scholarly journals THU0032 MODIFIED PEPTIDES AS A NOVEL IMMUNOTHERAPY FOR RHEUMATOID ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 229.1-229
Author(s):  
E. Araklioti ◽  
L. Herman ◽  
N. Q. N. Nguyen ◽  
R. Roohi Ahangarani ◽  
M. Erak ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Flow Cytometric Analysis ◽  
Class Ii ◽  
Research Support ◽  
Modified Peptides ◽  
Apoptotic Activity ◽  
Walloon Region

Background:Rheumatoid arthritis (RA) is a highly prevalent and severe systemic autoimmune disease associated with permanent disability and strong socio-economic burdens. Currently, there is no therapeutic treatment and RA patients rely on lifelong, costly treatments. Imcyse develops modified peptides eliciting antigen specific cytolytic CD4 T cells (cCD4+) that induce apoptosis of antigen presenting cells (APC) in a contact dependent manner. cCD4+ also induce apoptosis of autoantigen-specific bystander T-cells, activated by the same APC thus eliminating the risk of general immunosuppression. Peptides consist of MHC class II T cell epitopes of a target autoantigen modified in their flanking region by the addition of an amino acid sequence containing a thiol-disulphide oxidoreductase active motif1.Objectives:The goal of this study was to synthesize modified peptides from a target RA autoantigen and test their potency to generatein vitrospecific and cytolytic CD4+ T cells from RA patients.Methods:We designed modified peptides from a target RA autoantigen after in silico and in vitro assessment to identify MHC II core binding region, HLA class II binding capacities and physiochemical properties.CD4+ T cells were purified from PBMC of a newly diagnosed seropositive RA patient and co-cultured with autologous APC in the presence of the modified peptide. The CD4+ T cells were restimulated periodically. Peptide’s ability to generate specific CD4+ T cells was evaluated by flow cytometric analysis of the expression of surface activation marker CD154 (CD40L). The peptide specific CD4+ T cell lines were sorted based on their surface CD154 expression. Their pro-apoptotic activity was assessed after overnight (O/N) co-culture of CD4+ T cells with fluorescent tracer labelled autologous lymphoblastoid cells lines (LCL). Flow cytometry quantification of LCL apoptosis was measured by annexin V staining. MHC II restriction of CD4+ T cells was demonstrated by the addition of blocking antibodies against HLA-DR, DP or DQ molecules.Results:CD4+ T cells were in vitro expanded after six consecutive stimulations with the peptide. We investigated their specificity by flow cytometry and showed that 69% of CD4+ T cells that were stimulated O/N in the presence of the peptide expressed the activation marker CD154 versus 29% of CD4+ T cells that were stimulated in its absence. These cells were sorted based on CD154 expression following specific stimulation. Cell enrichment was then assessed by flow cytometric analysis. Data showed that more than 91% (background 3%) were peptide specific based on CD154 expression.After co-culture of CD4+ T cells with LCL, in independent experiments, Annexin V binding was detected on peptide loaded LCL, ranging from 69% to 89%, while when LCL were kept unloaded these values were between 30% and 55%, respectively, indicating that when specifically activated, these CD4+ T cells had pro-apoptotic activity. When both the peptide and blocking antibodies against HLA-DR, DP or DQ molecules added in the co-culture the pro-apoptotic activity was inhibited by 68%, 20% and 25%, respectively.Conclusion:The preliminary but very promising data show that our modified peptide generates peptide-specific CD4+ T cells with lytic properties that lyse target APC in an HLA class II specific manner. Our plan is to show that these CD4+ T cells can also induce apoptosis in bystander T cells and to further validate our results in additional RA donors.References:[1]Carlier, V. A., Vanderelst, L., Janssens, W. & Jacquemin, M. G. Increased Synapse Formation Obtained by T Cell Epitopes Containing a CxxC Motif in Flanking Residues Convert CD4 + T Cells into Cytolytic Effectors.7, (2012).Disclosure of Interests:Eleni Araklioti Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Ludivine Herman Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Ngoc Quynh Nhu Nguyen Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Roxana Roohi Ahangarani Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Milos Erak Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Bernard Lauwerys: None declared, Patrick Durez Speakers bureau: AbbVie, Bristol-Myers Squibb, Celltrion, Eli Lilly, Pfizer, Sanofi, Vincent Geenen: None declared, Aaron Winkler Shareholder of: Shareholder of Pfizer, Inc, Employee of: Full time employee of Pfizer, Inc, Marcelle Van Mechelen Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Luc Vander Elst Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Vincent Carlier Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region

Increased Expression Of IL-15 Promotes Cutaneous T-Cell Lymphomagenesis Via The Upregulation Of Histone Deacetylases: Evidence For Successful Preclinical Targeting

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1826-1826
Author(s):  
Anjali Mishra ◽  
Krista M.D. La Perle ◽  
Laura Sullivan ◽  
Gregory H Sams ◽  
Douglas P Curphey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Histone Deacetylases ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Lymphoid Cells ◽  
Cutaneous Lymphoma ◽  
Normal Donor

Abstract Cutaneous lymphoma is a heterogeneous group of neoplasms of skin-homing malignant lymphocytes. Cutaneous T-cell Lymphoma (CTCL) represents 70-80% of all cutaneous lymphoma and its pathogenesis is largely unknown. Previous studies have shown that interleukin (IL)-15 is a potent stimulant and growth factor for CTCL cells in vitro. In order to investigate the intrinsic levels of IL-15 in CTCL patients, malignant CD4+ T-cells were analyzed for expression of IL-15. Relative quantitation of IL-15 transcript in patient vs normal donor (ND) CD4+ cells showed overexpression of IL-15 in patients (fold increase mean ± SD = 5.36 ± 4.39, N=13, P<0.001). Increase in IL-15 transcript was directly proportional to disease severity in patients i.e. fold increase mean ± SD in IL-15 in Stage I =3.28 ± 1.42, N=3 each, P=0.0047 vs. Stage III patients = 7.42 ± 1.30, N=3 each, P=0.0073. Further, cutaneous lesions in patients stained positive for IL-15 protein in atypical lymphoid cells and Pautrier's microabscess. We next investigated the role of IL-15 in CTCL development using IL-15 transgenic (tg) mice. Within 4-6 weeks of birth, IL-15 tg mice developed extensive patch/plaque skin lesions, progressive alopecia, and severe pruritus. Adult IL-15 tg mice developed extensive involvement with cutaneous lymphoma that was fatal in 100% of the mice (P=0.0003). Antibodies staining revealed that CD4+ skin resident T-cells in IL-15 tg mice were CD3+CD62L-CD44hiCCR4+CLA+. Flow cytometric analysis of single cell suspension of skin showed ∼25-fold increase in CD3+ T-cells in IL-15 tg compared to WT controls (Mean ± SD of absolute number of cells= 3.80 ± 6.97, N=14 vs. 0.15 ± 0.26, N=8 respectively, P<0.001). Lymphoma cells from IL-15 tg mouse skin engrafted and mimicked the primary disease in immune deficient SCID mice upon adoptive transfer. CD4+ T-cells from CTCL patients showed increased histone deacetylases (HDAC) 1, HDAC2 and HDAC6 transcripts over ND CD4+ T-cells and immunoblot analysis of ND CD4+ T-cells exposed to 100ng/ml IL-15 showed upregulation of HDAC1, HDAC2 and HDAC6 ex vivo. IL-15 stimulation of ND CD4+ T-cells resulted in loss of expression of the downstream HDAC1/2 target tumor suppressor, p21 in vitro, and knock down of HDAC6 in IL-15 stimulated ND CD4+ T-cells inhibited their migration in vitro; suggesting that IL-15 mediated upregulation of HDAC6 is critical for T-cell migration. Considering these observations, we used specific novel HDAC inhibitors (HDACi) to target HDAC1/2 (JQ12) and/or HDAC6 (WT161) in IL-15 tg mice to determine if we could prevent CTCL in vivo. IL-15 tg mice were treated with 50mg/kg of either or both the inhibitors, 5 days/week for 4 weeks (n=4 each). While placebo treated IL-15 tg mice progressively developed lesions during the course of treatment, IL-15 tg mice treated with JQ12 and/or WT161 showed no clinical signs of disease. This was further corroborated by histopathology analysis of skin sections from treated mice (Figure 1). Thus, our data suggest that inhibiting HDAC1, HDAC2 and/or HDAC6 pathways inhibits the development of CTCL in IL-15tg mice. In addition to the prevention study, we assessed the ability of a novel pan-HDACi, AR42, to treat active and progressive disease in our model. IL-15 tg mice with established CTCL were randomized to receive either AR42 or placebo feed (n=6 each) for 12 days. The IL-15 tg mice treated with AR42 showed remarkable improvement compared to the placebo mice whose disease progressed. Histopathology analysis of the AR42-treated IL-15 tg mice showed an impressive clearance of the CD3+ and CD4+ atypical lymphocytic infiltrate compared to placebo-treated mice (Figure 2). In summary we provide evidence that IL-15 has a causal role in the pathogenesis of CTCL; that IL-15 tg mice provide a novel model for studying disease pathogenesis and for evaluating potential therapies; that HDACi targeting specific HDACs may be effective in preventing CTCL and a novel pan-HDACi can reverse severe dermatologic disease in this CTCL model. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures: No relevant conflicts of interest to declare.

Histone Deacetylase Inhibitors Induce Immuno-Dominant Suppression of Dendritic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 456-456 ◽  
Author(s):  
Pavan Reddy ◽  
Yoshinobu Maeda ◽  
Raimon Duran-Struuck ◽  
Oleg Krijanovski ◽  
Charles Dinarello ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Hdac Inhibitors ◽  
Class Ii ◽  
Wild Type ◽  
Tnf Α

Abstract We and others have recently demonstrated that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor with anti-neoplastic properties, reduces experimental acute graft-versus-host disease (GVHD). We have now investigated the mechanisms of action of two HDAC inhibitors, SAHA and ITF 2357, on allogeneic immune responses. Bone marrow derived dendritic cells (DCs) were preincubated with the HDAC inhibitors at nanomolar concentrations for 16–18 hours and stimulated with lipopolysaccharide (LPS). Pretreatment of DCs caused a significant reduction in the secretion of TNF-α, IL-12p70 and IL-6 compared to the untreated controls (P< 0.005). Similar effects were seen using human peripheral blood mononuclear cell derived DCs. Pre-treatment of both murine and human DCs also significantly reduced their in vitro stimulation of allogeneic T cells as measured by proliferation and IFN-γ production (P<0.01). We determined the in vivo relevance of these observations utilizing a mouse model where the responses of allogeneic donor bm12 T cells depended on the function of injected host B6 DCs would stimulate. Recipient Class-II −/− B6 (H-2b) received 11 Gy on day -1 and were injected with 4–5 x 106 wild type B6 DCs treated with SAHA or with media on days -1 and 0 and then transplanted with 2 x 106 T cells and 5 x 106 TCDBM cells from either syngeneic B6 or allogeneic bm12 donors. SAHA treatment of DCs significantly reduced expansion of allogeneic donor CD4+ T cells on day +7 after BMT compared to controls (P<0.05). SAHA treatment induced a similarly significant reduction in the expansion of CD8+ cells in Class I disparate [bm1→β2M−/−] model. In vitro, SAHA treatment significantly suppressed the expression of CD40 and CD80 but did not alter MHC class II expression. Surprisingly, when mixed with normal DCs at 1:1 ratio, SAHA treated DCs dominantly suppressed allogeneic T cell responses. The regulation of T cell proliferation was not reversible by addition of IL-12, TNF-α, IL-18, anti-IL-10 or anti-TGFβ, either alone or in combination. Suppression of allogeneic responses was contact dependent in trans-well experiments. To address whether the regulation of SAHA treated DCs required contact with T cells, we devised a three cell experiment where SAHA treated DCs lacked the capacity to present antigens to T cells. DCs from B6 MHC Class II deficient (H-2b) were treated with SAHA and co-cultured with wild type B6 (H-2b) DCs along with purified allogeneic BALB/c (H-2d) CD4+ T cells in an MLR. Allogeneic CD4+ T cells proliferated well, demonstrating the regulation to be dependent on contact between SAHA treated DCs and T cells. To address the in vivo relevance of this suppression, we utilized a well characterized [BALB/c →B6] mouse model of acute GVHD. Recipient B6 animals received 11Gy on day -1 and were injected with of 5 million host type SAHA treated or control DCs on days −1, 0, and +2. Mice were transplanted on day 0 with 2 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Injection of SAHA treated DCs resulted in significantly better survival (60% vs. 10%, P < 0.01) and significantly reduced serum levels of TNF-α, donor T cell expansion and histopathology of GVHD on day +7 after BMT compared to the controls. We conclue that HDAC inhibitors are novel immunomodulators that regulate DC function and might represent a novel strategy to prevent GVHD.

scholarly journals In Vitro Generation of Multi-Epitope Specific CD4+ T Helper Cells for Adoptive Immunotherapy of Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1890-1890
Author(s):  
Pawel Muranski ◽  
Scott Stegemann ◽  
Greg Whitehill ◽  
A John Barrett
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Cultures ◽  
Adoptive Immunotherapy ◽  
Tumor Antigen ◽  
Cd4 T Cells ◽  
Class Ii ◽  
T Helper ◽  
Th Cells

Abstract Introduction: Acute myeloid leukemia (AML) and other hematological malignancies that constitutively express MHC class II molecules represent the ideal target for leukemia-specific CD4+ T helper (Th) cells. CD4+ Th cells are central to the functioning of the immune system. They regulate adaptive immunity against infections and drive pathogenic responses in many autoimmune diseases. Our preclinical studies indicate that adoptively transferred antigen specific Th cells efficiently eradicate even advanced tumors in mice, showing functional advantage over their better characterized classical CD8+ cytotoxic counterparts. Unfortunately, the production of human class II-restricted Th cells is complex and challenging. Consequently, the clinical activity of tumor-specific Th cells has not been systematically evaluated and very little is known about their therapeutic potential in humans. Striking evidence of their enormous power comes from some anecdotal clinical reports of complete regression of metastatic cancers upon transfer of antigen-specific Th cells. Methods and Results: We investigated a reliable GMP-compatible method for in vitro expansion of antigen-specific CD4+ T cells targeting common leukemia associated antigens (LAAs) Willm's Tumor antigen 1 (WT1) and Preferentially Expressed Antigen in Melanoma (PRAME) for future adoptive immunotherapy in the setting of allogeneic stem cell transplantation (SCT). We hypothesized that the naïve CD4+ T cell compartment, rather than the bulk Th cell population could be a superior source for generating a tumor antigen specific cell product. To test this hypothesis bulk and naïve CD4+ T cells were isolated from the peripheral blood of normal donors by magnetic bead separation. Purified T cells were stimulated in vitro with autologous dendritic cells (DCs) pulsed with overlapping 15 amino-acid long peptide (pepmixes) spanning the length of both proteins. Two rounds of stimulation were performed in presence of IL-7, IL-15 and later +/- addition of IL-1, IL-6 and IL-23. IL-2 in low concentration was supplemented during the second round of stimulation. At the end of the second expansion the cells were tested for reactivity by intracellular cytokine production using flow cytometry (FACS) upon stimulation with cognate LAAs or irrelevant control pepmixes. In T cell cultures derived from naïve CD4+ T cells we observed robust induction of PRAME reactivity from majority of tested normal donors, while reactivity against WT1 was donor dependent. The frequency of LAAs in bulk CD4+ T cells was significantly lower in all cases, supporting the notion that pre-exiting memory Th cells have a competitive advantage over the LAA-specific precursor. T cell cultures supplemented with inflammatory cytokines demonstrated further enhancement of antigenic reactivity. Next we tested if LAA pepmix -stimulated T cells can recognize tumor targets. Naïve-derived PRAME and WT1 Th cells generated from normal SCT sibling donors produced IFN-γ, IL-2 and TNF-α upon exposure to fully HLA-matched AML blasts while no reactivity was seen in control CMV pp65-specific Th cells from the same donors. This observation suggests that LAA-specific CD4+ T cells induced with pepmixes have the ability to recognize physiologically-relevant tumor antigens. Conclusions: Here we report the feasibility of generating naïve-derived anti-leukemia CD4+ T cells from majority of normal donors. Removal of competing memory Th cells unmasks the LAA-specific reactivity, thus improving the reliability of the process. Importantly, these Th cells demonstrate highly-specific recognition of the tumor epitopes naturally processed by HLA-matched leukemic blasts, establishing the foundation for a future adoptive immunotherapy clinical trial in patients with hematological malignancy. Disclosures No relevant conflicts of interest to declare.

scholarly journals The CD28 ligand B7/BB1 provides costimulatory signal for alloactivation of CD4+ T cells.

1991 ◽  
Vol 173 (3) ◽  
pp. 759-762 ◽  
Author(s):  
L Koulova ◽  
E A Clark ◽  
G Shu ◽  
B Dupont
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
T Lymphocyte ◽  
Cd4 T Cells ◽  
B Lymphocyte ◽  
Interleukin 2 ◽  
Class Ii ◽  
Cell Surface Molecule

Activation via the T lymphocyte cell surface molecule CD28 provides a potent amplification signal for interleukin 2 (IL-2) production in several in vitro systems. The B lymphocyte activation antigen, B7/BB1, is a natural ligand for CD28. Here we investigate the role of CD28 and B7/BB1 in primary activation of CD4+ T lymphocytes stimulated with allogeneic B lymphoblastoid cell lines. A subset of peripheral CD4+ T cells that is unresponsive to crosslinking of CD3/T cell receptor (TCR) with CD3 monoclonal antibody (mAb) does proliferate in response to allogeneic B lymphoblasts. TCR binding to allogeneic major histocompatibility complex antigens was an absolute requirement for activation of these cells because mAbs to either CD3 or human histocompatibility leukocyte antigen (HLA) class II completely inhibited activation. CD28 and B7/BB1 antibodies inhibited T cell proliferation 90% and 84%, respectively. Similar results were obtained with the total CD4+ T lymphocyte population. Crosslinking of HLA-DR antigens on small, resting B cells induced rapid expression of B7/BB1, which peaked at 6 h and returned to baseline levels within 18 h. These data demonstrate that CD28-B7/BB1 binding provides an important early second signal for alloactivation of CD4+ T lymphocyte by B lymphoblasts. The results also suggest that T cells interacting with allogeneic resting B cells may induce B7/BB1 expression in the alloantigen-presenting cell as a consequence of interaction between the TCR and class II molecules.

scholarly journals CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4073-4082 ◽  
Author(s):  
Katharina N. Steinitz ◽  
Pauline M. van Helden ◽  
Brigitte Binder ◽  
David C. Wraith ◽  
Sabine Unterthurner ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Structural Features ◽  
Cd4 T Cell ◽  
T Cell Epitopes ◽  
Mhc Class Ii Molecules

Abstract Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-cell epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

scholarly journals Early CD4+ T-Cell Effector Alloreactivity Towards Multiple Mismatched HLA Class II Alleles Is Associated with Graft Predominance after Double Umbilical Cord Blood Transplantation (dUCBT)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 387-387
Author(s):  
Jan J. Cornelissen ◽  
Rebecca Wijers ◽  
Cornelis A.M. van Bergen ◽  
Judith Somers ◽  
Eric Braakman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Graft Failure ◽  
Class Ii ◽  
Median Number ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Kinetics Of

Abstract While dUCBT may be associated with less graft failure as compared to single UCBT, hematopoietic recovery following dUCBT generally originates from only a single graft, designated as the 'winning' graft. Graft predominance was suggested to be T-cell mediated, but is still incompletely understood. We recently showed that CD4+ T-cells rapidly expand after dUCBT and early CD4+ T-cell chimerism predicts for graft predominance (Somers et al BBMT 2012; Haematologica 2014). Given the frequent HLA class II allele mismatches between the 2 UCB units in dUCBT, we hypothesized that HLA class II-specific CD4+ T-cells from the 'winning' CBU may be responsible for rejection of the 'loser' CBU. In order to test that hypothesis, we evaluated whether 'wining' CD4+ T-cells specifically recognize individual HLA class II allele mismatches, expressed by the rejected graft. Patient T-cells were propagated in-vitro by 1. HLA unbiased polyclonal expansion (by K562 clone 2D11+CD3mAb and sequential addition of cytokines IL-7, Il-15 and IL-12), and 2. specific activation by HLA class II allele transduced HELA stimulator cells. In-vitro reactivity of propagated T cells was assessed in a co-culture with class II allele transduced HELA cells, and measured by analysis of T cell activation or effector markers by flow cytometry (FCM). 11 patients with poor-risk hematological malignancies were included, receiving 22 UCB units matched at HLA A, B, and DRB1 for 5/6 (n=7) or 4/6 (n=15) with the recipient. The median number (range) of class II allele mismatches between the 2 UCB units per transplant was 2 (1-6). In total, 33 different class II allele mismatches were tested, including 16 at HLA DRB1, 7 at DQB, and 10 at DP. Conditioning with TBI (4 Gy) combined with fludarabine and cyclophosphamide was applied and mycophenolate and ciclosporin were given for GVH-prophylaxis. All patients engrafted, at a median number of 26 days (range: 20-50) after transplantation (without G-CSF), and all developed complete single unit chimerism. Peripheral blood CD3+ T-cell numbers of samples taken at 1-6 months post UCBT were low (median: 0.207; range 0.030-0.699 x 10-9/L) with 74% (range, 8-96%) consisting of CD4+ T-cells. In all 11 patients, alloreactive CD4+ T-cells towards one or more mismatched class II alleles were detectable. In total, CD4+ alloreactivity towards 29 out of 33 (88%) mismatches was detected, including 15/16 for DRB1 (94%), 7/7 for DQ (100%), and 7/10 (70%) for DP alleles. All mismatched alleles but one (DPB1*04:01) elicited a CD4+ T-cell response. Stronger CD4+ T-cell reactivity was observed towards DRB1 and DQ. Analysis of activity towards matched, control alleles showed positivity in 2/11 (18%) combinations. Reactivity towards irrelevant third party alleles showed positivity in 3/17 (18%). The class II alloresponse was significantly higher for mismatched versus matched alleles (median fold increase over control: 7.4 (range 4.4-21.5) versus 1.4 (1.1-2.2), respectively). The highest alloreactive responses were observed in samples (n=14) taken at 1 month post UCBT (Figure). Alloreactive CD4+ T-cells upregulated CD137 and CD134, PD1 and the effector markers CD107 and Interferon-gamma. Collectively, these results demonstrate that specific effector alloreactivity by 'winner' CD4+ T-cells directed to multiple class II mismatched alleles was present in all patients, already at 1 month post UCBT. These results suggest that immediate cytotoxicity exerted by CD4+ T-cells from the 'winning' cord represent a novel mechanism of rapid rejection of the 'losing' unit after dUCBT following a non-ATG conditioning regimen. Furthermore, these observations suggest that the lower incidence of graft failure observed after dUCBT may, in part, result from an immunological, graft-potentiating effect evoked by mismatched class II alleles expressed by the rejected graft. Therefore, allele matching at HLA class II between the 2 units can be ignored in dUCBT. Class II mismatches between de UCB units might, alternatively, even be aimed for, but this should be confirmed in a prospective study. The potential graft versus leukemia effect of these alloreactive CD4+ T-cells is subject of ongoing investigation. Figure 1. Kinetics of anti-HLA class II allele CD4+ T-cell alloreactivity. Figure 1. Kinetics of anti-HLA class II allele CD4+ T-cell alloreactivity. Disclosures No relevant conflicts of interest to declare.

scholarly journals THU0035 OX40L EXPRESSING NEUTROPHILS INDUCE CD4 T FOLLICULAR AND PERIPHERAL HELPER CELL DIFFERENTIATION IN SYSTEMIC LUPUS ERYTHEMATOSUS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 230.2-231
Author(s):  
A. Pappalardo ◽  
E. Wojciechowski ◽  
I. Odriozola ◽  
I. Douchet ◽  
N. Merillon ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cells ◽  
B Cells ◽  
Lupus Erythematosus ◽  
Cd4 T Cells ◽  
Close Contact ◽  
Memory B Cells ◽  
Systemic Lupus ◽  
Research Support

Background:Neutrophils have been described as potent antigen-presenting cells able to activate T cells by MHC/TCR interaction and costimulatory molecules in tumor immunity. However, little is known about the direct interaction between neutrophils and CD4 T cells with respect to systemic lupus erythematosus (SLE). We have previously shown that OX40L expressed by monocytes from SLE patients promote the differentiation of naïve and memory cells into IL21 secreting T cells that are able to help B cells1,2.Objectives:In this study, we investigate OX40L expression on neutrophils from SLE patients and contribution of these OX40L+neutrophils in SLE pathogenesis to modulation of the B cell helper role of CD4 T cells.Methods:Surface expression of co-stimulatory molecules (OX40L, ICOSL, GITRL, 4-1BBL) on neutrophils from SLE patients and healthy donors (HD) was measured by flow cytometry (FC). Neutrophils from HD were stimulated with TLR7 or TLR8 agonists and IFNα after 5 hours of culture, OX40L expression was measured by FC and Western Blotting. CD4 T cells were cultured with the stimulated neutrophils for 3 days. At the end of the co-culture, percentages of IL21-expressing T follicular (Tfh) and peripheral helper (Tph) cells measured by FC. These generated T cells were also cultured in the presence of memory B cells. After 5 days of co-culture, plasmablast generation and Ig levels were assessed by FC and ELISA, respectively. Inhibition of OX40-OX40L interaction in vitro was achieved using ISB 830, a novel anti-OX40 mAb currently used in clinical trials.Results:Among the co-stimulatory molecules tested, percentages of OX40L+neutrophils in SLE (n=54) were increased compared to HD (n=25)(mean + SD: HD = 1,34%±1.62 vs SLE = 4,53%±8.1; p=0.29). OX40L expression positively correlated with SLE disease activity score (SLEDAI) (p = 0,04; r = 0,31) and with anti-DNA antibodies (p= 0,04, r = 0,33). Of note, the percentage of OX40L+neutrophils was higher in anti-sm-RNP+patients (n=16, mean= 9%±9.8), compared to anti-sm-RNP-patients (n=27, mean = 1,4%±2.5; p = 0,02). The percentage of OX40L+neutrophils was higher in patients with class III or IV lupus nephritis, and inflammatory infiltrate within the kidney biopsy disclosed OX40L+neutrophils, in close contact with T cells. Neutrophils from HD express OX40L with TLR8 agonist, or IFNα priming followed by TLR7 agonist. When memory CD4 T cells were cultured in the presence of TLR8-stimulated neutrophils, the proportion of IL21-expressing Tfh (CXCR5+PD1+) and Tph (CXCR5-PD1hi) were increased, compared to culture with unstimulated neutrophils. This process was dependent on OX40-OX40L interactions, since in vitro treatment with the anti-OX40 blocking antibody ISB 830, inhibited the differentiation of memory T cells into Tfh and Tph. Both generated Tfh and Tph were able to promote the differentiation of memory B cells into Ig-secreting plasmablasts.Conclusion:Our results disclose an unprecedented phenomenon where cross-talk between TLR7/8-activated neutrophils and CD4 lymphocytes operates through OX40L-OX40 costimulation, and neutrophils promote the differentiation of pro-inflammatory Tfh and Tph, as well as IL21 production. Therefore, OX40L/OX40 should be considered as a potentially therapeutic axis in SLE patients.References:[1]Jacquemin et al. Immunity 2015;[2]Jacquemin et al. JCI Insight 2018Disclosure of Interests:Angela Pappalardo Grant/research support from: Ichnos Sciences, Elodie Wojciechowski: None declared, Itsaso Odriozola: None declared, Isabelle Douchet: None declared, Nathalie Merillon: None declared, Andrea Boizard-Moracchini: None declared, Pierre Duffau: None declared, Estibaliz Lazaro: None declared, Marie-Agnes Doucey Employee of: Ichnos Sciences, Lamine Mbow Employee of: Ichnos Sciences, Christophe Richez Consultant of: Abbvie, Amgen, Mylan, Pfizer, Sandoz and UCB., Patrick Blanco Grant/research support from: Ichnos Sciences

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

scholarly journals Cd40-Independent Pathways of T Cell Help for Priming of Cd8+ Cytotoxic T Lymphocytes

2000 ◽  
Vol 191 (3) ◽  
pp. 541-550 ◽  
Author(s):  
Zhengbin Lu ◽  
Lingxian Yuan ◽  
Xianzheng Zhou ◽  
Eduardo Sotomayor ◽  
Hyam I. Levitsky ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cytotoxic T Lymphocyte ◽  
Cell Communication ◽  
Cd8 T Cell ◽  
Cd4 Help ◽  
T Cell Help

In many cases, induction of CD8+ CTL responses requires CD4+ T cell help. Recently, it has been shown that a dominant pathway of CD4+ help is via antigen-presenting cell (APC) activation through engagement of CD40 by CD40 ligand on CD4+ T cells. To further study this three cell interaction, we established an in vitro system using dendritic cells (DCs) as APCs and influenza hemagglutinin (HA) class I and II peptide–specific T cell antigen receptor transgenic T cells as cytotoxic T lymphocyte precursors and CD4+ T helper cells, respectively. We found that CD4+ T cells can provide potent help for DCs to activate CD8+ T cells when antigen is provided in the form of either cell lysate, recombinant protein, or synthetic peptides. Surprisingly, this help is completely independent of CD40. Moreover, CD40-independent CD4+ help can be documented in vivo. Finally, we show that CD40-independent T cell help is delivered through both sensitization of DCs and direct CD4+–CD8+ T cell communication via lymphokines. Therefore, we conclude that CD4+ help comprises at least three components: CD40-dependent DC sensitization, CD40-independent DC sensitization, and direct lymphokine-dependent CD4+–CD8+ T cell communication.

scholarly journals CD4+ T Cells from Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgenic Mice Are Not Diabetogenic and Can Delay Diabetes Transfer

2002 ◽  
Vol 196 (4) ◽  
pp. 481-492 ◽  
Author(s):  
Kristin V. Tarbell ◽  
Mark Lee ◽  
Erik Ranheim ◽  
Cheng Chi Chao ◽  
Maija Sanna ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Receptor ◽  
Glutamic Acid Decarboxylase ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
Acid Decarboxylase ◽  
Gad 65

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286–300 (p286) of GAD65. These mice have GAD65-specific CD4+ T cells, as shown by staining with an I-Ag7(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, and IL-10 when stimulated in vitro with GAD65 peptide 286–300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4+ T cells, or p286-tetramer+CD4+ Tcells, from GAD65 286–300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286–300-specific T cells have disease protective capacity and are not pathogenic.

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