TEMRA Cells Research Articles

CD8+ TEMRA Clones Cause Platelet Lysis in Immune Thrombocytopenia

Background Immune thrombocytopenia (ITP) is traditionally thought of as an antibody-mediated disease. However, there are a number of features to suggest alternative mechanisms of platelet destruction. In this study we use a multi-dimensional approach to explore the role of terminally differentiated effector memory CD8+ T cells (TEMRA). Methods We characterised 83 adults with chronic ITP and 30 age-matched controls using immunophenotyping, next generation sequencing of T cell receptor (TCR), single cell (sc) RNA sequencing and functional assays. Next generation DNA sequencing of TCRβ was carried out using the Illumina MiSeq platform. Analysis of the raw TCR sequences was performed using MiXCR. Single cell immune profiling (Gene expression and TCR) was done using Chromium Single Cell V(D)J and 5' Library kits from 10x Genomics. The constructed library was sequenced on the HiSeq platform (Illumina). Seurat v.2.3.4 was used for gene expression analysis. Interactions between CD8+ T cells and platelets were measured by co-culturing CD8+ T cells with autologous platelets overnight and by perfusing them on the autologous platelet covered VenaFluoro8+ microchip channel. Results We found an expansion of CD8+ CD45RA+ CD62L- TEMRA cells expressing IFN gamma, TNF alpha and granzyme B, with no evidence of physiological exhaustion (failure to upregulate TIM-3 or PD-1 expression) in ITP patients which correlated with disease activity. DNA TCR sequencing revealed that expansion of TEMRA cells in ITP was associated with reduced T cell repertoire diversity (p ≤ 0.0001) and lower Simpson's diversity index (p ≤ 0.05). Patients with ITP had a higher number of T cell clones occupying more than 5% of the T cell repertoire when compared to age matched controls (p ≤ 0.05). Expanded clones were not shared across ITP patients and displayed no known antigen specificities when compared to available databases of TCR sequences, providing evidence for the lack of virally driven clonal expansion. Clonally expanded CD8+ T cells persisted over many years in ITP patients and characterised disease severity. Longitudinal samples from 9 patients showed expanded clones and reduced T cell repertoire diversity during active disease (platelet count of less than 30 x 109/L) compared to stable disease (platelet count greater or equal to 30 x 109/L) (p ≤ 0.01; r = 0.56 - Figure 1 shows an example patient). To better characterise CD8+ T cells, we performed paired scRNA and scTCR sequencing on isolated whole CD8+ T cells from patients with active and stable disease. Paired sequencing enables transcriptomic characterisation of T cell clones. ScRNA sequencing revealed four distinct clusters in CD8+ T cells: naïve, TEMRA, central memory (CM) and natural killer T cells (NKT). By mapping scTCR CD8+ sequencing to single cell gene expression, we found that the largest expanded clones showed an aggregative distribution, indicating transcriptional homogeneity and were almost exclusively comprised of TEMRA cells (Figure 2). Consistent with immunophenotyping and DNA TCR sequencing, expanded TEMRA cell clones were expanded in active disease compared to stable disease. To assess the impact of these CD8+ T cells on platelets in ITP, PBMCs from patients with ITP and HC were passed across a platelet layer at flow rates consistent with venous blood flow. CD8+ T cells in ITP patients interacted with platelets 4x more frequently than HCs. Isolated CD8+ T cells were cultured over night with autologous platelets to further explore these interactions: CD8+ T cell:platelet aggregates were identified more frequently in the co-cultures from patients with ITP (p ≤ 0.05) and were inhibited by blocking MHC Class I (p ≤ 0.01). CD8+ T cells within the aggregates had increased CD107a expression compared to non-aggregated CD8+ T cells (p ≤ 0.01), indicating cytolytic activity and release of Granzyme and Perforin. Correspondingly, platelets in the aggregates showed increased CD62P expression, a marker of activation and impending death (p ≤ 0.001). Conclusions Here we demonstrate clonal expansion of disease-associated TEMRA CD8+ T cells in ITP. These cells bind to platelets and cause TCR-mediated platelet activation and death providing an antibody-independent mechanism of platelet destruction. This could serve as biomarker to direct treatment in patients with refractory ITP and targeting specific T-cell clones could be a novel therapeutic option in refractory ITP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

KLRG1 Depletion Is a Novel Therapeutic Strategy for Patients with Mature T and NK/T-Cell Lymphomas

Outcomes for patients with aggressive T-cell lymphomas (TCLs) remain dismal. Killer cell lectin-like receptor G1 (KLRG1) is a co-inhibitory immune checkpoint receptor expressed on late differentiated CD8+ T effector memory (TEM) and TEMRA cells. Monotherapy with KLRG1 neutralizing antibodiesextended survival in solid tumor models. Here, we report the therapeutic potential of a novel and distinct strategy characterized by depletion of KLRG1+ malignant cells in hematologic malignancies and specifically in TCLs. We defined the molecular epidemiology of KLRG1 by determining tumor cell-specific RNA and protein expression on T cell subsets from 8 healthy donors, 26 TCL lines, 15 patient-derived xenografts (PDXs), and 165 tumor and blood specimens representing various subtypes of T and NK/TCLs. Microarray andRNA-seqdata demonstrated highertranscript levels of KLRG1 in patients with T-LGLL and PDXs of T-PLL and HSTCL relative to other subtypes, which were corroborated by protein expression. We quantified tumor cell-specific protein expression of KLRG1 by IHC by generating H scores (% of KLRG1+ tumor cells × measure of staining intensity) ranging from 0-300 across 141 primary TCL biopsies. PTCL-NOS and AITL had heterogeneous KLRG1 expression, whereas ALCL, CTCL, and EATL demonstrated none. The majority of patients with T-PLL (median 180), δ/γ TCLs (median 140), and NK/TCLs (median 100) exhibited higher H scores relative to other subtypes. Up to 99% of the CD3+/CD8+/CD57+ and up to 97% of the CD3-/CD56+/CD57+/CD16+/CD94+ leukemic cells in the blood of patients with T-LGLL (n=12) and CLPD-NK (n=12) respectively expressed KLRG1 protein (Fig.1). None of the TCL cell lines expressed surface KLRG1. Therefore, SMZ1 cells (TP63 rearranged PTCL-NOS line) were stably transduced to express KLRG1. We utilized two human KLRG1 binding antibodies, 13F12F2 (a commercial mIgG2a Ab) and mAb208 (a humanized afucosylated mIgG2a Ab) for in vitro and in vivo studies. Antibody binding capacity assays confirmed a higher number of KLRG1 receptors per cell on SMZ1 KLRG1+ (175,800), HSTCL (23,586), and CD8+ T cells (16,326) compared with B-cell lymphoma cells (242). Competition binding assays demonstrated that concentrations of 0.1 ug/ml of mAb208 saturated receptors on SMZ1 KLRG1+ cells. mAb208 induced a 2-5-fold dose-dependent increase in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), but not apoptosis of SMZ1 KLRG1+ cells relative to SMZ1 KLRG1- WT and isotype control (CTL). This was exhibited across a variety of human and murine effectors such as human PBMCs, human and murine macrophages, and human and murine complements. Reporter-based murine FcγRIV ADCC bioassay demonstrated a dose-dependent 2-6 fold induction of bioluminescence by mAb208 in SMZ1 KLRG1+ cells relative to WT (p=<0.0001). In 4/4 tested T-LGLL patient samples, mAb208 induced ADCC of CD8+/CD57+ LGLs with autologous PBMCs (p=0.03) relative to CTL. Finally, mAb208 induced selective ADCC of KLRG1+ CD8+ TEM and TEMRA cells while sparing KLRG1- naive and central memory T cells in 3 healthy donors (p=0.06). Duvelisib (Duv), a dual PI3K-δ/γ inhibitor (PI3Ki), has demonstrated a high ORR of 50% in phase II trials in TCLs and its ability to reprogram macrophages to an M1 phenotype in TCL PDXs. Thus, we hypothesized that the combination of PI3Ki with anti-KLRG1 mAbs would enhance the clearance of apoptotic and opsonized KLRG1+ TCL cells by proinflammatory macrophages in several PTCL subtypes. We tested this combination in vivo using SMZ1 KLRG1+ subcutaneous xenografts in NSG mice. After 17 days of treatment, combination (mAb208 + Duv)- treated mice had a marked reduction in their mean tumor volumes (159 mm3) relative to mAb208 (1003 mm3 ; p=0.03), Duv (465.6 mm3 ; p=0.03) and CTL (2104 mm3 ; p=0.03) mice resulting in prolonged survival (Fig. 2). Studies in PDX models of T-PLL and HSTCL with varying levels of KLRG1 expression (30% and 80% respectively) have been initiated and will be reported at the meeting. Selective depletion of tumor cells with an anti-KLRG1 depleting mAb in specific subtypes of TCLs, T-LGLL, and CLPD-NK is effective in vitro and in vivo, and its combination with Duv has marked activity in an aggressive PTCL-NOS xenograft model. A multicenter phase I/II trial of the KLRG1 depleting antibody ABC008 (Abcuro) in patients with R/R T-LGLL has been initiated. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Eltrombopag Causes Cell Cycle Arrest of T Cells through Iron Chelation and Could Modulate the Immune Response in ITP

Background Thrombopoietin receptor agonists (TPO-RAs) are the recommended second line treatment in most guidelines for immune thrombocytopenia (ITP) allowing patients to avoid immunosuppressive treatments (Provan et al and Neunert et al 2019). Three different TPO-RAs are licensed for use in ITP. Real world evidence has shown that if patients do not respond to one agent, they may respond to another. This is thought to relate to different signalling pathways, and possibly one TPO-RA creating a stronger signal than another (Olnes et al, 2012; D'Arena et al, 2013). Nonetheless, it is difficult to fully explain responses on switching agents, especially a response to Eltrombopag (ELT) when Romiplostim (ROMI) has failed. In addition to being a TPO-RA, ELT is a small molecule peptide, which chelates cations, in particular iron and calcium. We have previously shown its ability to shuttle iron out of cells, and the impact this has on liver, cardiac and megakaryocyte cells in vitro (Roth et al, 2012; Vlachodimitropoulou et al, 2016, Liu et al 2022). We previously reported that ELT has an anti-proliferative effect on stimulated Jurkat and primary T cells, and demonstrated that patients on ELT have a reduced frequency of terminally differentiated effector (TEMRA) CD8 T cells compared to patients on ROMI (Sayed et al, ASH, 2019). We propose that this is related to an intracellular iron chelation effect, which could either explain or be additive to its megakaryocyte stimulating effect in ITP. In this study, we further explored the immunomodulatory effect of ELT on CD8 T cells in ITP. Methods Primary T cells were stimulated with Dynabeads Human T-Activator CD3/CD28 in the presence of ELT, ROMI, Deferoxamine (DFX) or human recombinant TPO (rhTPO) at increasing doses based on their calculated therapeutic serum levels. DFX and rhTPO act as positive control for iron chelation and TPO-R, respectively. Cell cycle was measured using Propidium Iodide (PI) and analysed using flow cytometry. To explore the in vivo effect of ELT, we measured CD8 TEMRA cells before and after treatment in a cohort of patients with ITP treated with ELT. Results Primary T cells treated with ELT resulted in cell cycle arrest to G1 phase. This effect was more pronounced with increasing doses of ELT. DFX exhibited similar effect on primary T cells, however, the induction of G1 phase cell cycle arrest was less potent than ELT. ROMI and rhTPO did not affect cell cycle progression of primary T cells (Figure 1). To confirm whether this cell cycle arrest was due to the iron chelating properties of ELT, we treated the arrested primary T cells with ammonium iron (III) for 24 hours, and found that cell cycle was able to be rescued. We further investigated the immunomodulatory effect of ELT in patients with ITP by assessing CD8 TEMRA cells (CD45RA+CD62L-) before and after ELT treatment. We found that patients who responded to ELT had a reduction in the frequency of TEMRA cells correlating to their platelet count. An example shown in Figure 2 of a patient who had previously failed ROMI, Mycophenolate Mofetil (MMF) and Rituximab, and subsequently responded to ELT. Prior to ELT, at low platelet count, patient presented with high frequency of CD8 TEMRA cells. Response to ELT resulted in an increased platelet count and a corresponding fall in CD8 TEMRA cells. Discussion Here we show that ELT suppresses the proliferation of T cells, through cell cycle arrest via its iron chelating properties. We propose that this could have a dual impact in patients with ITP, increasing platelet production as well as dampening down the immune response, hence explaining why switching to ELT could be effective if other TPO-RAs have failed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Granzyme B in circulating CD8+ T cells as a biomarker of immunotherapy effectiveness and disability in neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSD) are chronical inflammatory demyelinating diseases of the central nervous system (CNS) and the underlying mechanism remains unclear. Several recent studies have demonstrated that T cells play a pivotal role in the pathogenesis of NMOSD.In this study, we investigated CD8+ T cell phenotypes and levels of the cytotoxic protein granzyme B (GzmB), as well as their potential clinical application in NMOSD. In this study, 90 peripheral blood samples were collected from 59 NMOSD patients with seropositive anti-aquaporin-4 (AQP4) antibodies and 31 sex- and age-matched healthy donors (HDs). Flow cytometry was used to detect circulating levels of GzmB and CD8+ T cell subpopulations, including naïve (TN, CCD7+CD45RA+), central memory (TCM, CCD7+CD45RA-), effector memory (TEM, CCD7-CD45RA-), terminal differentiation effector memory cells (TEMRA, CCD7-CD45RA+) in both groups. The associations between GzmB levels in CD8+T cells and clinical characteristics of NMOSD were evaluated. NMOSD patients exhibited significantly decreased proportions of CD8+TN cells and increased proportions of highly differentiated CD8+T cells (TEMRA) compared with HDs. In addition, levels of GzmB in CD8+ T cells were markedly higher in NMOSD patients than in HDs. Moreover, we observed that high proportions of GzmB-expressing CD8+ T cells were more common in patients with a poor response to immunotherapies, and showed a good potential to distinguish poor responders from responders (ACU=0.89). Clinical correlation analysis indicated that high levels of GzmB in CD8+ T cells were not only related to severe disability but also significantly associated with increased serum levels of neurofilament light (NFL) and glial fibrillary acidic protein (GFAP). Multivariate linear regression analyses further suggested that GzmB expression in CD8+ T cells was predominantly associated with disability and immunotherapy effectiveness in NMOSD, independent of the sex, age, and disease phase. Transcription factor T-bet in CD8+ T cells were also significantly elevated in NMOSD and were associated with increasing number of circulating CD8+TEMRA cells and GzmB-expressing CD8+T cells. Our study support the involvement of GzmB-expressing CD8+ T cells in the inflammatory response in patients with NMOSD and provide a potential biomarker for disease immunotherapy effectiveness and disability progression.

Human, mouse scRNA-seq and flow cytometry data in ischemic heart failure models predict immune cell activation signatures with altered endothelial cell cross talk

Abstract Chronic ischemic Heart Failure (HF) with reduced ejection fraction is marked by adverse remodelling and sustained inflammation. The effects of monocytes and T cells in early state immune responses after myocardial infarction are known, to date their role in progression and maintenance of chronic HF remains elusive. Single cell RNA sequencing of peripheral immune cells from healthy and HF donors revealed a significant increase of the antigen presentation (i.e. HLA-DRB5) and co-stimulatory molecules (i.e. ICAM-1) on monocytes, while the elevation of the T effector memory population in HF patients shows enhanced T cell activation. Subsequent flow cytometry validation in 55 healthy and 110 HF patients confirmed that HF derived monocytes have elevated levels of HLA-DRB5 predicts increased ICAM-1 and TREM-1 (p&amp;lt;0.0001 for both), indicating enhanced T cell activation capacity. In addition, the activated fraction of both CD4+ and CD8+ T cells, including central memory, effector memory and TEMRA cells, is significantly enriched in the HF patients (p&amp;lt;0.0001 for both) with reduction of the naïve T cells. T cell receptor (TCR) sequencing of age matched healthy and HF donors showed an increase in the relative TCR clonality in HF patients, indicating an expansion of the circulating T cells. ScRNA-seq murine myocardial infarction (MI) time course data (day (d) 0/1/3/7/14/28/48 post-MI), found a progressive increase in Th17 cells and activated CD8+ T cells in the cardiac tissue at d48 (2.8x-, and 2.7x-fold, respectively) relative to d0. The T cell imbalance was marked by loss of immunosuppressive markers such as Lef1 (p=2e-30) in T cell clusters primarily populated by d48 relative to d0. Mechanistically the downstream effects of the immune cell activation were analysed in the serum of HF patients, where increased levels of sICAM-1, IL-6 and TNFRI levels (1.75x-, 5.30x-, 2.63x-fold, respectively) could be detected. Furthermore, treatment of endothelial cells with the conditioning media from HF-immune cells induced capability to adhere with monocytes in a co-culture system but also decreased the checkpoint inhibitor molecule PD-L1, a known blocker of autoreactive T cells. These data link HF with enhanced monocyte and T cell activation as well as T cell clonal expansion, along with increased T cell numbers in mouse hearts post-MI. These data suggest HF is may drive impaired resolution of inflammation in cardiac tissue and facilitate adverse remodelling and cardiac damage. Further studies will assess the specific role of chronic monocyte and T cell activation in the progression of heart failure. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG -Deutsche ForschungsgemeinschaftDZHK -Deutsches Zentrum für Herzkreislauferkrankungen

IFN-α with dasatinib broadens the immune repertoire in patients with chronic-phase chronic myeloid leukemia.

In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRβ sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.

Heterogenous CD8+ T Cell Maturation and 'Polarization' in Acute and Convalescent COVID-19 Patients.

Background. The adaptive antiviral immune response requires interaction between CD8+ T cells, dendritic cells, and Th1 cells for controlling SARS-CoV-2 infection, but the data regarding the role of CD8+ T cells in the acute phase of COVID-19 and post-COVID-19 syndrome are still limited. Methods.. Peripheral blood samples collected from patients with acute COVID-19 (n = 71), convalescent subjects bearing serum SARS-CoV-2 N-protein-specific IgG antibodies (n = 51), and healthy volunteers with no detectable antibodies to any SARS-CoV-2 proteins (HC, n = 46) were analyzed using 10-color flow cytometry. Results. Patients with acute COVID-19 vs. HC and COVID-19 convalescents showed decreased absolute numbers of CD8+ T cells, whereas the frequency of CM and TEMRA CD8+ T cells in acute COVID-19 vs. HC was elevated. COVID-19 convalescents vs. HC had increased naïve and CM cells, whereas TEMRA cells were decreased compared to HC. Cell-surface CD57 was highly expressed by the majority of CD8+ T cells subsets during acute COVID-19, but convalescents had increased CD57 on ‘naïve’, CM, EM4, and pE1 2–3 months post-symptom onset. CXCR5 expression was altered in acute and convalescent COVID-19 subjects, whereas the frequencies of CXCR3+ and CCR4+ cells were decreased in both patient groups vs. HC. COVID-19 convalescents had increased CCR6-expressing CD8+ T cells. Moreover, CXCR3+CCR6- Tc1 cells were decreased in patients with acute COVID-19 and COVID-19 convalescents, whereas Tc2 and Tc17 levels were increased compared to HC. Finally, IL-27 negatively correlated with the CCR6+ cells in acute COVID-19 patients. Conclusions. We described an abnormal CD8+ T cell profile in COVID-19 convalescents, which resulted in lower frequencies of effector subsets (TEMRA and Tc1), higher senescent state (upregulated CD57 on ‘naïve’ and memory cells), and higher frequencies of CD8+ T cell subsets expressing lung tissue and mucosal tissue homing molecules (Tc2, Tc17, and Tc17.1). Thus, our data indicate that COVID-19 can impact the long-term CD8+ T cell immune response.

Identification of Pathogenic Immune Cell Subsets Associated With Checkpoint Inhibitor-Induced Myocarditis.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies used to activate the immune system against tumor cells. Despite therapeutic benefits, ICIs have the potential to cause immune-related adverse events such as myocarditis, a rare but serious side effect with up to 50% mortality in affected patients. Histologically, patients with ICI myocarditis have lymphocytic infiltrates in the heart, implicating T cell-mediated mechanisms. However, the precise pathological immune subsets and molecular changes in ICI myocarditis are unknown. To identify immune subset(s) associated with ICI myocarditis, we performed time-of-flight mass cytometry on peripheral blood mononuclear cells from 52 individuals: 29 patients with autoimmune adverse events (immune-related adverse events) on ICI, including 8 patients with ICI myocarditis, and 23 healthy control subjects. We also used multiomics single-cell technology to immunophenotype 30 patients/control subjects using single-cell RNA sequencing, single-cell T-cell receptor sequencing, and cellular indexing of transcriptomes and epitopes by sequencing with feature barcoding for surface marker expression confirmation. To correlate between the blood and the heart, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing on MRL/Pdcd1-/- (Murphy Roths large/programmed death-1-deficient) mice with spontaneous myocarditis. Using these complementary approaches, we found an expansion of cytotoxic CD8+ T effector cells re-expressing CD45RA (Temra CD8+ cells) in patients with ICI myocarditis compared with control subjects. T-cell receptor sequencing demonstrated that these CD8+ Temra cells were clonally expanded in patients with myocarditis compared with control subjects. Transcriptomic analysis of these Temra CD8+ clones confirmed a highly activated and cytotoxic phenotype. Longitudinal study demonstrated progression of these Temra CD8+ cells into an exhausted phenotype 2 months after treatment with glucocorticoids. Differential expression analysis demonstrated elevated expression levels of proinflammatory chemokines (CCL5/CCL4/CCL4L2) in the clonally expanded Temra CD8+ cells, and ligand receptor analysis demonstrated their interactions with innate immune cells, including monocytes/macrophages, dendritic cells, and neutrophils, as well as the absence of key anti-inflammatory signals. To complement the human study, we performed single-cell RNA sequencing/T-cell receptor sequencing/cellular indexing of transcriptomes and epitopes by sequencing in Pdcd1-/- mice with spontaneous myocarditis and found analogous expansions of cytotoxic clonal effector CD8+ cells in both blood and hearts of such mice compared with controls. Clonal cytotoxic Temra CD8+ cells are significantly increased in the blood of patients with ICI myocarditis, corresponding to an analogous increase in effector cytotoxic CD8+ cells in the blood/hearts of Pdcd1-/- mice with myocarditis. These expanded effector CD8+ cells have unique transcriptional changes, including upregulation of chemokines CCL5/CCL4/CCL4L2, which may serve as attractive diagnostic/therapeutic targets for reducing life-threatening cardiac immune-related adverse events in ICI-treated patients with cancer.

Altered Transcriptional Regulation of Glycolysis in Circulating CD8+ T Cells of Rheumatoid Arthritis Patients.

Peripheral T lymphocytes of rheumatoid arthritis (RA) patients show pathological changes in their metabolic pathways, especially glycolysis. These changes may drive the increased proliferation and tissue invasiveness of RA T cells. In order to study the transcriptional regulation underlying these alterations, we analysed publicly available RNA sequencing data from circulating T lymphocyte subsets of healthy individuals, untreated RA patients, and patients undergoing treatment for RA. Differential co-expression networks were created using sample-wise edge weights from an analysis called “linear interpolation to obtain network estimates for single sample” (lionessR), and annotated using the Gene Transcription Regulation Database (GTRD). Genes with high centrality scores were identified. CD8+ effector memory cells (Tem) and CD8+CD45RA+ effector memory cells (Temra) showed large changes in the transcriptional regulation of glycolysis in untreated RA. PFKFB3 and GAPDH were differentially regulated and had high centrality scores in CD8+ Tem cells. PFKFB3 downregulation may be due to HIF1A post transcriptional inhibition. Tocilizumab treatment partially reversed the RA-associated differential expression of several metabolic and regulatory genes. MYC was upregulated and had high centrality scores in RA CD8+ Temra cells; however, its glycolysis targets were unaltered. The upregulation of the PI3K-AKT and mTOR pathways may explain MYC upregulation.

The generation of stem cell-like memory cells early after BNT162b2 vaccination is associated with durability of memory CD8+ Tcell responses.

COVID-19 vaccines elicit humoral and cellular immune responses. Durable maintenance of vaccine-induced immunity is required for long-term protection of the host. Here, we examine activation and differentiation of vaccine-induced CD8+ T cells using MHC class I (MHC-I) multimers and correlations between early differentiation and the durability of CD8+ T cell responses among healthcare workers immunized with two doses of BNT162b2. The frequency of MHC-I multimer+ cells is robustly increased by BNT162b2 but decreases 6 months post-second vaccination to 2.4%–65.6% (23.0% on average) of the peak. MHC-I multimer+ cells dominantly exhibit phenotypes of activated effector cells 1–2 weeks post-second vaccination and gradually acquire phenotypes of long-term memory cells, including stem cell-like memory T (TSCM) cells. Importantly, the frequency of TSCM cells 1–2 weeks post-second vaccination significantly correlates with the 6-month durability of CD8+ T cells, indicating that early generation of TSCM cells determines the longevity of vaccine-induced memory CD8+ T cell responses.

AGE, AUTOIMMUNITY, AND INFLAMMATION: THE CURIOUS CASE OF IMMUNOSENESCENCE AND INFLAMM-AGING

An ever-aging population has caused an increase in the prevalence of diseases which occur in the elderly like diabetes and cancer; and autoimmune disease like rheumatoid arthritis (RA). On the other hand, ageing also causes an increased susceptibility to infections, reactivation of latent infections and a poorer vaccine response. Together, this ageing-related decline in immunity is called immunosenescence and the associated ageing-related inflammation is called inflamm-aging.&#x0D; In this brief review, we describe the changes seen with ageing in innate and adaptive immunity and how these lead to the various peculiarities associated with ageing in the immune system. TEMRA cells, Senescence associated secretory phenotype (SASP) and exhausted T cells are the main changes that occur in ageing T cells. Age-associated B cells (ABCs) contribute to changes associated with autoimmunity in elderly. In the innate arm, the macrophages-led inflamm-aging cause an overall net pro-inflammatory state. However, the macrophages have reduced phagocytosis leading to accumulation of necrotic and apoptotic debris.&#x0D; We also attempt to explain how immunosenescence and inflamm-aging cause defective vaccine responses and an increased predisposition to autoimmune diseases. As the average life expectancy of the world continues to increase, this is not just a curiosity to study at whim, but an indispensable part of medicine in the near-future.

Abstract 2529: T-cell phenotype and differentiation vary in the tumor microenvironment of follicular lymphoma and are associated with patient outcome

Abstract Previous studies have found that the prevalence of subtypes of T cells may be associated with patient outcomes, but a comprehensive analysis of the immune profile in the TM of FL has not been done. In the present study, we identified groups of FL patients with discretely unique TMEs and determined whether the T-cell phenotypes in the TME differed among groups. Using a cohort of 82 FL patients with biopsy specimens collected before treatment, we defined the type of TME based on the content of major lineages (T, B, monocytes/macrophages and NK cells) determined by CyTOF analysis. Hierarchical clustering of this cohort stratified patients into 4 groups with different TME: group 1 (G1) included patients with a high percentage of monocyte/macrophages/NK cells; patients from G2 and G3 were enriched for intratumoral T and B cells, respectively. Patients with intermediate numbers of T and B cells were included in G4. CITRUS analysis revealed that T-cell clusters with phenotypes expressing KLRG1, CD57, PD-1dim and rich in TEMRA cells were significantly more abundant in G1 when compared to G2, G3 or G4. In contrast, T-cell clusters with phenotypes containing CD127, CD45RA, CCR7 or PD-1high cells were significantly less abundant in G1 when compared to other groups. When compared to G3 or G4, 2 classes of T cell clusters, all from CD4+ T cells, were significantly more (CD127+KLRG1-) or less (CD57+PD-1high) abundant in G2, respectively. Clusters with a phenotype rich in short-lived effector cells (SLEC) (from CD8+) were upregulated in G3 when compared to G4. These results suggest that patient groups with distinct TME exhibited variable T-cell phenotypes. To determine the role of T-cell differentiation in predicting patient outcome in FL, we identified T-cell subsets using tSNE plots based on the expression of T-cell maturation markers. We identified 18 subsets (S1-S18) of T cells (11 from CD4, 6 from CD8 and 1 from CD4-CD8-) in each sample. Four subsets - S4 (CD4+TN), S5 (CD4+TCM), S7 (CD8+TN) and S10 (CD8+MEPCs) - were considered to be naïve cells or cells in the early stages of differentiation. Four additional subsets - S8 (CD57+TFH), S9 (CD4+TEXH), S13 (CD8+SLECs) and S17 (CD4+PD-1+Treg) - were memory cells with expression of surface markers indicting late-stage differentiation. We found that the 4 subsets (S4: p=0.08, S5: p=0.01, S7: p=0.04 and S10: p&amp;lt;0.01) with an early-stage phenotype were associated with a favorable clinical outcome. In contrast, subsets (S8: p=0.02, S9: p=0.04, S13: p=0.07 and S17: p=0.06) with a late-stage differentiation phenotype had an unfavorable survival. Supporting this finding, we observed that increased numbers of CD45RA+ T cells correlated with a favorable survival. These results indicate that the differentiation stage may determine the role of T cells in predicting patient outcome in FL. Citation Format: Zhi-Zhang Yang, Hyo Jin Kim, Hongyan Wu, Xinyi Tang, Jordan Krull, Patrizia Mondello, Jose Villasboas, Anne Novak, Stephen Ansell. T-cell phenotype and differentiation vary in the tumor microenvironment of follicular lymphoma and are associated with patient outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2529.

CCL5-Secreting Virtual Memory CD8+ T Cells Inversely Associate With Viral Reservoir Size in HIV-1-Infected Individuals on Antiretroviral Therapy.

Recent studies highlighted that CD8+ T cells are necessary for restraining reservoir in HIV-1-infected individuals who undergo antiretroviral therapy (ART), whereas the underlying cellular and molecular mechanisms remain largely unknown. Here, we enrolled 60 virologically suppressed HIV-1-infected individuals, to assess the correlations of the effector molecules and phenotypic subsets of CD8+ T cells with HIV-1 DNA and cell-associated unspliced RNA (CA usRNA). We found that the levels of HIV-1 DNA and usRNA correlated positively with the percentage of CCL4+CCL5- CD8+ central memory cells (TCM) while negatively with CCL4-CCL5+ CD8+ terminally differentiated effector memory cells (TEMRA). Moreover, a virtual memory CD8+ T cell (TVM) subset was enriched in CCL4-CCL5+ TEMRA cells and phenotypically distinctive from CCL4+ TCM subset, supported by single-cell RNA-Seq data. Specifically, TVM cells showed superior cytotoxicity potentially driven by T-bet and RUNX3, while CCL4+ TCM subset displayed a suppressive phenotype dominated by JUNB and CREM. In viral inhibition assays, TVM cells inhibited HIV-1 reactivation more effectively than non-TVM CD8+ T cells, which was dependent on CCL5 secretion. Our study highlights CCL5-secreting TVM cells subset as a potential determinant of HIV-1 reservoir size. This might be helpful to design CD8+ T cell-based therapeutic strategies for cure of the disease.

Chronic Kidney Failure Provokes the Enrichment of Terminally Differentiated CD8+ T Cells, Impairing Cytotoxic Mechanisms After Kidney Transplantation.

Chronic kidney failure (KF) provokes the development of immune senescent CD8+ cytotoxic T cells, affecting the occurrence of graft rejection, viral infections, and malignancies after kidney transplantation. In this study, we analyzed the impact of KF, subsequent dialysis treatment, and kidney transplantation on the differentiation of CD8+CD31+CD45RA+CCR7+ recent thymic emigrant (CCR7+ RTE) Tregs/Tresps into CD8+CD31-CD45RA- memory (CD31- memory) Tregs/Tresps and its effect on the release of cytokines, Fas receptor, Fas ligand as well as cytotoxic mediators by naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (TEMRA) Tresps. We found that normal age-dependent differentiation of CD8+ Tregs/Tresps generally differs in the way that TEMRA cells only arise in Tresps. Compared to healthy controls, KF patients revealed an age-independently decreased frequency of CCR7+ RTE Tregs/Tresps, but increased frequencies of CCR7+ MN Tregs/Tresps and CD31- memory Tregs/Tresps, suggesting an increased differentiation via CD31+CD45RA- memory (CD31+ memory) Tregs/Tresps into CD31- memory Tregs/Tresps. Intensified differentiation via CD31+ memory Tresps increased the emergence of apoptosis-resistant CM Tresps with strong Fas ligand-mediated cytotoxicity. CCR7+ RTE Tresp proliferation generated TEMRA Tresps, secreting high levels of cytotoxic mediators. In dialysis and transplant patients, CD31+ TEMRA Tregs/Tresps accumulated, proposing an impaired CCR7+ RTE Treg/Tresp differentiation via CD31+ memory Tregs/Tresps into CD31- memory Tregs/Tresps. Increased percentages of CD31- TEMRA Tresps, but not of CD31- TEMRA Tregs, were observed in all patient groups, indicating impaired proliferation of CCR7+ RTE Tresps, but not of CCR7+ RTE Tregs, into CD31- memory Tregs/Tresps. In transplant patients, CCR7+ RTE Tregs accumulated, while frequencies of CCR7+ RTE Tresps were decreased, suggesting that the immunosuppressive therapy only prevented excessive CCR7+ RTE Treg differentiation but not that of CCR7+ RTE Tresps. Presumably, this caused the accumulation of TEMRA Tresps with decreased release of cytotoxic mediators, such as perforin. In conclusion, we propose that chronic KF affects both the differentiation of CD8+ Tregs and CD8+ Tresps. However, the immunosuppressive therapy after transplantation may successfully prevent excessive Treg differentiation, but not as suffciently that of Tresps. Therefore, the risk for graft rejection may be reduced, while the susceptibility for infections and malignancies may be increased in these patients.

T-cell phenotype varies in distinct tumor microenvironments and CD57+ TFH cells are associated with disease progression and inferior survival in follicular lymphoma

Abstract The tumor microenvironment (TME) plays a crucial role in mediating the tumor immune response, thereby affecting patient outcomes in follicular lymphoma (FL). Using CyTOF, we analyzed a cohort of 82 FL patients with biopsy specimens collected before the first treatment regimen was administered. Our results showed that there was a difference in the T-cell phenotype in patients with various TMEs. Patients with TMEs rich in monocyte/macrophages/NK cells had more CD8+ cells and tended to have fewer TN, but more TEMRA and TEXH cells. Patients enriched with T cells exhibited a T-cell phenotype skewed toward to TCM and patients with B cell dominance tended to have significant higher number of TFH cells. T-cell phenotypes with more terminally differentiated, senescent and exhausted cells were highly represented in patients with less T cells when compared to patients with more T cells. We observed that CD57+TFH cells was significantly more abundant in patients who had disease progression than patients who had complete response to therapy, suggesting a role for CD57+ TFH cells in promoting malignant cell growth in FL. Consistent with this finding, we observed that increased numbers of CD57+ TFH cells correlated with an inferior survival in FL. Using CITE-seq technology, we found that CD57+ TFH cells exhibited a substantially different transcriptome from CD57− TFH cells. Genes that were differentially upregulated in CD57+ TFH cells when compared to CD57− TFH cells included genes involved in cell survival, compromised inflammatory response, and metabolic activation. Taken together, our results indicate different tumor microenvironments among patient groups that is associated with variable T-cell phenotypes.

Epigenetic quantification of immunosenescent CD8+ TEMRA cells in human blood.

Age‐related changes in human T‐cell populations are important contributors to immunosenescence. In particular, terminally differentiated CD8+ effector memory CD45RA+ TEMRA cells and their subsets have characteristics of cellular senescence, accumulate in older individuals, and are increased in age‐related chronic inflammatory diseases. In a detailed T‐cell profiling among individuals over 65 years of age, we found a high interindividual variation among CD8+ TEMRA populations. CD8+ TEMRA proportions correlated positively with cytomegalovirus (CMV) antibody levels, however, not with the chronological age. In the analysis of over 90 inflammation proteins, we identified plasma TRANCE/RANKL levels to associate with several differentiated T‐cell populations, including CD8+ TEMRA and its CD28− subsets. Given the strong potential of CD8+ TEMRA cells as a biomarker for immunosenescence, we used deep‐amplicon bisulfite sequencing to match their frequencies in flow cytometry with CpG site methylation levels and developed a computational model to predict CD8+ TEMRA cell proportions from whole blood genomic DNA. Our findings confirm the association of CD8+ TEMRA and its subsets with CMV infection and provide a novel tool for their high throughput epigenetic quantification as a biomarker of immunosenescence.

Persistent Overactive Cytotoxic Immune Response in a Spanish Cohort of Individuals With Long-COVID: Identification of Diagnostic Biomarkers.

Long-COVID is a new emerging syndrome worldwide that is characterized by the persistence of unresolved signs and symptoms of COVID-19 more than 4 weeks after the infection and even after more than 12 weeks. The underlying mechanisms for Long-COVID are still undefined, but a sustained inflammatory response caused by the persistence of SARS-CoV-2 in organ and tissue sanctuaries or resemblance with an autoimmune disease are within the most considered hypotheses. In this study, we analyzed the usefulness of several demographic, clinical, and immunological parameters as diagnostic biomarkers of Long-COVID in one cohort of Spanish individuals who presented signs and symptoms of this syndrome after 49 weeks post-infection, in comparison with individuals who recovered completely in the first 12 weeks after the infection. We determined that individuals with Long-COVID showed significantly increased levels of functional memory cells with high antiviral cytotoxic activity such as CD8+ TEMRA cells, CD8±TCRγδ+ cells, and NK cells with CD56+CD57+NKG2C+ phenotype. The persistence of these long-lasting cytotoxic populations was supported by enhanced levels of CD4+ Tregs and the expression of the exhaustion marker PD-1 on the surface of CD3+ T lymphocytes. With the use of these immune parameters and significant clinical features such as lethargy, pleuritic chest pain, and dermatological injuries, as well as demographic factors such as female gender and O+ blood type, a Random Forest algorithm predicted the assignment of the participants in the Long-COVID group with 100% accuracy. The definition of the most accurate diagnostic biomarkers could be helpful to detect the development of Long-COVID and to improve the clinical management of these patients.

New prognostic criterion for the outcome of pregnancy in patients with threatening preterm birth.

To identify new criteria for predicting the outcome of pregnancy in women with threatened preterm labor based on the establishment of the characteristics of differentiation and functional activity of memory T-cells in the population of CD8 + T-lymphocytes. The study involved 56 women with threatened preterm labor at 22-34 weeks of pregnancy. Depending on the outcome of pregnancy, patients were divided into 3 groups: Group I included 22 women by premature labor; group II - 34 women whose pregnancy ended by deliver at term. By tricolor flow cytofluorometry method the content of peripheral Tn, Tcm, Tem and Temra cells in CD8+ population was studied. Statistical analysis was carried out using programs «Statistica for Windows 13.0», «Microsoft Excel 2019» and «MedCalc 19.3». Patients with the threat of preterm birth were characterized by a significant increase in the relative content of CD8+ T-lymphocytes in the peripheral blood compared to the control group (p=0.001). When comparing this indicator in the subgroups of women with the threat of preterm birth, it was found that preterm birth was associated with significantly higher relative CD8 + lymphocyte counts (p=0.03) compared to the indicators of the subgroup of women who gave birth on time. When evaluating the results obtained, it was found that the percentage of CD8+Temra GranzymeB-producing cells in the group of patients with threatened preterm birth, whose pregnancy ended in preterm birth, was significantly lower than in the group of women who gave birth on time (p=0.003). The content of Tcm, Tem memory cells producing GranzymeB in the subgroups of women with pregnancy outcomes of timely and preterm birth did not have significant differences compared to the control group. Thus, the prediction of preterm birth is possible with a value of GranzymeB-producing CD8 + Tem equal to 8.2% or less (sensitivity-87.9%, specificity-85%, accuracy-87.9%). Thus, the identification of a new criterion will make it possible to predict preterm birth in a timely manner and promote the choice of optimal pregnancy management tactics, reasonable prevention of fetal respiratory distress syndrome and transfer of a woman to an obstetric hospital designed to provide high-tech care to premature newborns.

T cell senescence and impaired CMV-specific response are associated with infection risk in kidney transplant recipients

Older kidney transplant recipients demonstrate increased rates of infection, and lower rates of rejection, compared with younger kidney transplant recipients. However, the mechanism behind this observation remains unknown. To develop a multifaceted view of age-associated immune dysfunction, we determined the function and phenotype of T cells predisposing to vulnerability to infection on a molecular level.Overlapping peptide pools representing the dominant CMV antigens were used to stimulate PBMC collected from 51 kidney transplant recipients, using cytokine secretion to determine specificity and intensity of response. Staphylococcal endotoxin B (SEB) was analyzed in parallel. To define immune cell subsets, we used single cell RNA sequencing (scRNAseq) to evaluate cellular surface markers and gene expression.We found increased frequency of SEB- and CMV-specific T cells was associated with freedom from infection, especially in older patients. Spatialized t-SNE analysis revealed decreased frequency of naïve T cells, increased frequency of TEMRA cells, and decreased frequency of IFNγ secreting T cells in patients with infection. Application of scRNAseq analysis revealed increased frequency of terminally differentiated T cells expressing NK-associated receptors and inhibitory markers.These findings offer unique insight into the mechanism behind vulnerability to infection in the kidney transplant recipient, revealing a specific T cell subtype of impaired antigen response and terminal effector phenotype as markers of T cell senescence.

Changes in the phenotype and function of mucosal-associated invariant T cells in neutrophilic asthma

Asthma is a chronic heterogeneous inflammatory disease. Most neutrophilic asthma (NA) cases are severe asthma involving many inflammatory cells and mediators, although the specific pathogenesis is not clear. Mucosal-associated invariant T (MAIT) cells as innate-like T lymphocytes play an important role in the immune response in asthma by producing cytokines. In this study, we evaluated the phenotype and function of circulating MAIT cells in patients with NA and inflammatory-related cytokines in plasma and induced sputum supernatants using flow cytometry. The results showed that the frequency of circulating MAIT cells in asthma patients, particularly NA patients, decreased significantly, and CD8+ MAIT and MAIT Temra cells also decreased significantly. Increased expression of CD69 and PD-1 on MAIT cells indicated excessive activation and depletion, leading to the decrease in MAIT cells. Levels of IL-17A and TNF-α secreted by MAIT cells of NA patients increased, whereas IFN-γ levels decreased, indicating that MAIT cells in NA are biased to the Th17 subtype. MAIT cells were also negatively correlated with clinical parameters, indicating that these cells are related to asthma severity. Pro-inflammatory cytokines in plasma and sputum supernatant increased to varying degrees, whereas IL-10 declined, corresponding with asthma severity. We speculate that increased IL-17A and TNF-α synergistically stimulated respiratory epithelial cells to secrete IL-6 and IL-8, thereby recruiting neutrophils to inflammatory sites and aggravating asthma symptoms. Therefore, MAIT cells could serve as a potential therapeutic target in NA immunity, thus providing a new strategy for the treatment of asthma.