Population Of CD4 Research Articles

Quantitative and Qualitative Perturbations of CD8+ MAITs in Healthy Mycobacterium tuberculosis-Infected Individuals.

CD8 T cells are considered important contributors to the immune response against Mycobacterium tuberculosis, yet limited information is currently known regarding their specific immune signature and phenotype. In this study, we applied a cell population transcriptomics strategy to define immune signatures of human latent tuberculosis infection (LTBI) in memory CD8 T cells. We found a 41-gene signature that discriminates between memory CD8 T cells from healthy LTBI subjects and uninfected controls. The gene signature was dominated by genes associated with mucosal-associated invariant T cells (MAITs) and reflected the lower frequency of MAITs observed in individuals with LTBI. There was no evidence for a conventional CD8 T cell–specific signature between the two cohorts. We, therefore, investigated MAITs in more detail based on Vα7.2 and CD161 expression and staining with an MHC-related protein 1 (MR1) tetramer. This revealed two distinct populations of CD8+Vα7.2+CD161+ MAITs: MR1 tetramer+ and MR1 tetramer−, which both had distinct gene expression compared with memory CD8 T cells. Transcriptomic analysis of LTBI versus noninfected individuals did not reveal significant differences for MR1 tetramer+ MAITs. However, gene expression of MR1 tetramer− MAITs showed large interindividual diversity and a tuberculosis-specific signature. This was further strengthened by a more diverse TCR-α and -β repertoire of MR1 tetramer− cells as compared with MR1 tetramer+. Thus, circulating memory CD8 T cells in subjects with latent tuberculosis have a reduced number of conventional MR1 tetramer+ MAITs as well as a difference in phenotype in the rare population of MR1 tetramer− MAITs compared with uninfected controls.

COVID-19: Studying the Global Pandemic – Foreword

COVID-19: Studying the Global Pandemic – Foreword

The role of sex in the innate and adaptive immune environment of metastatic colorectal cancer

BackgroundWomen with colorectal cancer (CRC) have a significant survival advantage over men. Sex influences on the tumour microenvironment (TME) are not well characterised, despite the importance of immune response in CRC. We hypothesised that sex-divergent immune responses could contribute to survival.MethodsUsing a murine model of metastatic CRC, we examined T cells, macrophages, and cytokines locally and systemically. TME and serum cytokines were measured by multiplex bead-based arrays, while FCA was used to identify cells and phenotypes. IHC provided spatial confirmation of T cell infiltration.ResultsFemales had increased survival and T cell infiltration. CD8, CD4 and Th2 populations correlated with longer survival. Males had increased serum levels of chemokines and inflammation-associated cytokines. Within the TME, males had lower cytokine levels than females, and a shallower cytokine gradient to the periphery. Female tumours had elevated IL-10+ macrophages, which correlated with survival.ConclusionsThese data demonstrate survival-associated differences in the immune response of males and females to metastatic CRC. Females showed changes in cytokine production accompanied by increased immune cell populations, biased toward Th2-axis phenotypes. Key differences in the immune response to CRC correlated with survival in this model. These differences support a multi-faceted shift across the TME.

Role of glutamine metabolism in CD8+PD-1+TIM-3+ T cells in ICI resistant melanoma.

e22055 Background: Immune checkpoint inhibitors (ICI) targeting checkpoints has achieved cures in half of stage IV melanoma patients yet there remains a critical need to identify why other half remain either ICI resistant or relapse. Recent studies show that multiple subpopulations of tumor-infiltrating CD8+PD-1+ T cells, previously known to be exhausted, are in fact metabolically active. We have identified a sub population of CD8+PD-1+ T cells resistant melanoma that are metabolically active, highly proliferative; and has high glutamine metabolic pathway. Rapidly proliferating cells utilize the glucose-lactic acid pathway for faster energy production and can metabolize glutamine for energy. A recent study showed that glutamine metabolism blockade improves immunotherapy response by enhancing cytotoxic T cell function. However, the role of glutamine in dysfunctional CD8+T cells has not be studied. Methods: Using patient-derived ICI resistant tumors (n = 8), tumor infiltrating lymphocytes (TILs) were isolated and sorted for total CD8+ T cells for single cell RNA sequencing, and then into CD8+PD-1+TIM-3+ T cells for bulk RNA analysis. FACS analysis was performed to assess the protein expression of SLC1A5 (a major glutamine transporter) and ki67. Seahorse Fuel Flex Test was employed to examine oxidative phosphorylation and mass spectrometry analysis was performed on sorted CD8+PD-1+TIM-3 TILs to assess the glutamine and lactic acid content. Results: A volcanic plot from bulk RNAseq data demonstrated an increase in glutamine pathway activity as well as the expression of SLC1A5 in CD8+PD-1+TIM3+ T cells compared to the peripheral CD8+ T cells. Similarly, scRNAseq analysis showed an increased expression of ki67 and SLC1A5 in MAD CD8+ T cells compared to the effector and naïve CD8+ T cells. FACS analysis of TILs gated for CD3+CD8+,showed an increased protein expression of SLC1A5 and ki67 in the PD-1+TIM-3+ population. A seahorse assay using sorted CD8+PD-1+ TIM3+ TILs from human samples confirmed an increased oxidative consumption rate (OCR) and increased dependency of glutamine metabolism in MAD CD8+ T cells compared to healthy CD8+ T cells. Importantly, mass spectrometry of CD8+PD-1+ TIM3+ T cells showed a significant increase in glutamine and lactate supporting our hypothesis that MAD CD8+ T cells have increased glutamine metabolism due to anaerobic glycolysis. Conclusions: Glutamine is a major metabolic source for rapidly proliferating CD8+PD-1+TIM-3+ dysfunctional T cells and inhibition of glutamine metabolism can be leveraged as immunotherapy in ICI resistant melanoma.

Heterogenous Populations of Tissue-Resident CD8+ T Cells Are Generated in Response to Infection and Malignancy

Tissue-resident memory CD8+ Tcells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ Tcells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ Tcell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted Tcells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

Assessing CD8+ cytotoxic T-cell dysfunction in obesity and its implications in anti-PD-1 blockade therapy

Abstract In chronic conditions, notably cancers, CD8+ cytotoxic T cells gradually lose their effector functions due to constant exposure to antigens and consequent upregulation of inhibitory receptors like PD-1. In this regard, PD-1 blockade therapy was shown to bolster cytotoxic T-cell response and improve disease prognosis in some cases. PD-1 mediated T cell dysfunction is also involved in obesity-induced tumorigenesis. In this study, we used a molecular cytometry approach to better understand the disturbances caused by obesity in immune cells. A detailed single-cell analysis of various cell compartments revealed a unique population of CD8+ cytotoxic T cells in the epididymal fat of mice consuming a high fat diet for 16 weeks. These cells showed high expression of proteins and/or mRNA transcripts for various inhibitory receptors, including PD-1. Based on these results, we created a 28-color panel for high-throughput cell analysis at multiple time points over the 16-week period, using a BD FACSymphony™. The frequency of CD8+PD-1+ T cells in the fat correlated with the numbers of inflammatory cells, suggesting that the dysfunctional phenotype resulted from a growth in inflammation over time. The functional state of the CD8+PD-1+ cells was also examined by challenging these cells with or without PD-1 blockade. Taken together the data delineated a refined application for a broad analysis of immune cells in the fat and provided insights into the effects of obesity-induced T-cell dysfunction in mice. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 laser product. BD, the BD Logo, FACSymphony is trademark of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

Tumor intrinsic mechanisms of immunosuppression in CD8 T cell function and response to lung cancer

Abstract Immunotherapies are effective in a subset of patients with non-small cell lung cancer (NSCLC), however the majority do not respond which highlights the ongoing need for novel therapeutics and biomarkers. Tumor cells can produce cytokines and chemokines that influence both the recruitment and function of immune cells. In patients with NSCLC, the microRNA-17~92 (miR-17~92) cluster was shown to be upregulated, and high levels of microRNA-17 in serum was associated with decreased survival. We aimed to identify whether tumor cell intrinsic mechanisms, like upregulation of miR-17~92, regulate chemokine or cytokine production to induce an immunosuppressive tumor microenvironment and inhibit CD8+ T cell anti-tumor function in the lung. We used the CIBERSORTX program to evaluate alterations to immune cell populations and survival in NSCLC patients with increased chemokine or miR-17~92 expression. We found high expression of miR-17 and low expression of IL-7Rα correlated with decreased survival in lung adenocarcinoma (LAC) patients. Furthermore, LAC patient’s tumor biopsies with high miR-17 had decreased IL-7Rα and increased populations of CD8+ T cells by CIBERSORTX. We will use in vitro and in vivo models of lung cancer to determine whether miR-17~92 upregulation and tumor cell chemokines and cytokines production drives immunosuppression in the tumor microenvironment. Understanding how lung tumor cells regulate cytokines and chemokines, and their putative control of exhaustion in CD8 T cells in the tumor microenvironment would guide selection of efficacious immunotherapy treatment.

PD-1hi CD8+ tissue-resident memory T cells balance immunity and fibrotic sequelae following influenza infection

Abstract CD8+ tissue-resident memory T (TRM) cells provide frontline immunity in mucosal tissues. The mechanisms regulating CD8+ TRM maintenance, heterogeneity, and protective and pathological functions are largely elusive. Here, we identify a population of CD8+ TRM cells that is maintained by major histocompatibility complex class I (MHC-I) signaling, and CD80 and CD86 co-stimulation after acute influenza infection. These TRM cells have both “exhausted-like” phenotypes and memory features, and provide heterologous immunity against secondary infection. Using recombinant influenza virus, we demonstrate that the development of these PD-1hi “exhausted-like” TRM cells is dependent on the doses of the antigen during primary infection. Furthermore, we showed that lung CD11c+ antigen presenting cells are required for sustaining these TRM cells at the memory stage. Our data suggest that continuous antigen presentation in situ is required for the development and maintenance of these “exhausted-like” TRM cells in the lung. Strikingly, PD-L1 blockade after the resolution of primary infection promotes the rejuvenation of these “exhausted-like” TRM cells, restoring protective immunity at the cost of promoting post-infection inflammatory and fibrotic sequelae. Thus, PD-1 serves to limit the pathogenic capacity of “exhausted-like” TRM cells at the memory phase under specific tissue environment with chronic low-levels of antigen presentation following viral clearance. Our data indicate that TRM cell “exhaustion” is the result of a tissue-specific cellular adaptation that balances fibrotic sequelae with protective immunity.

New early prognostic criteria for pregnancy outcome in women with recurrent miscarriage.

To determine the new criteria for predicting the outcome of pregnancy in women with habitual abortion based on features of differentiation of naive T cells and memory cells in a population of T-helper (CD4+) and cytotoxic T lymphocytes (CD8+). The study involved 61 women with threatened and habitual abortion in the first trimester of gestation. Depending on the outcome of pregnancy was allocated to 3 groups: I went to 39 women whose pregnancy ended in timely delivery; in II - 10 women whose pregnancies ended in premature birth; in III - 11 patients in whom there was a spontaneous miscarriage. Using three-color flow cytometry as peripheral venous blood in populations of CD8+ and CD4+ determined by the content Tn, Tcm, Tem and Temra cells. Statistical analysis was carried out in the program «MicrosoftOffice 2010», «Statistica for Windows 6.0» and MedCalc». When conducting a retrospective assessment, it was found that in the group of patients whose pregnancy ended in preterm delivery, the percentage of CD4+ Tem memory cells was significantly higher and CD4+ Tn lower than in the subgroup with timely delivery (p = 0.013 and p = 0.025, respectively ) In patients with early spontaneous miscarriage, the level of CD8+ Tn significantly decreased against the background of the growth of CD8+ Tem memory cells compared with the same parameters in patients with timely delivery (p = 0.040 and p = 0.014, respectively). Prediction of spontaneous abortion is possible up to CD4+ Tn equal to 34.2% or less (sensitivity - 100.0%, specificity - 56.4%, accuracy - 63.8%), premature birth - if the CD4+ Tn equal to 35, 2% or less (sensitivity - 66.7%, specificity - 74.4% accuracy - 72.9%). Thus, the new criteria will allow additional time to identify risk and assign adequate treatment aimed at prolonging the desired pregnancy.

CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Tumour grade significantly correlates with total dysfunction of tumour tissue-infiltrating lymphocytes in renal cell carcinoma

It is important to evaluate the clinical importance of both CD8 T cells and CD4 T cells expression simultaneously because they have crucial networks in tumour targeting immune responses. In 97 RCC patients, RNA sequencing and gene set enrichment analysis of both CD8 and CD4 T cells based on the expression levels of PD-1 and TIM-3 implied that the populations of PD-1+TIM-3+ CD8 T cells and PD-1lowTIM-3 + CD4 T cells were characterized as exhausted CD8 T cells and regulatory CD4 T cells, respectively. These populations of CD4 and CD8 T cells were significantly upregulated in the patients with RCC of higher WHO/ISUP grade (grades 3, 4) (P < 0.001). Moreover, the cytokine productivities of each population in both CD4 and CD8 T cells of the higher-grade patients were significantly lower than those of the lower-grade patients (P < 0.05). Multivariate analysis showed the prognosis of patients with metastatic RCC of higher WHO/ISUP grade treated by nivolumab to be significantly worse than that of patients with lower grade (P = 0.026). This study showed that tumour grade significantly correlated with dysfunction of both CD4+ and CD8+ TILs and the efficacy of nivolumab treatment.

Ovarian cancer cells with CD133+ phenotype is more resistant against Ngai Bun Boesenbergia pandurata extract than original ovarian cancer cells

Introduction: Ovarian cancer is one of the most common cancers in women. Due to the difficulty in early detection and treatment of ovarian cancer, many research studies and clinical trials have been developed to discover more efficient therapies. Besides Western medicine, traditional medicine has gained increased interest as a research field with potential to lead to the production of marketable therapeutic products. With the diversity of tropical plants in Asia, traditional medicine has been very popular and has served as a traditional therapy for generations. The Ngai bun (Boesenbergia pandurata) root is used not only as a food spice but also in ethnomedicine. This study aimed to compare the anti-tumor activity of Boesenbergia pandurata root extract against ovarian cancer cells and CD133+ ovarian cancer cells that were enriched from the original ovarian cancer cells.&#x0D; Methods: Crude extract of Boesenbergia pandurata roots were prepared in two kinds of solvents (methanol and chloroform). The ovarian cancer cells OVP-10 were used in this study. The population of CD133+ ovarian cancer cells (CD133+OVP-10) were sorted from the OVP-10 cancer cells. Both OVP-10 cells and CD133+OVP-10 cells were treated with these crude extracts. Adiposederived stem cells (ADSCs) were used as control normal cells for all assays. The anti-tumor activity of extracts were evaluated based on the IC50 values.&#x0D; Results: Based on the IC50 index, the chloroform extract had an anti-tumor activity higher than that of methanol extract, on both OVP-10 and CD133+OPV-10 cells (IC50 of methanol and chloroform extracts were 330.1 +/- 16.9 ug/mL and 246.5 +/- 21.2 ug/mL, respectively, for OVP-10 cells; IC50 of methanol and chloroform extracts were 411.8 +/- 83.7 ug/mL and 307 +/- 9.2 ug/mL respectively, for CD133+OVP-10 cells). The results also showed that CD133+OVP-10 cells were more resistant to chloroform extract than were OVP-10 cells (307 +/- 9.2 mg/mL vs. 246.5 +/- 21.2 mg/mL, respectively, for CD133+OVP-10 vs. OVP-10 cells, p &lt; 0.05).&#x0D; Conclusion: The chloroform extract of Boesenbergia pandurata roots displayed strong antitumor activity against ovarian cancer cells OVP-10 and CD133+OVP-10; the latter cells were found to be more resistant than the original ovarian cancer cells.&#x0D;

Analysis of Immune Cell Repopulation After Anti-thymocyte Globulin Administration for Steroid-Resistant T-cell–mediated Rejection

BackgroundAnti-thymocyte globulin (ATG) is a treatment option for steroid-resistant T-cell–mediated rejection after kidney transplantation. However, the extent to which immune-cell subsets can repopulate the peripheral blood is unknown. MethodsSix patients with steroid-resistant T-cell–mediated rejection were recruited and underwent analysis of their immune cells for 1 year after ATG administration. Multicolor flow cytometric analysis was used to evaluate the proportions of T cells, B cells, natural killer cells, and monocytes. ResultsT-cell repopulation from 24% to 75% occurred in the treatment group. The major repopulated cells were effector memory CD8+ T cells followed by effector memory CD4+ T cells. The population of effector memory CD8+ T cells with low expression of interleukin-7 receptor α increased over time. The population of regulatory T cells (eg, CD8+CD28-CD56+ T cells and CD4+CD25bright T cells) increased after ATG administration. However, the populations of other immune-cell subsets, including B cells, natural killer cells, and monocytes, were not significantly altered by ATG. ConclusionsOur findings on immune cell repopulation after ATG administration will enable future studies aiming to unravel the steroid-resistance mechanism underlying T-cell–mediated rejection.

JAK/STAT signaling controls the fate of CD8+CD103+ tissue-resident memory T cell in lupus nephritis

Autoimmune mediated inflammation and renal damage in lupus nephritis (LN) depends partly on the infiltration of lymphocytes in glomeruli and renal interstitium. Here we identified a population of CD8+ T cells with a CD103+-phenotype in the healthy kidneys of human and mouse. These cells were typically CD69+CD103+ tissue-resident memory T cells (TRM) in the kidney. CD8+ TRM cells were expanded in the kidneys of patients with LN or MRL/lpr mice. The expansion of renal CD8+ TRM cells correlated significantly with kidney disease activity. These cells were active in producing cytokines, perforin and granzyme B in the kidney of MRL/lpr mice. Importantly, renal CD8+ TRM cells underwent proliferation and self-renewal to maintain a stable TRM pool in the kidney of MRL/lpr mice, contributing to renal inflammation and damage. JAK/STAT signaling in the MRL/lpr mice was required for renal TRM self-renewal as well as maintenance of effector functions. Targeting JAK/STAT signaling by tofacitinib effectively suppressed effector functions and impaired the survival of renal TRM cells in the kidney, contributing to improved kidney function in MRL/lpr mice. These results provided evidences that renal CD8+ TRM cells play a role in the pathogenesis of LN. They could serve as a therapeutic target for LN.

T Cells Modified with CD70 as an Alternative Cellular Vaccine for Antitumor Immunity.

PurposeSuccessful tumor eradication primarily depends on generation and maintenance of a large population of tumor-reactive CD8 T cells. Dendritic cells (DCs) are well-known potent antigen-presenting cells and have applied to clinics as potent antitumor therapeutic agents. However, high cost and difficulty in obtaining sufficient amounts for clinical use are the crucial drawbacks of DC-based vaccines. Here, we aimed to develop T cell–based vaccine capable of eliciting potent antitumor therapeutic effects by providing effective costimulatory signals.Materials and MethodsAntigenic peptide-loaded T cells transfected with retrovirus encoding costimulatory ligands CD70, CD80, OX40L, or 4-1BBL were assessed for antigen-specific CD8 T-cell responses and evaluated antitumor effects along with immunization of a mixture of synthetic peptides, poly-IC and anti-CD40 antibodies (TriVax).ResultsT cells expressing CD70 (CD70-T) exhibited similar level of stimulatory functionality and therapeutic efficacy as DCs. Moreover, CD70-T prime followed by TriVax booster heterologous vaccination elicited therapeutic antitumor effect against B16 melanoma where mediated by CD8 T cells but not CD4 T cells or natural killer cells. The combination with programmed death-ligand 1 blockade led to potent therapeutic efficacy which exhibited increased tumor-infiltrating CD8 T cells. CD70-T pulsed with multi-antigenic peptide generated multiple antigen-specific polyvalent CD8 T cells that were capable of inhibiting tumor growth effectively. Moreover, CD70-T vaccination resulted in higher expansion and migration of adoptively transferred T cells into tumor sites and elicits enhanced therapeutic effects with peptide-based booster immu-nization.ConclusionThese results imply that T cells endowed with CD70 enable the design of effective vaccination strategies against solid cancer, which may overcome current limitations of DC-based vaccines.

From the Editor's Desk…

From the Editor's Desk…

A31 A Reservoir of Tumor-Specific CD8 T Cells in Lung Cancer Resides in the Draining Lymph Node

Recent work has described the population of CD8 T cells that respond to anti-PD-1 therapy (marked by TCF1 and PD-1), but it remains unclear how these T cells are maintained within the immunosuppressive tumor microenvironment (TME) of lung cancer. To understand this, we developed a genetically engineered model in which Kras-G12D expressing p53 deficient lung adenocarcinomas express a known neoantigen called the iNversion INduced Joined neoAntigen (NINJA). NINJA allows us to follow neoantigen-specific CD8 T cells over the course of tumor development. We find that ∼20% of tumor-specific T cells in early 8-wk tumors are TCF1+, but by 17-20 wks, this TCF1+ has significantly shrunk, and there has been a concomitant increase in the expression of markers of T-cell terminal differentiation (Tim3). This is consistent with the idea that T cells receive signals in the TME that drive terminal differentiation and restrict responses to immunotherapy. We reasoned that if the signals driving terminal differentiation were provided in the TME, neoantigen-specific T cells in the tumor-draining lymph node (dLN) may remain less differentiated over the course of tumor development. Analysis of tumor-specific T cells in the dLNs of 8-wk and 17-wk tumors showed that they were mostly TCF1+. Moreover, single-cell transcriptional analyses suggested that these cells were less differentiated than their counterparts in tumors. T-cell receptor (TCR) signals are a major driver of terminal differentiation, and we observed that tumor-specific T cells in the dLN were not receiving TCR signals, while all T cells in the TME were positive for TCR signals. This suggested at least two models for how antitumor T cells function: 1) tumor-reactive T cells in dLNs and TME could function independently of one another, or 2) tumor-reactive T cells might have a role in sustaining the antitumor T-cell response over the course of lung-tumor development through migration. Consistent with the latter model, TCR sequencing of dLN and TME neoantigen-specific T cells showed a close clonal relationship: 13 of the top 15 clones in the TME were present in the dLN, and the hierarchy of clonal dominance was maintained. This was also true in 17-wk tumor-bearing mice, suggesting that the population of tumor-specific CD8 T cells in the dLN serves as a reservoir of less differentiated cells that can continuously replenish the T cells in the TME, helping to sustain the antitumor T-cell response over the course of tumor development. Critically, it is unclear whether current immunotherapeutic strategies leverage this reservoir of T cells, raising the possibility that this population of dLN tumor-specific T cells could be targeted to provide significant additional benefit for patients receiving immunotherapy.

Functions of human liver CD69+CD103-CD8+ T cells depend on HIF-2α activity in healthy and pathologic livers

Human liver CD69+CD8+ T cells are ~95% CD103- and ~5% CD103+. Although CD69+CD103+CD8+ T cells show tissue residency and robustly respond to antigens, CD69+CD103-CD8+ T cells are not yet well understood. Liver perfusate and paired peripheral blood were collected from healthy living donors and recipients with cirrhosis during liver transplantation. Liver tissues were obtained from patients with acute hepatitis A. Phenotypic and functional analyses were performed by flow cytometry. Gene expression profiles were determined by microarray and quantitative reverse transcription PCR. PT-2385 was used to inhibit hypoxia-inducible factor (HIF)-2α. Human liver CD69+CD103-CD8+ T cells exhibited HIF-2α upregulation with a phenotype of tissue residency and terminal differentiation. CD103- cells comprised non-hepatotropic virus-specific T cells as well as hepatotropic virus-specific T cells, but CD103+ cells exhibited only hepatotropic virus specificity. Although CD103- cells were weaker effectors on a per cell basis than CD103+ cells, following T cell receptor or interleukin-15 stimulation, they remained the major CD69+CD8+ effector population in the liver, surviving with less cell death. An HIF-2α inhibitor suppressed the effector functions and survival of CD69+CD103-CD8+ T cells. In addition, HIF-2α expression in liver CD69+CD103-CD8+ T cells was significantly increased in patients with acute hepatitis A or cirrhosis. Liver CD69+CD103-CD8+ T cells are tissue resident and terminally differentiated, and their effector functions depend on HIF-2α. Furthermore, activation of liver CD69+CD103-CD8+ T cells with HIF-2α upregulation is observed during liver pathology. The immunologic characteristics and the role of CD69+CD103-CD8+ T cells, which are a major population of human liver CD8+ T cells, remain unknown. Our study shows that these Tcells have a terminally differentiated tissue-resident phenotype, and their effector functions depend on a transcription factor, HIF-2α. Furthermore, these T cells were activated and expressed higher levels of HIF-2α in liver pathologies, suggesting that they play an important role in immune responses in liver tissues and the pathogenesis of human liver disease.

Overexpression of CD6 and PD-1 Identifies Dysfunctional CD8+ T-Cells During Chronic SIV Infection of Rhesus Macaques.

Effective CD8+ T-cell responses play an important role in determining the course of SIV/HIV viral infection. Here we identified a unique population of dysfunctional CD8+ T-cells in lymphoid tissues and bronchoalveolar lavage (BAL) of rhesus macaques with chronic SIV infection characterized by co-expression of CD6 and PD-1. The frequency of CD6 and PD-1 co-expressing CD8+ T-cells was significantly increased in lymphoid tissues and BAL during chronic SIV infection compared to pre-infection levels. These CD6+PD-1+CD8+ T-cells displayed impaired proliferation, cytokine secretion and cytotoxicity compared to their CD6−PD-1+CD8+ T cell counterparts. The frequency of CD8+PD-1+ and CD8+CD6−PD-1+ T-cells in the lymph node and bone marrow did not correlate with SIV viral load, whereas the frequency of CD8+CD6+PD-1+ T-cells positively correlated with SIV viral load in these tissues highlighting the contribution of CD6 to disease progression. CD6+PD-1+CD8+ T-cells expressed elevated levels of SHP2 phosphatase compared to CD6−PD-1+CD8+ T-cells providing a potential mechanism by which CD6 may induce T-cell dysfunction during chronic SIV infection. Combined targeting of CD6 and PD-1 effectively revived the CD8+ T-cell proliferative response in vitro suggesting a strategy for potential therapeutic benefit.

Generation of highly activated, antigen-specific tumor-infiltrating CD8+ T cells induced by a novel T cell-targeted immunotherapy

ABSTRACT The induction of tumor-targeted, cytotoxic T lymphocytes has been recognized as a key component to successful immunotherapy. DPX-based treatment was previously shown to effectively recruit activated CD8+ T cells to the tumor. Herein, we analyze the unique phenotype of the CD8+ T cells recruited into the tumor in response to DPX-based therapy, and how combination with checkpoint inhibitors impacts T cell response. C3-tumor-bearing mice were treated with cyclophosphamide (CPA) for seven continuous days every other week, followed by DPX treatment along with anti-CTLA-4 and/or anti-PD-1. Efficacy, immunogenicity, and CD8+ T cells tumor infiltration were assessed. The expression of various markers, including checkpoint markers, peptide specificity, and proliferation and activation markers, was determined by flow cytometry. tSNE analysis of the flow data revealed a resident phenotype of CD8+ T cells (PD-1+TIM-3+CTLA-4+) within untreated tumors, whereas DPX/CPA treatment induced recruitment of a novel population of CD8+ T cells (PD-1+TIM-3+CTLA-4−) within tumors. Combination of anti-CTLA-4 (ipilimumab) with DPX/CPA versus DPX/CPA alone significantly increased survival and inhibition of tumor growth, without changing overall systemic immunogenicity. Addition of checkpoint inhibitors did not significantly change the phenotype of the newly recruited cells induced by DPX/CPA. Yet, anti-CTLA-4 treatment in combination with DPX/CPA enhanced a non-antigen specific response within the tumor. Finally, the tumor-recruited CD8+ T cells induced by DPX/CPA were highly activated, antigen-specific, and proliferative, while resident phenotype CD8+ T cells, seemingly initially exhausted, were reactivated with combination treatment. This study supports the potential of combining DPX/CPA with ipilimumab to further enhance survival clinically.