Flow Cytometry Analysis of Peripheral Blood CD4+/CD25+/FOXP3+ T Regulator Cells from 11 Patients Treated with Ipilimumab Following Relapse of Malignancy after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3240-3240
Author(s):  
Jiehua Zhou ◽  
Ruikun Zhong ◽  
Sue Corringham ◽  
Teresa Sapp ◽  
Robert Soiffer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Single Dose ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Activated T Cells ◽  
Hematopoietic Stem

Abstract We conducted a multi-center dose-escalation trial to assess whether CTLA-4 blockade is safe and efficacious as immunotherapy in patients with relapse of malignancy following allogeneic hematopoietic stem cell transplantation (allo-HCT). We hypothesized that since CTLA-4 is a negative regulator of effector T-cell activation, inhibition of CTLA-4 ligation with a blocking human monoclonal antibody (ipilimumab) will augment graft-versus-malignancy. Here we report on the effects of a single dose of ipilimumab on in vivo T cell subsets from 11 patients treated at the highest dose level (3 mg/kg) and compare these findings with those in normal volunteer donor lymphocytes. We analyzed the expression of intracellular CTLA-4 and FOXP3 on CD4+/CD25+ Treg cells, intracellular cytokines and surface markers for T-cell activation on peripheral T cells from 11 patients and 9 normal donors. Peripheral blood mononuclear cells (PBMC) from patients before (day 0) and after the treatment at day 1, 3, 7, 14 and 30 were stained with a panel of antibodies and analyzed by flow cytometry. Lymphocytes from normal donors at time zero and 3 days after culture in IL-2 (200u/ml) were used as controls. The pre-treatment expression of intracellular CTLA-4 was significantly higher in CD4+ T cells from patients than normal controls and was increased further after antibody treatment from 7.1±3.8% at day 0 to 18.2±7.1% at day 30 (p=0.02). Although the expression of FOXP3 in CD4+/CD25+ T cells was higher in patients than in normal donors (6.3±4.8% compared with 2.0±1.6%, p=0.02), there was no significant change in the levels following ipilimumab infusion. The expression of CD4+/CD25high in the patients was 7.7±2.8%, higher than the normal donors (2.3±1.1%). However, 6/11 cases had increased expression while others had decrease or no change, overall there was no statistically significant change. CD4+/CD25low activated T cells were elevated in 10/11 patients before ipilimumab (42.1±8.5%). Their levels were not affected by CTLA-4 blockade. CD8+/CD69+ activated T-cells were significantly increased in 8/11 patients within the 30 days after ipilimumab treatment but typically returned to baseline values on longer follow-up. CD4+/CD69+ and CD4+/HLA-DR+ T-cells were unchanged following ipilimumab treatment. These data show that a single dose of ipilimumab enhances levels of some subsets of activated T cells without a significant effect on cells with a T-regulatory phenotype.

scholarly journals Type I Interferon Signaling before Hematopoietic Stem Cell Transplantation Lowers Donor T Cell Activation Via Reduced Allogenicity of Recipient Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4431-4431
Author(s):  
Erik Thiele Orberg ◽  
Julius Clemens Fischer ◽  
Sascha Göttert ◽  
Florian Bassermann ◽  
Hendrik Poeck
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Type I ◽  
Hematopoietic Stem ◽  
Conditioning Therapy

Background: Recent studies highlight immunoregulatory functions of type I interferons (IFN-I) during the pathogenesis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We demonstrated that selective activation of IFN-I pathways including RIG-I/MAVS and cGAS/STING prior to allo-HSCT conditioning therapy can ameliorate the course of GVHD. However, direct effects of IFN-Is on immune cells remain ill characterised. Methods: We applied selective RIG-I agonists (3pRNA) to stimulate IFN-I production in murine models of conditioning therapy with total body irradiation (TBI) and GVHD. Results: Using IFNAR1-deficient donor T and hematopoietic donor cells, we found that endogenous and RIG-I-induced IFN-Is do not reduce GVHD by acting on these respective cell types. However, 3pRNA applied before conditioning therapy reduced the ability of CD11c+ recipient cells to stimulate proliferation and interferon gamma expression of allogeneic T cells. Consistently, RIG-I activation before TBI reduced the proliferation of transplanted T-cells after allo-HSCT. The reduced allogenicity of CD11c+ recipient cells was dependent on IFN-I signalling. Notably, this immunosuppressive function of DCs was restricted to a scenario of genotoxic tissue damage as neither RIG-I activation and IFN-I induction in naive (non-irradiated) mice altered allogeneic T cell activation. Conclusion: Our findings uncover a hitherto unknown IFN-I- and context dependent immunosuppressive function of dendritic cells. This needs to be considered in the development of IFN-I based therapeutic approaches to modulate donor T cell activation after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Donor CTLA-4 Gene Polymorphism Associations with Acute GvHD After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2010-2010
Author(s):  
Irena Frydecka ◽  
Lidia Karabon ◽  
Edyta Pawlak-Adamska ◽  
Anna Tomkiewicz ◽  
Tomasz Wrobel ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Hematopoietic Stem

Abstract Abstract 2010 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been widely carried out as a therapy for several hematological malignances and non malignant disorders. Graft–versus -host disease is one of the major complications after allo-HSCT with main cause of morbidity and mortality. Donor T lymphocytes play the crucial role in alloimmune recognition and their ability to detect non –self antigens can lead to aGvHD. The effective recognition and activation of naïve T-cells requires two independent signals. The first, an antigen-specific signal, is sent via the T-cell receptor (TCR) on T-cells. The second signal, termed co-stimulation, is critical for allowing full activation, sustaining cell proliferation, preventing anergy and/or apoptosis, inducing differentiation to effector cells. CD28 is the primary T-cell co-stimulatory molecule. Cytotoxic T-cell antigen (CTLA-4) is a homologous molecule of CD28 which plays an inhibitory role in the early and late stages of T-cell activation. CTLA-4 ligation provides a negative signal for regulation of the cell cycle and inhibits the activity of the transcriptional factors: nuclear factor-kB (NF-kB), nuclear factor of activated T-cells (NF-AT), and activator protein 1 (AP-1). Moreover, CTLA-4 binds to CD28 ligands (CD80 and CD86) with higher affinity and avidity and in that way also inhibits T-cell activation. Since co-stimulatory and down regulatory molecules synthesis depend on the rate of gene transcription and/or translation, polymorphisms in the corresponding genes might result in abnormal expression, function as well as dysregulated trafficking of these molecules within cellular compartments. The human CTLA-4 gene is located on 2q33 which is susceptibility region for autoimmune diseases. The aim of this study is to investigate the associations between polymorphisms in CTLA-4 gene: CTLA-4c.49A>G (rs231775), CTLA-4g.319C>T (rs5742909), CTLA-4g6230G>A (CT60, rs3087243), CTLA-4g.10223G>T (Jo31, rs11571302) in donors of HSTC and occurrence of aGvH disease in recipients after allogeneic hematopoietic stem cell transplantation. Altogether 136 donors of HSCT (58- related donors, 88 haploidentical unrelated donors) were genotyped for all polymorphisms using allelic discrimination methods with the TaqManÒ SNP Genotyping Assay. In patients without aGvHD and in patients with aGvHD grade I-IV the similar distribution of alleles and genotypes for all investigated polymorphisms in donors was observed. However, we have noticed trend toward increased frequency of CT60 [G] donor allele among recipients with aGvHD I-IV (0.48 vs. 0.39, p=0.1, OR 1.49, 95% CI: 0.90–2.49) compared to recipients without aGvHD in whole group of patients. In patients transplanted from related donor also increased risk of aGvHD grade I-IV was observed for CT60 [G] donor allele (0.75 vs. 0.55, p=0.09, OR 2.11, 95%CI: 0.88–5.26). In contrary the frequencies of CT60 [G] donor allele in patients transplanted from unrelated donors are similar in recipients with and without aGvH symptoms. Haplotype estimation analysis indicated that donor haplotype CTLA-4c.49A>G[A], CTLA-4g.319C>T[C], CT60 [A], Jo31 [T] tended to be protective against aGvHD grade I-IV in whole studied group of patients (0.28 vs. 0.40, p=0.06, OR 0.60, 95% CI: 0.36–1.02) This association reach statistical significance in recipients of related transplantation (0.18 vs. 0.43, p=0.01, OR 0.29, 95% CI: 0.14–0.97) Our study indicated that donor CT60 polymorphism might be associated with occurrence of aGvHD, especially in recipients transplanted from HLA-identical sibling donors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rapid Flow Cytometry Method for Quantitation of LFA-1-Adhesive T Cells

2006 ◽  
Vol 13 (3) ◽  
pp. 403-408 ◽  
Author(s):  
Brian Crucian ◽  
Mayra Nelman-Gonzalez ◽  
Clarence Sams
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Subsets ◽  
Activated T Cells ◽  
Peripheral Blood Mononuclear ◽  
Simple Method ◽  
Rapid Flow

ABSTRACT Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an “adhesive” state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-μm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3+ CD4+ or CD8+ CD19− CD16− CD45RO+ CD62L+ CD27+ CD57−. A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.

Demonstration of a Direct Immunomodulatory Role for Vitamin D in Human T Cells Using Flow Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2577-2577
Author(s):  
Richard W. Joseph ◽  
Tae Kon Kim ◽  
Lisa St. John ◽  
Jahan Khalili ◽  
Uday R Popat ◽  
...  
Keyword(s):  
Vitamin D ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
Activation Markers

Abstract Clinical and epidemiological studies have demonstrated an increasingly stronger link between Vitamin D deficiency and a broad array of illnesses characterized by inflammation, including autoimmune diseases, coronary artery disease, and cancers. Vitamin D is a steroid hormone that exerts the majority of its biologic effects via the binding of the intracellular Vitamin D receptor (VDR). While upregulation of VDR has been demonstrated in activated bulk T cells using traditional approaches (e.g., western blotting), such assays cannot precisely define VDR distribution and kinetics. To overcome these limitations, we developed what we believe to be the first flow cytometric assay to quantify VDR expression at a single-cell level. We used a primary antibody against VDR (mouse monoclonal IgG2a to human VDR) in permeabilized T cells followed by a labeled secondary antibody. We detected a positive cell population using flow cytometry that was sharply increased following activation, consistent with upregulation of VDR confirmed by immunoblotting of sorted cells. We then applied this validated assay to define the kinetics of VDR upregulation in activated T cells. We stimulated PBMC with PMA:Ionomycin (P:I) for varying intervals and assessed intracellular VDR using flow cytometry. VDR is significantly upregulated by 15 min after stimulation, reaches a plateau after 6 hr, and may remain elevated for up to 7 d. We compared VDR to classical early and late T cell activation markers (CD69 and CD25, respectively), and we found that VDR was upregulated as consistently as (but even earlier than) CD69, and that VDR and CD25 were both consistently upregulated at later intervals (p<0.0001). To examine the association between VDR expression and proliferation, we stimulated CFSE-labeled T cells with OKT3 (2mg/ml) for 5 d and found that proliferating T cells expressed a significantly higher level of VDR than resting T cells, which maintained baseline VDR expression (p<0.0001). To assess the association between T cell cytokine production and VDR expression, we stimulated T cells with (P:I) for 6 hr in the presence of brefeldin A, and we confirmed that all cytokine-producing cells (TNFα, IL-2, IFNγ) were contained within the VDR-high population. We then assessed whether physiologic concentrations of Vitamin D could inhibit T cell proliferation in vitro. We stimulated CFSE-labeled PBMC with either OKT3 or irradiated allogeneic dendritic cells (DC) in the presence or absence of physiologic concentrations of calcitriol (50 nm) for 5 to 7 d. The presence of calcitriol during OKT3 stimulation resulted in significantly reduced cell division (p=0.004, n=5). Using a previously validated phenotype to demarcate activated alloreactive CD4+ T cells (CD4hiCD38+), we demonstrated that physiologic calcitriol supplementation decreased alloreactive activation following 7 d stimulation with allogeneic DC (p=0.0003, n=10). In conclusion, VDR is a consistent and specific early and late marker of T cell activation, suggesting a direct role for the Vitamin D axis in immunoregulation. Furthermore, physiological concentrations of Vitamin D can inhibit T cell proliferation induced by polyclonal stimuli, including allogeneic DC. These data provide confirmation for a direct immunoregulatory role for Vitamin D and suggest that further mechanistic and clinical studies may yield novel therapeutic strategies for inflammatory conditions, including graft-versus-host disease.

Phase I Study of a Neutralizing Monoclonal Anti-CTLA4 Antibody (MDX-010) in Patients with Relapse of Malignancy after Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2017-2017 ◽  
Author(s):  
Asad Bashey ◽  
Bridget Medina ◽  
Sue Corringham ◽  
Mildred Pasek ◽  
Ewa Carrier ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Phase I ◽  
T Cell Activation ◽  
Cell Activation ◽  
Hematopoietic Stem ◽  
Anti Cancer ◽  
Grade 3

Abstract Relapse of malignancy is an important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HCT). Malignant cells may evade adoptive immunotherapy post allo-HCT by mechanisms including lack of co-stimulation and direct or indirect inhibition of T-cell activation. CTLA-4 is a homologue of CD28 which functions as a negative regulator of T-cell activation. Blockade of CTLA-4 using neutralizing antibodies has demonstrated potent anti-cancer effects in animal models. Phase I/II clinical trials of CTLA-4 blockade in advanced tumors have demonstrated durable tumor regressions as well as immune breakthrough phenomena. Although CTLA-4 blockade may augment graft-versus-malignancy following allo-HCT, GVHD and other immune complications may also be increased. We report the results of a phase I dose-escalation trial of a neutralizing human monoclonal anti-CTLA-4 antibody (MDX-010) in patients with relapse of malignancy following allo-HCT. Eligibility criteria included allo-HCT ≥90 days previously, > 50% donor T-cell chimerism, no prior grade 3/4 GVHD, no prophylaxis/therapy for GVHD for ≥ 6 weeks. Patients received a single dose of MDX-010 over 90 min. DLI at a dose of 5 x 10e6 CD3 cells/kg was allowed 8 weeks following MDX-010 if no GVHD occurred and progression of malignancy (PD) was present. Twelve patients (8M, 4F; median age 45; CML=2, CLL=1, AML=1, Hodgkins disease [HD] =3 Myeloma [MM]=3, Renal Ca =1, Breast Ca=1) have been treated (4 at dose-level 0.1 mg/kg, 3 at 0.33 mg/kg, 4 at 0.66 mg/kg and 1 at 1.0 mg/kg). Median time between BMT and MDX-010 infusion was 10.3 months (4–79). Four patients received additional DLI. MDX-010 was well tolerated in this setting. No infusional toxicity was seen. No patient has developed clinically significant GVHD following MDX-010 alone. One patient developed grade II acute GVHD of the skin 12 weeks following DLI. Two possible immune breakthrough events were documented: grade 3 polyarthropathy 14 weeks following MDX-010, but also 6 weeks post DLI, which resolved with corticosteroid therapy, (AML, dose 0.1mg/kg, RhF+ pre-MDX-010); grade 1 chemical hyperthyroidism with thyroid-stimulating antibody 6 weeks post MDX-010 (CLL, 0.66 mg/kg). Three patients have demonstrated possible anti-cancer responses following MDX-010 alone (partial remission of AML refractory to prior therapies at 0.1 mg/kg dose, molecular remission of CML maintained off imatinib at 0.1 mg/kg dose, stabilization of previously progressive MM (0.33mg/kg) and one patient developed regression of malignancy (HD) following additional DLI (0.66mg/kg). With a median follow-up of 195d (lead f/u 570d) from MDX-010 infusion three patients have died (PD), 8 are alive and 1 is in CR and 1 is in PR. Pharmacokinetic and correlative science data will be presented. This study shows tolerability with possible anti-tumor effects at the dose levels

scholarly journals T Cell Activation By OKT3 Is a Function of the Percent Monocytes in PBMC of Healthy Donors and MDS Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4870-4870
Author(s):  
Alison Tarke ◽  
Valentina Ferrari ◽  
Hannah Fields ◽  
Luca Ferrari ◽  
Franco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Transplant ◽  
Activation Markers ◽  
Hematopoietic Stem ◽  
Hla Dr ◽  
Healthy Donors

Background: Myelodysplastic Syndromes (MDS) are a heterogeneous hematologic malignancy characterized by bone marrow failure and cytopenias. The median survival rate for patients with higher-risk MDS who fail standard-of-care chemotherapy with hypomethylating agents (HMAs) is less than 6 months, and the only curative treatment for these patients is hematopoietic stem cell transplant (HSCT). Over the past 10 years, immunotherapy as a cancer treatment has achieved variable levels of success in different tumor types. There are currently 22 active clinical trials of immunotherapies for MDS (www.clinicaltrials.gov; 7/30/19), including our phase I clinical trial with a personalized adoptive cellular therapy targeting MDS patient neoantigens (NCT 03258359). Because MDS patients are frequently monocytopenic and the existing literature is inconsistent regarding the ability of MDS patients' monocytes to support T cell activation, we compared the activation of MDS T cells with those of healthy donors in the presence of autologous monocytes. Methods: Peripheral blood mononuclear cells (PBMC) from 5 healthy donors and 7 higher-risk MDS patients were cryopreserved after Ficoll separation. These PBMC were thawed and aliquoted into 6 replicate wells of 200,000 cells in 96-well u-bottom plates in R-10 culture medium. Half of the wells were treated with 25 ng/mL OKT3 and 200 U/mL IL-2. After 48 hours at 37˚C with 5% CO2, the wells were collected for analysis by flow cytometry. Beads were used to detect T cell activation induced secretion of IFNg, TNFa, IL-4, IL-10, and IL-17 in the supernatant and fluorescent antibodies were used to phenotype viable cells for CD3, CD4, CD8, and the T cell activation markers, CD69, CD25, CTLA-4, PD-1, and HLA-DR. Results: We measured a higher release of IFNg and TNFa in donor PBMC compared to MDS patients after OKT3/IL-2 activation, p < 0.01 and 0.04, respectively by 2-way ANOVA. The expression of CD69, CD25, HLA-DR, and CTLA4 increased variably on activated T cells from donors or MDS patients, but expression of CD4+CD25+ was more frequent on donor T cells after activation (p = 0.03). Activation also resulted in a higher frequency of PD-1 expression on donor CD4+ and CD8+ T cells than on MDS T cells (p < 0.01 and < 0.01, respectively). Interestingly, on both MDS and normal T cells the percentage of CD8+PD1+ activated cells correlated strongly with the percent of CD14+ monocytes present in the PBMC (R2 = 0.92 and 0.60 respectively; Fig 1a and 1b). We designed further experiments to test whether this was a patient intrinsic phenomenon, or if the absolute number of CD14+ monocytes in the PBMC was associated with different levels of PD1 expression upon T cell activation. First, we separated CD14+ cells from the PBMC of a patient with MDS using magnetic beads. Then CD14+ cells were added back to the CD14-depleted PBMC at a final percent of 0.5, 5, 10, 20, 35, 70, or 100% of the original amount. Unmodified PBMC was included as a control and all cells were stimulated with OKT3 and IL-2 or left in R-10 medium without stimulus. After 24, 48, and 70 hours, samples were collected to analyze by flow cytometry for CD3, CD4, CD8, CD14, and PD1 expression. The results show that an increasing percent of monocytes corresponded to the increased expression of PD1 on CD8+ and CD4+ T cells. Conclusion: Our results show that there are variable reductions in markers of T cell activation and cytokine secretion in MDS patients compared to healthy donors. We also observed that the fold increase in activation induced PD-1 expression was well correlated with the percent of CD14+ monocytes in the PBMC of both MDS patients and healthy donors. Direct experimentation revealed that this correlation is a cause-effect relationship. We are continuing to investigate the role of monocytes in T cell activation in MDS patients. Disclosures Bejar: Celgene: Consultancy; Takeda Pharmaceuticals: Research Funding; AbbVie/Genentech: Consultancy, Honoraria; Astex/Otsuka: Consultancy; Modus Outcomes: Consultancy; Daiichi-Sankyo: Consultancy. Lane:PersImmune, Inc.: Employment.

scholarly journals Novel self-amplificatory loop between T cells and tenocytes as a driver of chronicity in tendon disease

2021 ◽  
pp. annrheumdis-2020-219335
Author(s):  
Emma Garcia-Melchor ◽  
Giacomo Cafaro ◽  
Lucy MacDonald ◽  
Lindsay A N Crowe ◽  
Shatakshi Sood ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
T Cell Activation ◽  
Cell Activation ◽  
Conditioned Media ◽  
Tissue Remodelling ◽  
Activated T Cells ◽  
T Cell Migration

ObjectivesIncreasing evidence suggests that inflammatory mechanisms play a key role in chronic tendon disease. After observing T cell signatures in human tendinopathy, we explored the interaction between T cells and tendon stromal cells or tenocytes to define their functional contribution to tissue remodelling and inflammation amplification and hence disease perpetuation.MethodsT cells were quantified and characterised in healthy and tendinopathic tissues by flow cytometry (FACS), imaging mass cytometry (IMC) and single cell RNA-seq. Tenocyte activation induced by conditioned media from primary damaged tendon or interleukin-1β was evaluated by qPCR. The role of tenocytes in regulating T cell migration was interrogated in a standard transwell membrane system. T cell activation (cell surface markers by FACS and cytokine release by ELISA) and changes in gene expression in tenocytes (qPCR) were assessed in cocultures of T cells and explanted tenocytes.ResultsSignificant quantitative differences were observed in healthy compared with tendinopathic tissues. IMC showed T cells in close proximity to tenocytes, suggesting tenocyte–T cell interactions. On activation, tenocytes upregulated inflammatory cytokines, chemokines and adhesion molecules implicated in T cell recruitment and activation. Conditioned media from activated tenocytes induced T cell migration and coculture of tenocytes with T cells resulted in reciprocal activation of T cells. In turn, these activated T cells upregulated production of inflammatory mediators in tenocytes, while increasing the pathogenic collagen 3/collagen 1 ratio.ConclusionsInteraction between T cells and tenocytes induces the expression of inflammatory cytokines/chemokines in tenocytes, alters collagen composition favouring collagen 3 and self-amplifies T cell activation via an auto-regulatory feedback loop. Selectively targeting this adaptive/stromal interface may provide novel translational strategies in the management of human tendon disorders.

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