scholarly journals Reversal of infectious mononucleosis-associated suppressor T cell activity by D-mannose.

1983 ◽  
Vol 158 (4) ◽  
pp. 1048-1060 ◽  
Author(s):  
G Tosato ◽  
S E Pike ◽  
R M Blaese
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Infectious Mononucleosis ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Cell Activity ◽  
Pokeweed Mitogen ◽  
Suppressor T Cells

Epstein-Barr virus-induced infectious mononucleosis (IM) is associated with the activation of suppressor T lymphocytes that profoundly inhibit immunoglobulin (Ig) production in vitro. We have examined the nature of signals operating in the interaction between IM suppressor T cells and their targets, and explored the possibility that a lectin-like receptor molecule and its specific sugar might provide specificity to this interaction. When D-mannose or some of its derivatives, including alpha-methyl-D-mannoside, mannose-6-phosphate, and mannan, were added to suppressed cultures containing IM T lymphocytes and pokeweed mitogen (PWM)-stimulated normal mononuclear cells, a significant enhancement of Ig production was observed. These sugars had little or no effect on Ig production by the PWM-stimulated responder cells alone and thus the enhanced Ig production could be attributed to the reversal of suppression in the co-cultures by these sugars. This was further confirmed by the observation that the sugars were effective only if present during the first 24 h of culture, a time when IM suppressor T cells exert their principal effect. The effect of sugars on Ig production by suppressed cultures was similar to that achieved by decreasing by about fourfold the number of IM T cells in culture. The effect of the sugars is unlikely to represent a form of nonspecific toxicity, since inhibited cultures become responders in the presence of the sugar. Furthermore, toxicity restricted to the suppressor T cells is unlikely, since preincubation of the T cells with the sugars did not reduce their subsequent ability to suppress in secondary indicator cultures. In addition, there was no correlation between the effect of the sugars on T cell proliferation and their effect on T cell-mediated suppression. The reversal of suppression by sugars was dose dependent and demonstrated stereo-specificity in that L-mannose was without effect while D-mannose reversed suppression. These data indicate that D-mannose and some of its derivatives consistently reverse suppression of Ig production by IM T cells and strongly suggest a role for saccharides as critical components in the cellular receptors involved in certain physiologic immune cell interactions.

scholarly journals Regulatory functions of hapten-reactive helper and suppressor T lymphocytes. II. Selective inactivation of hapten-reactive suppressor T cells by hapten-nonimmunogenic copolymers of D-amino acids, and its application to the study of suppressor T-cell effect on helper T-cell development

1977 ◽  
Vol 146 (1) ◽  
pp. 91-106 ◽  
Author(s):  
T Hamaoka ◽  
M Yoshizawa ◽  
H Yamamoto ◽  
M Kuroki ◽  
M Kitagawa
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Development ◽  
Cell Activity ◽  
Helper T Cells ◽  
Suppressor T Cells ◽  
Helper T Cell ◽  
Suppressor T Cell ◽  
Suppressor T Lymphocytes

An experimental condition was established in vivo for selectively eliminating hapten-reactive suppressor T-cell activity generated in mice primed with a para-azobenzoate (PAB)-mouse gamma globulin (MGG)-conjugate and treated with PAB-nonimmunogenic copolymer of D-amino acids (D- glutamic acid and D-lysine; D-GL). The elimination of suppressor T-cell activity with PAB-D-GL treatment from the mixed populations of hapten- reactive suppressor and helper T cells substantially increased apparent helper T-cell activity. Moreover, the inhibition of PAB-reactive suppressor T-cell generation by the pretreatment with PAB-D-GL before the PAB-MGG-priming increased the development of PAB-reactive helper T-cell activity. The analysis of hapten-specificity of helper T cells revealed that the reactivity of helper cells developed in the absence of suppressor T cells was more specific for primed PAB-determinants and their cross-reactivities to structurally related determinants such as meta-azobenzoate (MAB) significantly decreased, as compared with the helper T-cell population developed in the presence of suppressor T lymphocytes. In addition, those helper T cells generated in the absence of suppressor T cells were highly susceptible to tolerogenesis by PAB-D- GL. Similarly, the elimination of suppressor T lymphocytes also enhanced helper T-cell activity in a polyclonal fashion in the T-T cell interactions between benzylpenicilloyl (BPO)-reactive T cells and PAB- reactive T cells after immunization of mice with BPO-MGG-PAB. Thus inhibition of BPO-reactive suppressor T-cell development by the BPO-v-GL- pretreatment resulted in augmented generation of PAB-reactive helper T cells with higher susceptibility of tolerogenesis to PAB-D-GL. Thus, these results support the notion that suppressor T cells eventually suppress helper T-cell activity and indicate that the function of suppressor T cells related to helper T-cell development is to inhibit the increase in the specificity and apparent affinity of helper T cells in the primary immune response. The hapten-reactive suppressor and helper T lymphocytes are considered as a model system of T cells that regulate the immune response, and the potential applicability of this system to manipulating various T cell-mediated immune responses is discussed in this context.

Delayed in Vitro Immunoglobulin Production by Cord Lymphocytes

PEDIATRICS ◽  
1980 ◽  
Vol 65 (3) ◽  
pp. 497-500
Author(s):  
Yukiaki Miyagawa ◽  
Kenichi Sugita ◽  
Atsushi Komiyama ◽  
Taro Akabane
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Mononuclear Cells ◽  
Pokeweed Mitogen ◽  
Immunoglobulin Production ◽  
Mitogen Stimulation ◽  
Functional Immaturity ◽  
Adult Cells

Pokeweed mitogen-induced immunoglobulin (Ig) production by cord lymphocytes was studied in vitro by Ig-secreting plaque-forming cell (Ig-PFC) assay. Although adult mononuclear cells generated all of IgM-, IgG-, and IgA-PFC, cord mononuclear cells generated only IgM-PFC when cultured for seven days. The number of cord IgM-PFC was 102 ± 26/104 mononuclear cells, being about one fourth of that of adult IgM-PFC. When cultured for 14 days, cord mononuclear cells formed increased numbers of IgM-PFC in contrast to adult cells, and yielded IgG-PFC as well, indicating delayed Ig production. Cord T cells were much less effective at helping adult B cells to differentiate into Ig-PFC as compared with adult T cells. Substitution of adult T cells for cord T cell markedly improved the response of cord B cells. The present study demonstrates Ig secretion by cord lymphocytes in response to pokeweed mitogen stimulation. The results further indicate that the delayed Ig production by cord lymphocytes is largely due to functional immaturity of the T cells.

scholarly journals In vitro regulation of immunoglobulin synthesis after human marrow transplantation. II. Deficient T and non-T lymphocyte function within 3- 4 months of allogeneic, syngeneic, or autologous marrow grafting for hematologic malignancy

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 844-850 ◽  
Author(s):  
RP Witherspoon ◽  
LG Lum ◽  
R Storb ◽  
ED Thomas
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Hematologic Malignancy ◽  
Plaque Assay ◽  
Pokeweed Mitogen ◽  
Lymphocyte Function ◽  
Peripheral Blood Mononuclear ◽  
Immunoglobulin Secretion

Abstract Immunoglobulin secretion was studied in 37 patients between 19 and 106 days after allogeneic HLA-identical (30 patients), allogeneic one HLA- haplotype-identical (three patients), syngeneic (three patients), or autologous (one patient) marrow grafting. E rosette-positive (T) and E rosette-negative (non-T) peripheral blood mononuclear cells were cocultured with pokeweed mitogen for 6 days. Polyvalent immunoglobulin secretion was determined by counting plaque forming cells in a reverse hemolytic plaque assay. The number of antibody secreting cells in cocultures of autologous T and non-T lymphocytes was low in 40 of 44 tests conducted on samples from the 37 patients. Mononuclear or non-T cells from 38 of 40 tests failed to produce antibody when cultured with normal helper T cells. T cells from 23 of 37 tests failed to help normal non-T cells secrete antibody. T lymphocytes from 23 of 41 tests suppressed antibody production greater than 80% by normal T and non-T cells. The suppressor cells were radiosensitive in 17 of the 25 tests. The abnormal function of lymphocyte subpopulations in patients during the first 3 mo after syngeneic, allogeneic or autologous marrow grafting was similar regardless of the type of graft or the presence of acute graft versus host disease.

scholarly journals Antigen-specific suppressor T-cell activity in genetically restricted immune spleen cells.

1978 ◽  
Vol 148 (5) ◽  
pp. 1271-1281 ◽  
Author(s):  
C W Pierce ◽  
J A Kapp
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Activity ◽  
Antibody Responses ◽  
Spleen Cells ◽  
Suppressor T Cells ◽  
Specific Suppressor T Cells ◽  
Immune Spleen Cells

Virgin spleen cells develop comparable primary antibody responses in vitro to syngeneic or allogeneic macrophages (Mphi) bearing the terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT), whereas immune spleen cells primed with syngeneic or allogeneic GAT-Mphi develop secondary responses preferentially when stimulated with GAT-Mphi syngeneic to the GAT-Mphi used for priming in vivo. These restrictions are mediated by products of the I-A subregion of the H-2 complex and are operative at the level of the GAT-Mphi-immune helper T-cell interactions. To investigate why these immune spleen cells fail to develop a significant antibody response to GAT-Mphi other than those used for in vivo immunization and determine the mechanism by which the restriction is maintained, spleen cells from virgin and syngeneic or allogeneic GAT-Mphi-primed mice were co-cultured in the presence of GAT-Mphi of various haplotypes. Antibody responses to GAT developed only in the presence of GAT-Mphi syngeneic to the Mphi used for in vivo priming; responses in cultures with GAT-Mphi allogeneic to the priming Mphi, whether these Mphi were syngeneic or allogeneic with respect to the responding spleen cells, were suppressed. The suppression was mediated by GAT-specific radiosensitive T cells. Thus, development of GAT-specific suppressor T cells appears to be a natural consequence of the immune response to GAT in responder as well as nonresponder mice. The implications of stimulation of genetically restricted immune helper T cells, and antigen-specific, but unrestricted, suppressor T cells after immunization with GAT-Mphi in vivo are discussed in the context of regulatory mechanisms in antibody responses.

G-CSF Activated Granulocytes Inhibit Experimental Graft-versus-Host Disease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4989-4989
Author(s):  
Zilton F.M. Vasconcelos ◽  
Julia Farache ◽  
Bruna M. Santos Grad ◽  
Tereza S. Palmeira Grad ◽  
Luis Fernando Bouzas ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Cell Activity ◽  
Control Group ◽  
Low Density ◽  
Graft Versus Host ◽  
The One

Abstract Acute Graft versus host diseas (aGVHD) is a major complication of stem cell transplantation. The disease is mediated by T cells and a higher incidence/severity would be expected when higher numbers of T cells are inoculated. However, the incidence of aGVHD in PBST, which carries about 10 times more T cells then BMT, is not higher than the one found in later. This finding indicates a modulatory role for G-CSF over T cell activity. We had previously shown that T cells from G-CSF treated PBSC donors do not produce g-IFN nor IL-4 and that this inhibition was mediated by low density, G-CSF activated, granulocytes. In order to test if in fact G-CSF activated granulocytes could inhibit disease, we first checked if G-CSF could generate low density granulocytes, in vivo and in vitro. Indeed, either in vivo(21mg /day - 5 days) or in vitro (150 ng -12hs) with G-CSF generates low density granulocytes which co-purify with the mononuclear cells in the ficoll® gradient. Moreover, as we had shown in humans, these low density cells, inhibit the production of g-IFN by anti-CD3 activated T cells on flow cytometry studies (17%-T cells alone versus 3% T cells with granulocytes 1:1). Radiation quimaeras were set with (B6 X BALB/c)F1 as hosts reconstituted with T cell depleted C57Bl6 bone marrow, in the presence or absence of nylon wool selected spleen cells (NWSC), as T cell source, from normal or G-CSF treated mice. As previously shown by others, NWSC from G-CSF treated mice diminishes the incidence of acute disease on day 20 post-transplant, from 75 to 25%. In order to investigate if this inhibition was dependent on the activated granulocytes present in the NWSC from G-CSF treated mice, granulocytes were depleted with anti-GR1 and complement. In this case, the incidence of disease is the same or even higher (75% experiment#1 and 100% in experiment #2) than the one observed on the control group (NWSC from control mice). These results strongly suggest that activated granulocytes could indeed inhibit aGVHD. We then generated activated granulocytes in vitro, by treating spleen derived high density granulocytes with 150ng of G-CSF for 12 hs. After the incubation period, a new ficoll® gradient was performed and the low density cells were obtained. T cell contamination on the second gradient was eliminated by anti-CD4 and CD8 complement lysis. These activated granulocytes were inoculated together with NWSC from control mice in the radiation quimaeras at a 1:1 ratio. In this case 100% disease inhibition was observed when compared to the positive control group, where 75% of the animals got sick. Our data indicate that activated granulocytes are the major mediators of the G-CSF immunossupressive effects and that these cells can be used as a novel immune modulator in clinical transplantation to prevent acute GVHD.

scholarly journals In vitro regulation of immunoglobulin synthesis after human marrow transplantation. II. Deficient T and non-T lymphocyte function within 3- 4 months of allogeneic, syngeneic, or autologous marrow grafting for hematologic malignancy

Blood ◽  
1982 ◽  
Vol 59 (4) ◽  
pp. 844-850 ◽  
Author(s):  
RP Witherspoon ◽  
LG Lum ◽  
R Storb ◽  
ED Thomas
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Mononuclear Cells ◽  
Hematologic Malignancy ◽  
Plaque Assay ◽  
Pokeweed Mitogen ◽  
Lymphocyte Function ◽  
Peripheral Blood Mononuclear ◽  
Immunoglobulin Secretion

Immunoglobulin secretion was studied in 37 patients between 19 and 106 days after allogeneic HLA-identical (30 patients), allogeneic one HLA- haplotype-identical (three patients), syngeneic (three patients), or autologous (one patient) marrow grafting. E rosette-positive (T) and E rosette-negative (non-T) peripheral blood mononuclear cells were cocultured with pokeweed mitogen for 6 days. Polyvalent immunoglobulin secretion was determined by counting plaque forming cells in a reverse hemolytic plaque assay. The number of antibody secreting cells in cocultures of autologous T and non-T lymphocytes was low in 40 of 44 tests conducted on samples from the 37 patients. Mononuclear or non-T cells from 38 of 40 tests failed to produce antibody when cultured with normal helper T cells. T cells from 23 of 37 tests failed to help normal non-T cells secrete antibody. T lymphocytes from 23 of 41 tests suppressed antibody production greater than 80% by normal T and non-T cells. The suppressor cells were radiosensitive in 17 of the 25 tests. The abnormal function of lymphocyte subpopulations in patients during the first 3 mo after syngeneic, allogeneic or autologous marrow grafting was similar regardless of the type of graft or the presence of acute graft versus host disease.

scholarly journals Characterization of the Role of Regulatory T Cells (Tregs) in Inducing Progression of Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 502-502 ◽  
Author(s):  
Yawara Kawano ◽  
Michele Moschetta ◽  
Katsutoshi Kokubun ◽  
Pavlo Lukyanchykov ◽  
Esilida Sula Karreci ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rna Sequencing ◽  
Mouse Models ◽  
Immune Cells ◽  
Immune Cell ◽  
Cell Activity ◽  
Treg Depletion

Abstract Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis. Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry. Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs. Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs. Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals Simultaneous Induction of Multiple Antigen-Specific Cytotoxic T Lymphocytes in Nonhuman Primates by Immunization with a Mixture of Four Plasmodium falciparum DNA Plasmids

1998 ◽  
Vol 66 (9) ◽  
pp. 4193-4202 ◽  
Author(s):  
Ruobing Wang ◽  
Denise L. Doolan ◽  
Yupin Charoenvit ◽  
Richard C. Hedstrom ◽  
Malcolm J. Gardner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Peripheral Blood Mononuclear ◽  
Effector Cells ◽  
Cell Responses

ABSTRACT CD8+ T cells have been implicated as critical effector cells in protective immunity against malaria parasites developing within hepatocytes. A vaccine that protects against malaria by inducing CD8+ T cells will probably have to include multiple epitopes on the same protein or different proteins, because of parasite polymorphism and genetic restriction of T-cell responses. To determine if CD8+ T-cell responses against multiple P. falciparum proteins can be induced in primates by immunization with plasmid DNA, rhesus monkeys were immunized intramuscularly with a mixture of DNA plasmids encoding four P. falciparumproteins or with individual plasmids. All six monkeys immunized with PfCSP DNA, seven of nine immunized with PfSSP2 DNA, and five of six immunized with PfExp-1 or PfLSA-1 DNA had detectable antigen-specific cytotoxic T lymphocytes (CTL) after in vitro restimulation of peripheral blood mononuclear cells. CTL activity was genetically restricted and dependent on CD8+ T cells. By providing the first evidence for primates that immunization with a mixture of DNA plasmids induces CD8+ T-cell responses against all the components of the mixture, these studies provide the foundation for multigene immunization of humans.

scholarly journals THU0046 A PIPELINE TO STUDY ANTIGEN-SPECIFIC CD4+ T CELLS IN RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 235.1-236
Author(s):  
R. Kumar ◽  
N. Yoosuf ◽  
C. Gerstner ◽  
S. Turcinov ◽  
K. Chemin ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Tenascin C ◽  
Tetramer Staining ◽  
Tcr Repertoire ◽  
Citrullinated Antigen

Background:Autoimmunity to citrullinated autoantigens forms a critical component of disease pathogenesis in rheumatoid arthritis (RA). Presence of anti-citrullinated protein antibodies (ACPAs) in patients has high diagnostic value. Recently, several citrullinated antigen specific CD4+T cells have been described. However, detailed studies of their T-cell receptor usage and in-vivo profile suffer from the disadvantage that these cells are present at very low frequencies. In this context, we here present a pipeline for TCR repertoire analysis of antigen-specific CD4+T cells from RA patients, including both citrulline and influenza (control) specificities using in-vitro peptide challenge induced-cell expansion.Objectives:To enable studies of the T cell repertoire of citrullinated antigen-specific CD4+T cells in rheumatoid arthritisMethods:Peripheral blood mononuclear cells (PBMCs) (n=7) and synovial fluid mononuclear cells (SFMCs) (n=5) from HLA-DR*0401-postive RA patients were cultured in the presence of citrullinated Tenascin C peptide cocktails or influenza peptides (positive control). Citrulline reactive cells were further supplemented with recombinant human IL-15 and IL-7 on day 2. All cultures were replenished with fresh medium on day 6 and rIL-2 was added every 2 days from then. Assessment of proportion of peptide-HLA-tetramer positive cells was performed using flow cytometry whereby individual antigen-specific CD4+T cells were sorted into 96-well plates containing cell lysis buffer, followed by PCR-based alpha/beta TCR sequencing. TCR sequencing data was demultiplexed and aligned for TCR gene usage using MiXCR. Some tetramer positive cells were sorted into complete medium containing human IL-2 and PHA for expansion of antigen-specific cells. Cells were supplemented with irradiated allogenic PBMCs (30 times number of antigen specific cells). Clones of antigen specific CD4+T cells were further subjected to tetramer staining to confirm expansion of cells.Results:As evidenced by increase in frequency of tetramer positive CD4+T cells, in vitro peptide stimulation resulted in expansion of both influenza specific (Fig. 1a) and citrullinated antigen specific (Fig. 1b) CD4+T cells. Polyclonal in-vitro expansion of tenascin C tetramer positive sorted cells followed by tetramer staining further confirmed antigen specificity and enrichment for antigen specific CD4+T cells after polyclonal stimulation (Fig.1c). TCR repertoire analysis in PB and SF dataset from the first patient showed clonal expansion of influenza specific cells in both sites. Synovial fluid had more diversity of expanding clones as compared to paired PB, with few expanded clones being shared among SF and PB. We observed a more diverse TCR repertoire in citrulline specific CD4+T cells. We also observed sharing of TCR alpha chains among different citrulline specific CD4+T cell clones.Fig. 1In-vitroexpansion of antigen specific CD4+T cells:Conclusion:This method provides a highly suitable approach for investigating TCR specificities of antigen specific CD4+T cells under conditions of low cell yields. Building on this dataset will allow us to assess specific features of TCR usage of autoreactive T cells in RA.PBMCs were cultured in presence of (a) influenza (HA, MP54) and (b) citrullinated tenascin peptides. The proportion of antigen specific CD4+T cells was assessed using HLA-class II tetramer staining. We observed an increase in frequency of (a) Infleunza specific cells (red dots in upper left and lower right quadrants) and (b) citrullinated tenascin C specific cells (red dots in lower right quadrant), at day 13 post culture as compared to day 3. (c) Sorting of citrullinated tenascin specific CD4+T cells, followed by PHA expansion resulted in visible increase in proportion of citrullinated tenascin specific CD4+T cells.Disclosure of Interests:Ravi kumar: None declared, Niyaz Yoosuf: None declared, Christina Gerstner: None declared, Sara Turcinov: None declared, Karine Chemin: None declared, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica

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