scholarly journals B7-H4 Inhibits the Development of Primary Sjögren’s Syndrome by Regulating Treg Differentiation in NOD/Ltj Mice

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Xu Zheng ◽  
Qikai Wang ◽  
Xiang Yuan ◽  
Yingbo Zhou ◽  
Hui Chu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
Salivary Glands ◽  
Inflammatory Cytokines ◽  
Mrna Levels ◽  
Lymphocyte Infiltration ◽  
Flow Cytometry Analysis ◽  
Treg Differentiation

Background. This study is aimed at exploring the role of B7-H4 in the pathogenesis of primary Sjögren’s syndrome (pSS) in NOD/Ltj mouse. Methods. B7-H4 expression in salivary glands was examined by IHC, and lymphocyte infiltration was showed by H&E. Next, anti-B7-H4 mAb or immunoglobulin isotype was injected into NOD/Ltj mice. Cytokine levels were measured by quantitative RT-PCR, and immunoglobulins were measured by ELISA. T cell subsets were analyzed by flow cytometry. Last, we treated NOD/Ltj mice with B7-H4Ig and control Ig. CD4+Foxp3+ T cells were assessed by immunohistochemistry. Two-tailed Student’s t-tests were used to detect the statistical difference in various measures between the two groups. Results. B7-H4 expression was remarkably reduced in salivary glands of NOD/Ltj mice at 15 weeks compared with the NOD/Ltj mice at 8 weeks. Anti-B7-H4 mAb treatment increased lymphocyte infiltration in salivary glands. Inflammatory cytokines including IL-12, IL-18, IL-1α, TNF-α, IFN-α, and BAFF were upregulated markedly in anti-B7-H4 mAb-treated mice compared to IgG isotype-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb-treated mice had lower levels of CD4+Foxp3+/CD4+ T cells in spleen. Moreover, Foxp3 mRNA levels of salivary glands were diminished in anti-B7-H4 mAb-treated mice. Flow cytometry analysis showed that anti-B7-H4 mAb inhibited CD4+Foxp3+/CD4+ T cell production, while B7-H4Ig would promote naïve CD4+ T into Treg differentiation. Administration with B7-H4Ig displayed significantly decreased lymphocyte infiltration in salivary glands and low levels of total IgM and IgG in serum. Analysis of inflammatory cytokines in salivary glands after B7-H4Ig treatment revealed that the mRNA levels of IL-12, IL-6, IL-18, IL-1α, TNF-α, and IFN-α were significantly downregulated in B7-H4Ig-treated mice compared to control Ig treatment. B7-H4Ig-treated mice had significantly higher levels of CD4+Foxp3+/CD4+ T cells in spleen. IHC in salivary gland revealed that CD4+Foxp3+ T cells of B7-H4Ig treatment mouse were more than control Ig treatment. Conclusions. Our findings implicate that B7-H4 has a protective role for salivary gland epithelial cells (SGECs) and therapeutic potential in the treatment of pSS.

scholarly journals Combination immunotherapy induces distinct T-cell repertoire responses when administered to patients with different malignancies

2020 ◽  
Vol 8 (1) ◽  
pp. e000368
Author(s):  
Jason Cham ◽  
Li Zhang ◽  
Serena Kwek ◽  
Alan Paciorek ◽  
Tao He ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Metastatic Melanoma ◽  
Flow Cytometry Analysis ◽  
Cell Repertoire ◽  
Gm Csf ◽  
Cell Counts ◽  
Melanoma Patients ◽  
Circulating T Cells

BackgroundCTLA-4 blockade with ipilimumab is Food and Drug Administration-approved for melanoma as a monotherapy and has been shown to modulate the circulating T-cell repertoire. We have previously reported clinical trials combining CTLA-4 blockade with granulocyte-macrophage colony-stimulating factor (GM-CSF) in metastatic melanoma patients and in metastatic castration resistant prostate cancer (mCRPC) patients. Here, we investigate the effect that cancer type has on circulating T cells in metastatic melanoma and mCRPC patients, treated with ipilimumab and GM-CSF.MethodsWe used next-generation sequencing of T-cell receptors (TCR) to compare the circulating T cells of melanoma and mCRPC patients receiving the same treatment with ipilimumab and GM-CSF by Wilcoxon rank sum test. Flow cytometry was utilized to investigate specific T-cell populations. TCR sequencing results were correlated with each T-cell subpopulation by Spearman’s rank correlation coefficient. Of note, 14 metastatic melanoma patients had samples available for TCR sequencing and 21 had samples available for flow cytometry analysis; 37 mCRPC patients had samples available for sequencing of whom 22 have TCR data available at both timepoints; 20 of these patients had samples available for flow cytometry analysis and 16 had data available at both timepoints.ResultsWhile melanoma and mCRPC patients had similar pretreatment circulating T-cell counts, treatment induces greater expansion of circulating T cells in melanoma patients. Metastatic melanoma patients have a higher proportion of clones that increased more than fourfold after the treatment compared with mCRPC patients (18.9% vs 11.0%, p=0.017). Additionally, melanoma patients compared with mCRPC patients had a higher ratio of convergent frequency (1.22 vs 0.60, p=0.012). Decreases in clonality induced by treatment are associated with baseline CD8+ T-cell counts in both patient groups, but are more pronounced in the melanoma patients (r=−0.81, p<0.001 vs r=−0.59, p=0.02).Trial registration numbersNCT00064129;NCT01363206.

scholarly journals A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Maria E. Lund ◽  
Christopher B. Howard ◽  
Kristofer J. Thurecht ◽  
Douglas H. Campbell ◽  
Stephen M. Mahler ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Cytolytic Activity ◽  
Activated T Cells ◽  
Bispecific T Cell Engager

Abstract Background Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. Methods The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. Results Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. Conclusions This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

scholarly journals Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus

2020 ◽  
Vol 21 (11) ◽  
pp. 4180
Author(s):  
Jae Wook Jung ◽  
Jin Hong Chun ◽  
Jung Seok Lee ◽  
Si Won Kim ◽  
Ae Rin Lee ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Viral Infection ◽  
T Cell Subsets ◽  
Olive Flounder ◽  
Flow Cytometry Analysis ◽  
Color Flow

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

scholarly journals IL-21 Receptor Blockade Shifts the Follicular T Cell Balance and Reduces De Novo Donor-Specific Antibody Generation

2021 ◽  
Vol 12 ◽  
Author(s):  
Yeqi Nian ◽  
Zhilei Xiong ◽  
Panpan Zhan ◽  
Zhen Wang ◽  
Yang Xu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Germinal Center ◽  
De Novo ◽  
Antibody Mediated Rejection ◽  
Treg Differentiation ◽  
The Impact

Donor-specific antibodies (DSAs) play a key role in chronic kidney allograft injury. Follicular T helper (Tfh) cells trigger the humoral alloimmune response and promote DSA generation, while T-follicular regulatory (Tfr) cells inhibit antibody production by suppressing Tfh and B cells. Interleukin (IL)-21 exerts a distinct effect on Tfh and Tfr. Here, we studied whether blocking IL-21R with anti-IL-21R monoclonal antibody (αIL-21R) changes the Tfh/Tfr balance and inhibits DSA generation. First, we investigated the impact of αIL-21R on CD4+ T cell proliferation and apoptosis. The results showed that αIL-21R did not have cytotoxic effects on CD4+ T cells. Next, we examined Tfh and regulatory T cells (Tregs) in an in vitro conditioned culture model. Naïve CD4+ T cells were isolated from 3-month-old C57BL/6 mice and cultured in Tfh differentiation inducing conditions in presence of αIL-21R or isotype IgG and differentiation was evaluated by CXCR5 expression, a key Tfh marker. αIL-21R significantly inhibited Tfh differentiation. In contrast, under Treg differentiation conditions, FOXP3 expression was inhibited by IL-21. Notably, αIL-21R rescued IL-21-inhibited Treg differentiation. For in vivo investigation, a fully mismatched skin transplantation model was utilized to trigger the humoral alloimmune response. Consistently, flow cytometry revealed a reduced Tfh/Tfr ratio in recipients treated with αIL-21R. Germinal center response was evaluated by flow cytometry and lectin histochemistry. We observed that αIL-21R significantly inhibited germinal center reaction. Most importantly, DSA levels after transplantation were significantly inhibited by αIL-21R at different time points. In summary, our results demonstrate that αIL-21R shifts the Tfh/Tfr balance toward DSA inhibition. Therefore, αIL-21R may be a useful therapeutic agent to prevent chronic antibody mediated rejection after organ transplantation.

Flow cytometry analysis of T cells and continuous T-cell lines from autoimmune MRL/l mice

Nature ◽  
1981 ◽  
Vol 289 (5795) ◽  
pp. 298-300 ◽  
Author(s):  
D. E. Lewis ◽  
J. V. Giorgi ◽  
Noel L. Warner
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Flow Cytometry Analysis ◽  
T Cell Lines

CD4+ T Cell Functions Are Potently Suppressed By The Janus Kinase 1/2 (JAK1/JAK2) Inhibitor Ruxolitinib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2281-2281 ◽  
Author(s):  
Sowmya Parampalli Yajnanarayana ◽  
Isabelle Cornez ◽  
Annkristin Heine ◽  
Peter Brossart ◽  
Dominik Wolf
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
Cd4 T Cells ◽  
Cell Function ◽  
Cd4 T Cell ◽  
Jak2 Inhibitor ◽  
Dose Dependent ◽  
Pro Inflammatory Cytokines

Abstract Introduction Recent discoveries of activating JAK mutations in patients with myeloproliferative diseases (MPNs) coupled with the so far known biology of JAKs in cytokine signaling provided the rationale for targeting these kinases in MPNs. Ruxolitinib (INCB018424) is the first JAK1/JAK2 inhibitor approved for treatment of patients with myelofibrosis (MF). Although ruxolitinib shows limited anti-clonal activity, a profound improvement of quality of life and splenomegaly in MF patients is observed and linked to a substantial reduction of MF-associated circulating pro-inflammatory cytokines and pro-angiogenic factors. JAK/STAT-signalling is known to be involved in the regulation of various immune cells including CD4+ T cells, which critically orchestrate inflammatory responses. To better understand how ruxolitinib is modulating CD4+ T cell response, we here provide an in depth analysis of CD4+ T cell function upon ruxolitinib exposure. Methods Highly purified CD4+ T cells isolated from healthy human PBMC from buffy coats were stimulated for 4 days with i) plate bound anti-CD3, ii) plate bound anti-CD3 and soluble anti-CD28 antibodies, iii) IL-2 in the presence of increasing concentrations of ruxolitinib (0.1µM – 10µM) or the respective vehicle control (DMSO). Phenotype and function were analyzed by flow cytometry. Cytokine production was quantified either by intracellular staining and subsequent flow cytometry or by flow-based bead assays (Human Th1/Th2 11plex FlowCytomix Multiplex). Proliferation was detected by CFSE dilution analysis using FACS. CD4+CD62L+ T cells obtained from C57BL/6 mice were isolated by using the CD4+CD62L+ T Cell Isolation Kit (Miltenyi Biotec) and subsequently differentiated into TH1, TH2, TH9, TH17 and iTreg. Polarization into the different CD4+ T cell subsets was induced by cytokine/antibody cocktails (TH1: IL-12 and anti-IL4; TH2: IL-4 and anti-IL12; TH9: IL-4, TGF-β and anti-IFNγ; iTreg: IL-2 and TGFβ; TH17: IL-6, TGFβ, IL-1b, anti-IFNγ and anti-IL4) together with anti-CD3 and anti-CD28. For analysis of apoptosis/necrosis induction, annexin/propidium iodide staining was applied. Signalling events were analyzed by phospho-flow technology to evaluate ruxolitinib-mediated changes of TCR- and/or cytokine-induced signalling cascades (using pS6, pSTAT1, pSTAT3, pSTAT5, pERK, pAKT, pP38, pFos, pJun and pZAP70 antibodies). Results CD4+ T cell proliferation is significantly and dose-dependently suppressed by ruxolitinib when T cells were activated by each of the three conditions tested. Of note, we could not detect any changes in the viability of ruxolitinib-exposed CD4+ T cells. In line with previous studies, production of pro-inflammatory cytokines such as IL-1β, IL-5, IL-6 and TNF-α were dose-dependently inhibited in ruxolitinib-exposed CD4+ T cells, although expression of the pro-inflammatory IL-8 was increased in a dose-dependent manner. Interestingly, despite the complete proliferation block, we also observed an increase in IL-2 and IFNγ particularly at the lower ruxolitinib concentrations (0.1μM) followed by a dose dependent reduction at higher dose-levels (10µM). After short-term activation of ruxolitinib-exposed CD4+ T cells by anti-CD3 and anti-CD28, proximal TCR signaling events (phosphorylation of SLP76 and ZAP70) were not affected, whereas a clear down-regulation of IL-2 induced STAT5 phosphorylation could be detected. After wash-out the ruxolitinib-induced inhibitory effects on CD4+ T cell function were fully reversible, as shown by induction of the T cell activation markers CD25 and CD69. Finally, we differentiated murine CD4+ naïve T cells into the various T Helper cell subsets and could provide clear evidence that the differentiation capacity of naïve CD4+ T cells into TH1, TH9, TH17 and iTreg was markedly reduced, whereas inhibition of Th2 differentiation was only marginally affected. The anti-inflammatory effects of ruxolitinib are currently tested in a TH9-dependent lung inflammation model in mice. Conclusion We could show that ruxolitinib potently affects CD4+ T cell biology. These data provide a rationale for testing JAK inhibitors in diseases triggered by hyperactive CD4+ T cells, such as autoimmune diseases. However, they also provide an explanation for the increased infection rates (i.e. viral reactivation and urinary tract infection) seen in ruxolitinib-treated patients. Disclosures: Wolf: Novartis: Honoraria, Research Funding.

scholarly journals Purification of Plasmodium Sporozoites Enhances Parasite-Specific CD8+T Cell Responses

2016 ◽  
Vol 84 (8) ◽  
pp. 2233-2242 ◽  
Author(s):  
Zachary P. Billman ◽  
Annette M. Seilie ◽  
Sean C. Murphy
Keyword(s):  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
Salivary Glands ◽  
Gradient Centrifugation ◽  
T Cell Responses ◽  
Inhibitory Effect ◽  
Content Type ◽  
Cell Responses ◽  
Lower Magnitude

Malaria infection caused byPlasmodiumparasites continues to cause enormous morbidity and mortality in areas where it is endemic, and there is no licensed vaccine capable of inducing sterile protection. Hyperimmunization with attenuated whole sporozoites can induce sterile protective immune responses targeting preerythrocytic antigens. Most animal models of hyperimmunization rely on sporozoites dissected from mosquito salivary glands and injected without further purification. In BALB/c mice, repeated small doses ofP. yoeliisporozoites progressively expand the population of sporozoite-specific CD8+T cells. In this study, large secondary doses of unpurified sporozoites unexpectedly led to contraction of sporozoite-specific CD8+T cell responses in sporozoite-primed mice. While sporozoite-primed CD8+T cells alternatively can be expanded by secondary exposure toListeria monocytogenesexpressing recombinantPlasmodiumantigens, such expansion was potently inhibited by coinjection of large doses of unpurified sporozoites and by uninfected salivary glands alone. Purification of sporozoites away from mosquito salivary gland debris by density gradient centrifugation eliminated salivary gland-associated inhibition. Thus, the inhibitory effect appears to be due to exposure to uninfected mosquito salivary glands rather than sporozoites. To further assess the effect of salivary gland exposure on later sporozoite vaccinations, mice were immunized with uninfected salivary glands from a single mosquito. Compared to naive mice, salivary gland presensitization reduced subsequent liver burdens by 71%. These data show that a component(s) in mosquito salivary glands reduces liver infection, thereby limiting antigen dose and contributing to lower-magnitude T cell responses. These findings suggest that sporozoite immunogenicity studies be performed using purified sporozoites whenever feasible.

scholarly journals 373 Enhancement of TCR-engineered T-cells targeting MAGE-A4 antigen by co-expression of CD8α and inhibition of AKT signaling during ex vivo T-cell expansion

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A401-A401
Author(s):  
Emily Schmidt ◽  
Katerina Mardilovich ◽  
Natalie Bath ◽  
Gareth Betts ◽  
William Spinner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Memory Phenotype ◽  
Post Infusion

BackgroundAutologous Specific Peptide Enhanced Affinity Receptor (SPEAR) T-cells targeting MAGE-A4 can be effective treatment for solid tumors.1–3 To improve efficacy, we developed a next-generation SPEAR-T cell targeting MAGE-A4 co-expressing CD8α (ADP-A2M4CD8). ADP-A2M4CD8 is under investigation in the Phase 1 SURPASS trial (NCT04044859). Enhancements have also been made to the manufacturing process with an AKT inhibitor (AKTi) during ex vivo expansion to provide a greater proliferative potential and enhanced memory phenotype.4MethodsSPEAR-T cells were manufactured using a Lentiviral vector with CD8α and MAGE-A4 targeted TCR genes. AKTi was added during ex vivo expansion. T-cell attributes were evaluated, including markers of differentiation (flow cytometry), capacity for in vitro tumor lysis (Incucyte) and changes to gene expression (scRNASeq) initially assessed with the first-gen product. Post-infusion, the presence of transduced T-cells in the peripheral circulation (PCR) and levels of inflammatory cytokines in serum (MesoScale Discovery Assay [MSD]) were evaluated.ResultsAs of May 24, 2021, 18 patients with 9 different primary tumor types were evaluable. Twelve pts received product that had AKTi during manufacture. Five patients had objective responses (RECIST), and 10 had stable disease. Responses occurred at lower MAGE-A4 expression levels and lower transduced T-cell doses relative to the first-gen product targeting MAGE-A4.1 CD4 T-cells from manufactured ADP-A2M4CD8 demonstrated direct in vitro tumor cell killing similar to CD8+ T-cells (Incucyte). scRNASeq gene expression profiles of first-gen ADP-A2M4 product manufactured with AKTi revealed the AKTi-expanded T-cells had a greater proliferation or an enhanced memory phenotype; scRNASeq analyses are ongoing for the ADP-A2M4CD8 product.An increase in IL-12 levels (MSD) in serum post-infusion suggests that endogenous immune cells are being activated, further resulting in increased levels of IFN gamma (MSD) secretion relative to patients who received first-gen product. Manufacturing with AKTi resulted in T-cells with a less differentiated phenotype (flow cytometry), and post-infusion was associated with enhanced antigen-specific serum cytokine responses, increased proliferative state (i.e., elevated levels of IL-2), and higher persistence of T-cells in peripheral blood by PCR.ConclusionsSPEAR T-cells targeting MAGE-A4 expressing cancers have been enhanced by co-expressing CD8α and adding AKTi during manufacture. These enhanced products improve CD4+ T-cell killing, release more inflammatory cytokines, proliferate more robustly with an early memory phenotype, and better engage the patient‘s endogenous immune system when compared to first-gen products or next-gen manufactured without AKTi.Trial RegistrationNCT04044859ReferencesHong, et al. ASCO 2020.D’Angelo, et al. ASCO 2021.Hong, et al. SITC 2020.Mardilovich, et al. SITC 2020.

scholarly journals AB0133 INCREASED SOLUBLE SEMAPHORIN 4D/CD100 IN THE PLASMA OF SJÖGREN’S SYNDROME AND ITS EFFECTS ON HUMAN SALIVARY GLAND CELL AND CD4+ T CELL

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1367.1-1367
Author(s):  
S. E. Kang ◽  
S. U. Kim ◽  
R. H. Kim ◽  
H. J. Yoo ◽  
Y. J. Lee ◽  
...  
Keyword(s):  
T Cells ◽  
Salivary Gland ◽  
T Cell ◽  
Tight Junction ◽  
Sjogren's Syndrome ◽  
Healthy Controls ◽  
Human Salivary Gland ◽  
Semaphorin 4D ◽  
Aqp5 Expression ◽  
Ifn Γ

Background:Semaphorin 4D (SEMA4D) / CD100, known as a subfamily of axonal guidance proteins, has also been reported to act as an immunoregulator in several infectious and inflammatory diseases [1]. Sjögren’s syndrome (SS) is a systemic autoimmune disease that primarily affects the exocrine glands by infiltrated lymphocytes resulting in dryness of mouth and eyes. IL-17 was reported to impair the integrity of tight junction barrier and attenuate the expression of aquaporin 5 (AQP5), causing salivary gland dysfunction in SS [2].Objectives:This study was aimed to evaluate the role of SEMA4D in patients with SS and investigate the effect of SEMA4D on human salivary gland epithelial cell (SGEC) and T cell.Methods:Soluble SEMA4D levels in plasma were measured by enzyme-linked immunosorbent assay (ELISA) from patients with SS, non-SS sicca and healthy controls. Immortalized human SGECs, originated from acini (NS-SV-AC) and duct (NS-SV-DC), were used to evaluate the effects of SEMA4D. CD4+T cells from human peripheral blood were isolated to determine the secretion of cytokines in response to SEMA4D. IFN-γ and IL-17 were used to determine the effects on AQP5 expression of SGEC.Results:The levels of soluble SEMA4D in plasma were increased in patients with SS (median [interquartile range]: 1221.3 [393.5] pg/mL) compared to non-SS sicca (940.2 [355.1] pg/mL,p= 0.006) or healthy controls (909.5 [108.0] pg/mL,p <0.0001). The levels of soluble SEMA4D in plasma were correlated with the levels of several autoantibodies including anti-SSA (Spearman’s rho = 0.358,p= 0.006), anti-SSB (rho = 0.350,p= 0.007), and anti-muscarinic receptor 3 (M3R) Ab (rho = 0.495,p< 0.001), and also correlated with total IgG (rho = 0.431,p= 0.002). SEMA4D-stimulated SGECs showed decreased expression of tight junctions such as occludin and Zo-1. CD4+T cells secreted IFN-γ (p= 0.025), IL-17 (p= 0.028), and IL-21 (p= 0.007) with SEMA4D stimulation. IFN-γ and IL-17 decreased AQP5 expression in SGECs.Conclusion:SEMA4D contributed to decreased expression of tight junction in SGECs. SEMA4D induced production of IFN-γ and IL-17 in CD4+T cells and these cytokine decreased AQP5 expression in SGECs.References:[1]Worzfeld T, Offermanns S. Nat Rev Drug Discov. 2014;13(8):603-21.[2]Bhattarai KR, Junjappa R, Handigund M, Kim HR, Chae HJ. Autoimmun Rev. 2018;17(4):376-90.Disclosure of Interests:None declared

scholarly journals 588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A623-A623
Author(s):  
Yannick Rakké ◽  
Lucia Campos Carrascosa ◽  
Adriaan van Beek ◽  
Valeska de Ruiter ◽  
Michael Doukas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Colorectal Carcinoma ◽  
Liver Metastases ◽  
Mismatch Repair ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Human Tumour ◽  
Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

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