scholarly journals Hypoxia Supports Differentiation of Terminally Exhausted CD8 T Cells

2021 â—½  
Vol 12 â—½  
Author(s):  
Nadia Bannoud â—½  
Tomás Dalotto-Moreno â—½  
Lucía Kindgard â—½  
Pablo A. García â—½  
Ada G. Blidner â—½  
...  
Keyword(s):  
T Cells â—½  
Cd8 T Cells â—½  
Clinical Effectiveness â—½  
Fine Tuning â—½  
Reciprocal Regulation â—½  
Hypoxic Conditions â—½  
Functional Features â—½  
Endothelial Growth Factor â—½  
The Impact

Hypoxia, angiogenesis, and immunosuppression have been proposed to be interrelated events that fuel tumor progression and impair the clinical effectiveness of anti-tumor therapies. Here we present new mechanistic data highlighting the role of hypoxia in fine-tuning CD8 T cell exhaustion in vitro, in an attempt to reconcile seemingly opposite evidence regarding the impact of hypoxia on functional features of exhausted CD8 T cells. Focusing on the recently characterized terminally-differentiated and progenitor exhausted CD8 T cells, we found that both hypoxia and its regulated mediator, vascular endothelial growth factor (VEGF)-A, promote the differentiation of PD-1+ TIM-3+ CXCR5+ terminally exhausted-like CD8 T cells at the expense of PD-1+ TIM-3- progenitor-like subsets without affecting tumor necrosis factor (TNF)-α and interferon (IFN)-γ production or granzyme B (GZMB) expression by these subpopulations. Interestingly, hypoxia accentuated the proangiogenic secretory profile in exhausted CD8 T cells. VEGF-A was the main factor differentially secreted by exhausted CD8 T cells under hypoxic conditions. In this sense, we found that VEGF-A contributes to generation of terminally exhausted CD8 T cells during in vitro differentiation. Altogether, our findings highlight the reciprocal regulation between hypoxia, angiogenesis, and immunosuppression, providing a rational basis to optimize synergistic combinations of antiangiogenic and immunotherapeutic strategies, with the overarching goal of improving the efficacy of these treatments.

Efficient Induction and Isolation of CMV-Specific CD8+ T Cells from CMV Seronegative Donors for the Treatment of CMV Reactivation in CMV Seropositive Patients Transplanted with a CMV Seronegative Donor.

Blood â—½  
2007 â—½  
Vol 110 (11) â—½  
pp. 1053-1053 â—½  
Author(s):  
Inge Jedema â—½  
Marian van de Meent â—½  
Pim L.J. van der Heiden â—½  
Erik W.A. Marijt â—½  
Pauline Meij â—½  
...  
Keyword(s):  
T Cells â—½  
Cd8 T Cells â—½  
Memory T Cells â—½  
Immunomagnetic Bead â—½  
Healthy Donors â—½  
Donor T Cells â—½  
The Impact â—½  
Cmv Reactivation

Abstract Cytomegalovirus (CMV) disease is a significant cause of morbidity and mortality after allogeneic stem cell transplantation (allo-SCT) in CMV seropositive (CMV+) patients. In a recent cohort of CMV+ patients, we investigated the impact of donor CMV serostatus on the severity of CMV reactivation after T cell depleted allo-SCT. A high incidence of CMV related mortality was seen in patients transplanted with a CMV- donor (5/20) whereas no CMV-related deaths occurred after transplantation with a CMV+ donor (0/20). In most CMV+ patients transplanted with a CMV+ donor reconstitution with CMV specific (memory) T cells was found. We recently performed a phase I/II clinical study using isolated CMV-specific CD8+ memory donor T cells for the treatment of patients with persistent CMV reactivation despite seropositivity of the donor. In this study we demonstrated the feasibility of isolating and selecting CMV-specific CD8+ memory T cells from CMV+ donors using the interferon-gamma (IFNg) capture assay and CliniMACS isolation after peptide stimulation of the CMV-specific donor T cells. We have illustrated the in-vivo potential of these T cells after adoptive transfer in 5 patients, resulting in clearance of the CMV load. However, no suitable method was available for the induction of primary immune responses against CMV for the treatment of persistent CMV reactivation in patients transplanted with a CMV- donor. In the current study we investigated the possibility to induce and isolate CMV-specific T cells from CMV- healthy donors by in-vitro priming and selection. We used as responder cells CD14- CD45RO- PBMC from HLA-A1, A2, A3, B7, or B8 positive CMV- healthy donors (n=10). By CD45RO depletion we removed the majority of regulatory T cells (Tregs) capable of inhibiting the initiation of the response. Mature monocyte-derived dendritic cells (DCs) were loaded with a cocktail containing 1μg of each relevant CMV pp65, pp50, or IE1 derived 9-mer peptide, depending on the HLA type of the donor. Naïve donor T cells were cocultured in a 10/1 ratio with peptide loaded DCs. From day 4 on 5 ng/mL IL-7 and IL-15 was added to the culture. At day 10, the responses were specifically restimulated with peptide loaded autologous PBMC. Cytokines were refreshed twice weekly. At day 20 CMV-specific CD8+ T cells were detected and isolated by specific tetramer staining and flowcytometric cell sorting, by specific pentamer staining and immunomagnetic bead isolation, or further enriched by another restimulation, followed by isolation of CD137 expressing T cells at day 21. In 10/10 CMV seronegative donors CMV specific T cells could be detected at day 20 of the immune response in frequencies ranging from 0.01–0.4%. These tetramer positive cells could be isolated and expanded both in bulk cultures and clonally. Functional CMV-specific T cells against all 3 major immunogenic CMV proteins pp65, pp50, and IE1 were detected and isolated with different dominant responses detected in different donors. In conclusion, we have developed a method for the in-vitro induction and isolation of functional CMV-specific CD8+ T cells from CMV- donors. This may allow the treatment of serious CMV-related complications in CMV+ patients transplanted with a CMV- donor.

Prolonged Thrombocytopenia Is Associated With Increases Of CD8+ CX3CR1+ Cells In The Bone Marrow After Allogeneic Hematopoietic Stem Cell Transplantation

Blood â—½  
2013 â—½  
Vol 122 (21) â—½  
pp. 5494-5494
Author(s):  
Xiaohui Zhang â—½  
Guoxiang Wang â—½  
Honghu Zhu â—½  
Yanrong Liu â—½  
Daihong Liu â—½  
...  
Keyword(s):  
Bone Marrow â—½  
T Cells â—½  
Stem Cell â—½  
Hematopoietic Stem Cell Transplantation â—½  
Stem Cell Transplantation â—½  
Cell Transplantation â—½  
Cd8 T Cells â—½  
Hematopoietic Stem â—½  
The Impact

Abstract Prolonged thrombocytopenia is a common complication after allogeneic hematopoietic stem cell transplantation(allo-HSCT),which is associated with a high mortality and poor prognosis. The aim of this study was to assess the impact of the CD8+CX3CR1+ T cells on the development and maturation of megakaryocytes in patients with the prolonged thrombocytopenia after allo-HSCT in order to identify the risk factors related to thrombocytopenia after allo-HSCT. The changes in CD8+ T cells and their homing receptors CX3CR1, CXCR4 and VLA-4 in bone marrow of patients( N=89) with and without (N=94 ) prolonged thrombocytopenia following allo-HSCT and the impact of activated CD8+ T cells on apoptosis and ploidy of megakaryocytes in vitro ware determined. The percentage of CD8+CX3CR1+ T cells was significantly higher in prolonged thrombocytopenia patients than control (P<0.001).The increase in CD8+ T cells was not observed in peripheral blood. Patients with prolonged thrombocytopenia exhibited a marked increase in the number of low ploidy(≤8) megakaryocytes compared to those without(P<0.05). Depletion of CD8+ T cells increased apoptosis of megakaryocytes (P<0.05), which was corrected by reconstitution of CD8+ T cells (P<0.05). We demonstrated that prolonged thrombocytopenia is associated with increased expression of CX3CR1 and recruitment of CD8+ T cells into the bone marrow. Activated CD8+ T cells suppress apoptosis of megakaryocytes in vitro. Our findings shed light on the prolonged thrombocytopenia is associated with increases of CD8+ CX3CR1+ cells in the bone marrow after allo-HSCT patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Generation and Characterization of B7-H4/B7S1/B7x-Deficient Mice

2006 â—½  
Vol 26 (17) â—½  
pp. 6403-6411 â—½  
Author(s):  
Woong-Kyung Suh â—½  
Seng Wang â—½  
Gordon S. Duncan â—½  
Yoshiyuki Miyazaki â—½  
Elizabeth Cates â—½  
...  
Keyword(s):  
T Cells â—½  
T Cell â—½  
Leishmania Major â—½  
Inflammatory Responses â—½  
Fine Tuning â—½  
Th2 Cells â—½  
The Impact â—½  
Deficient Mice

ABSTRACT Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.

scholarly journals 5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

10.1155/2014/418292 â—½  
2014 â—½  
Vol 2014 â—½  
pp. 1-12 â—½  
Author(s):  
Thomas Stübig â—½  
Anita Badbaran â—½  
Tim Luetkens â—½  
York Hildebrandt â—½  
Djordje Atanackovic â—½  
...  
Keyword(s):  
T Cells â—½  
T Cell â—½  
Cd8 T Cells â—½  
Cell Phenotype â—½  
Target Cells â—½  
T Helper 1 â—½  
Cell Immunity â—½  
The Impact

Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigatedin vitroandin vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells.In vivodata confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.

scholarly journals Thymosin alpha-1 Protected T Cells from Excessive Activation in Severe COVID-19

2020 â—½  
Author(s):  
Kuai Yu â—½  
Yongjian Wu â—½  
Jingjing He â—½  
Xuefei Liu â—½  
Bo Wei â—½  
...  
Keyword(s):  
T Cells â—½  
Immune Responses â—½  
Cd8 T Cells â—½  
Severe Disease â—½  
Adaptive Immune Responses â—½  
Activated T Cells â—½  
Adaptive Immune â—½  
The Impact â—½  
Excessive Activation

Abstract Two typical features of uncontrolled inflammation, cytokine storm and lymphopenia, are associated with the severity of coronavirus disease 2019 (COVID-19), demonstrating that both innate and adaptive immune responses are involved in the development of this disease. Recent studies have explored the contribution of innate immune cells to the pathogenesis of the infection. However, the impact of adaptive immunity on this disease remains unknown. In order to clarify the role of adaptive immune response in COVID-19, we characterized the phenotypes of lymphocytes in PBMCs from patients at different disease stages using single-cell RNA sequencing (scRNA-seq) technology. Dynamics of the effector cell levels in lymphocytes revealed dysregulated adaptive immune responses in patients with severe disease. A new cluster of excessively activated CD8 T cells (Tea) was further identified, which displayed exhausted phenotypes and diminished function of antigen recognition. Interestingly, expression of PTMA, the proprotein of Tα1, was significantly increased in a group of highly proliferating CD8 T cells with memory stem cell features. We further showed that Tα1 significantly promoted the proliferation of activated T cells in vitro and relieved the lymphopenia in COVID-19 patients. Our data suggest that protection of T cells from excessive activation might be critical for the prevention of severe COVID-19.

scholarly journals Hypermethylation at the CXCR5 gene locus limits trafficking potential of CD8+ T cells into B-cell follicles during HIV-1 infection

Blood Advances â—½  
2022 â—½  
Author(s):  
Funsho J Ogunshola â—½  
Werner Smidt â—½  
Anneta F Naidoo â—½  
Thandeka Prudence Nkosi â—½  
Thandekile Ngubane â—½  
...  
Keyword(s):  
T Cells â—½  
Hiv Infection â—½  
B Cell â—½  
Cd8 T Cells â—½  
Transcriptional Start Site â—½  
Small Subset â—½  
Dna Hypermethylation â—½  
Infected Cells â—½  
The Impact

CD8+ T-cells play an important role in HIV control. However, in human lymph nodes (LNs), only a small subset of CD8+ T-cells expresses CXCR5, the chemokine receptor required for cell migration into B cell follicles, which are major sanctuaries for HIV persistence in individuals on therapy. Here, we investigate the impact of HIV infection on follicular CD8+ T-cells (fCD8s) frequencies, trafficking pattern and CXCR5 regulation. We show that, although HIV infection results in a marginal increase of fCD8s in LN, the majority of HIV-specific CD8+ T-cells are CXCR5 negative (non-fCD8s) (p&lt;0.003). Mechanistic investigations using ATAC-seq showed that non-fCD8s have closed chromatin at the CXCR5 transcriptional start site (TSS). DNA bisulfite sequencing identified DNA hypermethylation at the CXCR5 TSS as the most probable cause of closed chromatin. Transcriptional factor footprints analysis revealed enrichment of transforming growth factors (TGFs) at the TSS of fCD8s. In-vitro stimulation of non-fCD8s with recombinant TGF-β resulted in significant increase in CXCR5 expression (fCD8s). Thus, this study identifies TGF-β signaling as a viable strategy for increasing fCD8s frequencies in follicular areas of the LN where they are needed to eliminate HIV infected cells, with implications for HIV cure strategies.

scholarly journals POS0362 INVESTIGATING THE ANKYLOSING SPONDYLITIS-ASSOCIATED REGULATORY SNPS AT THE RUNX3 LOCUS WITH A FUNCTIONAL GENOMICS APPROACH

2021 â—½  
Vol 80 (Suppl 1) â—½  
pp. 411.2-411
Author(s):  
M. Vecellio â—½  
P. B. Wordsworth â—½  
C. J. Cohen
Keyword(s):  
T Cells â—½  
Ankylosing Spondylitis â—½  
Cd8 T Cells â—½  
Risk Allele â—½  
Therapeutic Drug â—½  
Genetic Associations â—½  
Nurd Complex â—½  
Mobility Shift â—½  
The Impact

Background:Of the >100 genetic associations with ankylosing spondylitis (AS), RUNX3 transcription factor (TF) involved in diverse immunological processes, is robustly (10−15) associated.1 The biggest challenge is to understand the mechanism behind this association. We demonstrated the association between AS and the single nucleotide polymorphism (SNP) rs4648889 located in a 2kb regulatory locus upstream of the RUNX3 promoter can be explained by allele-specific effects on TF recruitment that alter gene expression, specifically in CD8+ T-cells.2 We recently dissected the full plethora of TFs affected by rs4648889 AS-associated allele showing the NuRD complex and IRF5 differentially bound and having a crucial role in CD8+ T-cells function3. Nevertheless, other post-GWAS approaches are needed to elucidate the function of RUNX3 AS-associated SNPs (i.e. the lead SNP rs6600247).Objectives:The purpose of this work is to define the functional effect of rs6622047 in CD8+ T-cells and monocytes. In specific, we expect to define the impact of AS-associated allele to TF binding and to evaluate chromosome looping between rs6600247 and the RUNX3 promoter.Methods:The epigenetic landscape of SNP rs6600247 was defined using Roadmap database. In vitro functional studies were performed to characterize the effects of this SNP on TFs binding. Chromosome conformation capture (3C) provided critical functional evidence for looping among AS-associated SNPs and the RUNX3 promoter.Results:(1) In silico data revealed a c-MYC ChIP-seq peak in GM12878 lymphoblastoid cells overlapping rs6600247; (2) Mobility shift assays (EMSAs) and WB-EMSAs showed reduced DNA/protein binding in the presence of the AS-risk allele in CD14+ monocytes. C-MYC binding-site is disrupted and binding abolished in the presence of the AS-risk allele; (3) 3C experiments indicate low interaction frequency between SNP rs6600247 and RUNX3 promoter.Conclusion:The enhancer upstream the RUNX3 gene has a plausible functional role in AS, probably by regulating gene transcription and DNA looping. These observations are critically important in defining dysregulated pathways and potential therapeutic drug targets.References:[1]IGAS et al. Nat Genet. 2013 Jul;45(7):730-8.[2]Vecellio M. et al, Ann Rheum Dis. 2016 Aug;75(8):1534-40.[3]Vecellio et al Arthritis Rheumatol 2020. doi: 10.1002/art.41628.Acknowledgements:We thank Professor Julian Knight for his constructive advices and comments to the present work.Disclosure of Interests:None declared

scholarly journals Functional Exhaustion of HBV-Specific CD8 T Cells Impedes PD-L1 Blockade Efficacy in Chronic HBV Infection

2021 â—½  
Vol 12 â—½  
Author(s):  
Sara Ferrando-Martinez â—½  
Angie Snell Bennett â—½  
Elisabete Lino â—½  
Adam J. Gehring â—½  
Jordan Feld â—½  
...  
Keyword(s):  
Flow Cytometry â—½  
T Cells â—½  
Cd8 T Cells â—½  
Ex Vivo â—½  
Peptide Pool â—½  
Clinical Effectiveness â—½  
T Cell Response â—½  
Functional Cure â—½  
Chronic Hbv

BackgroundA functional cure for chronic HBV could be achieved by boosting HBV-specific immunity. In vitro studies show that immunotherapy could be an effective strategy. However, these studies include strategies to enrich HBV-specific CD8 T cells, which could alter the expression of the anti-PD-1/anti-PD-L1 antibody targets. Our aim was to determine the efficacy of PD-L1 blockade ex vivo.MethodsHBV-specific CD8 T cells were characterized ex vivo by flow cytometry for the simultaneous analysis of six immune populations and 14 activating and inhibitory receptors. Ex vivo functionality was quantified by ELISpot and by combining peptide pool stimulation, dextramers and intracellular flow cytometry staining.ResultsThe functionality of HBV-specific CD8 T cells is associated with a higher frequency of cells with low exhaustion phenotype (LAG3-TIM3-PD-1+), independently of the clinical parameters. The accumulation of HBV-specific CD8 T cells with a functionally exhausted phenotype (LAG3+TIM3+PD-1+) is associated with lack of ex vivo functionality. PD-L1 blockade enhanced the HBV-specific CD8 T cell response only in patients with lower exhaustion levels, while response to PD-L1 blockade was abrogated in patients with higher frequencies of exhausted HBV-specific CD8 T cells.ConclusionHigher levels of functionally exhausted HBV-specific CD8 T cells are associated with a lack of response that cannot be restored by blocking the PD-1:PD-L1 axis. This suggests that the clinical effectiveness of blocking the PD-1:PD-L1 axis as a monotherapy may be restricted. Combination strategies, potentially including the combination of anti-LAG-3 with other anti-iR antibodies, will likely be required to elicit a functional cure for patients with high levels of functionally exhausted HBV-specific CD8 T cells.

Treg Induced Impairment of DC Function and Its Impact on GvH Reactions

Blood â—½  
2014 â—½  
Vol 124 (21) â—½  
pp. 3815-3815
Author(s):  
Mavin Emily â—½  
Lindsay Nicholson â—½  
Rafez Ahmed â—½  
Matthew Collin â—½  
Anne Dickinson â—½  
...  
Keyword(s):  
T Cells â—½  
T Cell â—½  
Cd8 T Cells â—½  
Tissue Damage â—½  
Conflicts Of Interest â—½  
Cd8 T Cell â—½  
Target Tissue â—½  
T Cell Priming â—½  
The Impact

Abstract Promising results from murine models and early stage clinical trials have shown that adoptive transfer of regulatory T cells (Treg) prevents graft-versus-host disease (GvHD). However, the primary target of Treg mediated protection against GvHD is yet to be fully defined. We have previously shown that the presence of Treg during effector T cell priming is able to ameliorate cutaneous GvH reactions in vitro by blocking effector cell migration. This has led to the hypothesis that Treg modulation of dendritic cells (DC) could be a key mechanism by which Treg exert their protective role in GvHD. DC are fundamental for the initiation of allo-reactive immune responses and are critical in GvHD pathogenesis. We investigated the effect of Treg on the phenotypic profile and allo-reactive functions of DCs. Furthermore, the impact of Treg treatment on the ability of DCs to induce GvH target tissue damage was examined for the first time using an in vitro human GvHD skin explant model. Immature, mature and Treg treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC respectively). The three moDC populations were generated using the well-established 6 day culture with GM-CSF and IL-4 followed by 24 hour LPS maturation. Treg were added on day 3 of moDC culture. Im-DC, mat-DC and Treg-DC were harvested on day 7 and tested in parallel as stated below. Prior to functional assays Treg-moDC were isolated by FACS sorting via FSC/SSC/CD3neg gating to remove Treg present in the co-culture. Our results revealed that Treg-DC displayed a semi-mature phenotype with CD83, CD80 and CD86 expression significantly lower than mat-moDC (p<0.005) but significantly higher than im-moDC (P<0.05) whilst HLA-DR levels were comparable to mat-moDC but significantly higher than imDC (p<0.005). Treg-DC also expressed CCR7 comparable to that of im-DC but markedly higher than mat-DC (p=0.052). Distinct morphology of Treg-DC, defined by Giemsa staining, corroborated their semi-mature status. Data from FITC-dextran uptake showed a significant reduction in antigen-capture capacity of Treg treated im-DC compared to untreated im-DC (p=0.047). The Treg mediated functional impairment of DCs was also associated with significantly higher expression of LAP-TGFβ1 on Treg-DC when compared to that of mat-DC (P<0.05). Treg-DC were also markedly defective in stimulating activation and proliferation of allo-reactive CD8 T cells, detected by CD25 expression and CFSE dilution respectively (p=0.009, p=0.046). Interestingly the presence of Treg throughout the entire allo-response induction resulted in a more potent reduction in activation and proliferation than when only the DC were treated with Treg (p=0.009, p=0.0085). Furthermore, allo-reactive CD8 T cells primed with Treg treated moDC were less able to mediate cutaneous GvH tissue damage (Figure 1). In conclusion, attenuation of DC signature and function is key for Treg mediated protection against GvHD. However, isolated DC modulation by Treg was less effective in suppressing CD8 T cell allo-responses compared to the continued presence of Treg during the CD8 T cell priming, activation and proliferation, suggesting that Treg exert most effective function via multidimensional modulation on the DC, T cells and DC-T cell interactions simultaneously. Figure 1. Skin histopathology induced by CD8 T cells primed with im-DC, mat-DC and Treg-DC Figure 1. Skin histopathology induced by CD8 T cells primed with im-DC, mat-DC and Treg-DC Disclosures No relevant conflicts of interest to declare.

scholarly journals Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression

2020 â—½  
Vol 79 (8) â—½  
pp. 1044-1054 â—½  
Author(s):  
Abdulla Watad â—½  
Hannah Rowe â—½  
Tobias Russell â—½  
Qiao Zhou â—½  
Lisa K Anderson â—½  
...  
Keyword(s):  
T Cells â—½  
T Cell â—½  
Cd8 T Cells â—½  
Cd4 T Cells â—½  
Influenza A â—½  
Orphan Nuclear Receptor â—½  
Healthy Human â—½  
Cell Reactivity â—½  
The Impact

BackgroundThe human enthesis conventional T cells are poorly characterised.ObjectivesTo study the biology of the conventional T cells in human enthesis.MethodsCD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated.ResultsImmunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-β compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion.ConclusionsHealthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.

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