   persistent T cell memory in primates. J Clin Invest 2008;118:294-305.
                                                                                 https://doi.org/10.1172/jci32103
The authors declare no conflict of interest.                                10
                                                                                 Carrasco J, Godelaine Dl, Van Pel A, et al. CD45RA on human
                                                                                 CD8 T cells is sensitive to the time elapsed since the last antigenic
Funding                                                                          stimulation. Blood 2006;108:2897-2905. https://doi.org/10.1182/
This research did not receive any specific grant from fund-                      blood-2005-11-007237
ing agencies in the public, commercial, or not-for-profit
                                                                            11
                                                                                 Lund VJ, Mackay IS. Staging in rhinosinusitis. Rhinology
                                                                                 1993;31:183-184.
sectors.                                                                    12
                                                                                 Bhattacharyya N, Fried MP. The accuracy of computed tomography in
                                                                                 the diagnosis of chronic rhinosinusitis. Laryngoscope 2003;113:125-
Author contributions                                                             129. https://doi.org/ 10.1097/00005537-200301000-00023
AMZ, OEB, MM Osman contributed to the study concep-                         13
                                                                                 Onerci M, Elsurer C, Guzel EE, et al. Distribution of inflammatory
tion and design. MM Osman was responsible for patients’                          cells, adhesion molecules, intermediate filaments, and chemokine re-
                                                                                 ceptors in subgroups of nasal polyp patients. Am J Rhinol Allergy
recruitment. Material preparation and data collection were                       2011;25:e176-e180. https://doi.org/10.2500/ajra.2011.25.3635
accomplished by all authors and data analysis was per-                      14
                                                                                 Hao J, Pang Y-T, Wang D-Y. Diffuse mucosal inflammation in nasal
formed by OEB and MMO. The first draft of the manu-                              polyps and adjacent middle turbinate. Otolaryngol Head Neck Surg
                                                                                 2006;134:267-275. https://doi.org/10.1016/j.otohns.2005.09.026
script was written by OEB and MMO and AMZ, OEB and                          15
                                                                                 Pant H, Hughes A, Miljkovic D, et al. Accumulation of effector mem-
MMO commented on previous versions of the manuscript,                            ory CD8+ T cells in nasal polyps. Am J Rhinol Allergy 2013;27:e117-
read and approved the final manuscript.                                          e126. https://doi.org/10.2500/ajra.2013.27.3958
                                                                            16
                                                                                 Ickrath P, Kleinsasser N, Ding X, et al. Characterization of T-cell
Ethical consideration                                                            subpopulations in patients with chronic rhinosinusitis with nasal
                                                                                 polyposis. Allergy Rhinol 2017;8:139-147. https://doi.org/10.2500/
The Faculty of Medicine Ethics Committee, Assiut Uni-                            ar.2017.8.0214
versity, reviewed and accepted the study protocol (IRB.                     17
                                                                                 Derycke L, Eyerich S, Van Crombruggen K, et al. Mixed T helper
No. 17300596), according to the latest revision of the Dec-                      cell signatures in chronic rhinosinusitis with and without polyps. PloS
                                                                                 One 2014;9:e97581. https://doi.org/10.1371/journal.pone.0097581
laration of Helsinki. All patients gave written informed
                                                                            18
                                                                                 Bartemes KR, Choby G, O’Brien EK, et al. Mass cytometry reveals
consent to participate in the study.                                             unique subsets of T cells and lymphoid cells in nasal polyps from pa-
                                                                                 tients with chronic rhinosinusitis (CRS). Allergy 2021;76:2222-2226.
                                                                                 https://doi.org/10.1111/all.14720
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