CD8 T-cell Exhaustion Research Articles

Abstract A076: T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status

Abstract Epithelial ovarian cancer is the most lethal gynecologic malignancy, with 5-year survival rates of 46%. High grade serous carcinoma (HGSC) is the most frequent subtype of epithelial ovarian cancer. Approximately 50% of cases are homologous recombination deficient (HRD). Whereas maintenance treatment by PARP inhibitors has significantly improved the prognosis of patients with HRD tumors, clinical outcome in this malignancy is still poor. Despite the immunogenic nature of HGSC, in which T-cell infiltration correlates to improved clinical outcome following surgery and chemotherapy, the disease exhibits low response rates to immune checkpoint blockade (ICB) monotherapy in advanced line settings. We have previously identified epithelial malignancies including HGSC, CD4 and CD8 populations of tumor-infiltrating and antigen-specific T cells that display features of terminal exhaustion. These populations were key players in the response to anti-PD-1 in patients with epithelial tumors. Here, we set out to characterize tumor-infiltrating T cells in a cohort of 80 HGSC patients treated at our institution from whom we collected tumor samples prior to therapy in which HRD status was assessed. We used multiplex immunofluorescence of FFPE tumor samples to assess infiltration by CD4 and CD8 T cells and multiparametric flow cytometry of frozen viable cell suspensions to assess CD4 and CD8 T-cell exhaustion. The link between biological parameters and clinical outcome was assessed by univariable and multivariable analyses. Assessment of the T-cell infiltrate by immunofluorescence showed no difference in the absolute numbers, per tumor or stroma mm2, of CD8 or CD4 T cells between HRD and homologous recombination proficient (HRP) tumors. Instead, analysis of exhausted CD8 and CD4 T cells showed an increased frequency of terminally exhausted PD-1HighTIM-3+ CD8 T cells in HRD tumors. This population, which expresses the ectonucleotidase CD39, was significantly correlated to the presence of T regulatory cells (Treg), which agreed with previous studies showing comigration of effector and regulatory T cells in ovarian cancer. In addition, terminally exhausted CD4 T cells, expressing the immune checkpoints PD-1 and TIM-3 as well as CD39, were enriched in HRD tumors. Among HRD tumors, no difference was observed between the exhaustion profile in tumors presenting deleterious BRCA1/2 mutations compared to those with other mutations in the homologous recombination pathway. Univariable analyses showed exhaustion parameters to be significant predictors of progression-free survival (PFS). The frequencies of exhausted CD4 and CD8 T cells stood out as predictors of PFS in multivariable analyses. Altogether, these results provide the first evidence of a link between T-cell exhaustion and HRD status and reveal T-cell exhaustion as an independent predictor of disease outcome. They also underline the major role played by the antitumor immune response in HGSC and warrant the development of combination immunotherapies despite the lack of response to ICB monotherapy. Citation Format: Anna Salvioni, Mathilde Del, Marie Michelas, Pierre Vuattoux, Clara-Maria Scarlata, Carlos Martinez-Gomez, Nathalie Van Acker, François-Xavier Frenois, Guillaume Bataillon, Jean-Pierre Delord, Alejandra Martinez, Maha Ayyoub. T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A076.

Identification of Proteome-Based Immune Subtypes of Early Hepatocellular Carcinoma and Analysis of Potential Metabolic Drivers

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide, ranking fourth in frequency. The relationship between metabolic reprogramming and immune infiltration has been identified as having a crucial impact on HCC progression. However, a deeper understanding of the interplay between the immune system and metabolism in the HCC microenvironment is required. In this study, we used a proteomic dataset to identify three immune subtypes (IM1-IM3) in HCC, each of which has distinctive clinical, immune, and metabolic characteristics. Among these subtypes, IM3 was found to have the poorest prognosis, with the highest levels of immune infiltration and T-cell exhaustion. Furthermore, IM3 showed elevated glycolysis and reduced bile acid metabolism, which was strongly correlated with CD8 T cell exhaustion and regulatory T cell accumulation. Our study presents the proteomic immune stratification of HCC, revealing the possible link between immune cells and reprogramming of HCC glycolysis and bile acid metabolism, which may be a viable therapeutic strategy to improve HCC immunotherapy.

Investigating the Role of MK2 in Head and Neck Squamous Cell Carcinoma Growth, Metastasis and STING Pathway Activation

Our prior work demonstrated that inhibition of MAPKAPK2 (MK2) can enhance radiation (RT)-mediated in vivo head and neck squamous cell carcinoma (HNSCC) tumor control and survival in preclinical immune incompetent models. The cytosolic DNA sensor cyclic GMP-AMP synthetase (cGAS) and its downstream adaptor protein, stimulator of interferon genes (STING), are conserved proteins within the innate immune signaling pathways and are important for mediating host defense against microbial infection and can play a role in anti-cancer immunity. We hypothesized that loss of MK2 enhances radiation-induced cGAS-STING pathway activation leading to improved tumor control and survival. MK2 shRNA knockdown human (Tu167, CAL27) and MK2 Cas9/CRISPR knockout (KO) syngeneic murine (Ly2, MLM3) HNSCC cell lines were treated with 10 Gy irradiation. Micronuclei were quantitated by DAPI-immunofluorescence (IF). Protein changes in cGAS-STING were evaluated by immunoblot. Inflammatory cytokine production including Type I IFNβ1 were evaluated by RT-qPCR. Ly2 and MLM3 cells were orthotopically or flank engrafted into immune competent mice (Balb/c, C57Bl/6, respectively) for animal tumor control-survival studies. Tumor immune cell infiltrate was examined using FACS and immunohistochemistry. Selected drug studies using the MK2 inhibitor, ATI-450, were performed with RT. Loss of MK2 in HNSCC (Tu167, CAL27, Ly2, MLM3) treated with RT led to a significant increase in micronuclei formation compared to control cells. MK2-enhanced micronuclei generation following RT could be inhibited with the actin filament polymerization inhibitor, cytochalasin B. RT treatment of MK2 shRNA cells led to increased cGAS and phospho-STING levels compared to either treatment alone. IFNβ1 levels were significantly higher in HNSCC cell lines treated with RT and with MK2 inhibited by an MK2 inhibitor (ATI-450) or genetic reduction compared to either treatment alone. In-vivo implantation of MLM3 cells into C57Bl/6 comparing control vs MK2 KO tumors treated with ±8 Gy RT demonstrated improved mouse survival favoring RT+MK2 KO over RT, MK2 KO or parental (63, 58, 58.5, 35 days, respectively). FACS analysis of MLM3 WT v KO tumors 3 days post RT showed an overall increase in the number of CD3/CD8 T-cells infiltrating into the tumor in all groups except for parental tumors. Further analysis demonstrated that loss of MK2 reversed CD8 T-cell exhaustion and when combined with radiotherapy led to increased CD8 T-cell activation. Furthermore, activated CD4 and CD8 T cells were reduced in WT+RT cells compared to WT tumors whereas no reduction was seen in the KO or KO+RT. HNSCC tumor MK2 inhibition enhances RT-mediated micronuclei formation and subsequent cGAS-STING-IFNβ1 levels. Loss of HNSCC MK2 leads to increased CD4-CD8 T-cell infiltration into the tumor and this effect is enhanced following RT. Targeting tumor MK2 may facilitate improved tumor control.

Field Cancerization Is Associated with Tumor Development, T-cell Exhaustion, and Clinical Outcomes in Bladder Cancer

BackgroundField cancerization is characterized by areas of normal tissue affected by mutated clones. Bladder field cancerization may explain the development and recurrence of bladder cancer and may be associated with treatment outcomes. ObjectiveTo investigate the predictive and prognostic roles of field cancerization in patients with high-risk non–muscle-invasive bladder cancer (NMIBC) treated with bacillus Calmette-Guérin (BCG). Design, setting, and participantsWe conducted comprehensive genomic and proteomic analyses for 751 bladder biopsies and 234 urine samples from 136 patients with NMIBC. The samples were collected at multiple time points during the disease course. Field cancerization in normal-appearing bladder biopsies was measured using deep-targeted sequencing and error correction models. Outcome measurements and statistical analysisEndpoints included the rates of recurrence and progression. Cox regression and Wilcoxon rank-sum and Fisher’s exact tests were used. Results and limitationsA high level of field cancerization was associated with high tumor mutational burden (p = 0.007), high tumor neoantigen load (p = 0.029), and high tumor-associated CD8 T-cell exhaustion (p = 0.017). In addition, high field cancerization was associated with worse short-term outcomes (p = 0.029). Nonsynonymous mutations in bladder cancer–associated genes such as KDM6A, ARID1A, and TP53 were identified as early disease drivers already found in normal-appearing bladder biopsies. Urinary tumor DNA (utDNA) levels reflected the bladder tumor burden and originated from tumors and field cancerization. High levels of utDNA after BCG were associated with worse clinical outcomes (p = 0.027) and with disease progression (p = 0.003). High field cancerization resulted in high urinary levels of proteins associated with angiogenesis and proliferation. Limitations include variation in the number of biopsies and time points analyzed. ConclusionsField cancerization levels are associated with tumor development, immune responses, and clinical outcomes. utDNA measurements can be used to monitor disease status and treatment response. Patient summaryMolecular changes in the tissue lining the bladder result in tumor recurrence. Urinary measurements may be used to monitor bladder cancer status and treatment responses.

Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) orchestrates a suppressive tumor microenvironment that fosters immunotherapy resistance. Tumor-associated macrophages (TAMs) are the principal immune cell infiltrating PDA and are heterogeneous. Here, by employing macrophage fate-mapping approaches and single-cell RNA sequencing, we show that monocytes give rise to most macrophage subsets in PDA. Tumor-specific CD4, but not CD8, Tcells promote monocyte differentiation into MHCIIhi anti-tumor macrophages. By conditional major histocompatibility complex (MHC) class II deletion on monocyte-derived macrophages, we show that tumor antigen presentation is required for instructing monocyte differentiation into anti-tumor macrophages, promoting Th1 cells, abrogating Treg cells, and mitigating CD8 Tcell exhaustion. Non-redundant IFNγ and CD40 promote MHCIIhi anti-tumor macrophages. Intratumoral monocytes adopt a pro-tumor fate indistinguishable from that of tissue-resident macrophages following loss of macrophage MHC class II or tumor-specific CD4 Tcells. Thus, tumor antigen presentation by macrophages to CD4 Tcells dictates TAM fate and is a major determinant of macrophage heterogeneity in cancer.

The development of CD8 T-cell exhaustion heterogeneity and the therapeutic potentials in cancer.

CD8+ T cells are essential lymphocytes with cytotoxic properties for antitumor immunotherapy. However, during chronic infection or tumorigenesis, these cells often become dysfunctional with a gradually depleted ability to release cytokines and the exhibition of reduced cytotoxicity, the state referred to as "T-cell exhaustion" (Tex). This unique state was characterized by the increasing expression of inhibitory checkpoint receptors, and interventions targeting immune checkpoint blockades (ICBs) have been considered as a promising strategy to stimulate T-cell killing. Recent investigations have demonstrated that exhausted T cells not only display functional, metabolic, transcriptional, and epigenetic differences but also comprise a heterogeneous group of cells. In this review, we summarize the current findings on dynamic differentiation process during Tex heterogeneity development in cancer and chronic infection. We discuss how the responses to immunotherapy are determined by these distinct subsets and highlight prospective approaches for improving the efficacy of ICB therapy for cancer by leveraging the heterogeneity of T cells.

ITK inhibition improves the response to immune checkpoint blockade in solid tumors

Abstract Cytotoxic CD8+ T cell (CTL) exhaustion is driven by persistent T cell receptor (TCR) stimulation in chronic infections and tumors. The exhausted state of T cells is characterized by impaired effector function and proliferative capacity, upregulation of multiple inhibitory receptors and altered transcriptional profiles. The phenotypic and epigenetic changes of these cells hinder effective antitumor immunity and immunotherapy. Therefore, reinvigorating exhausted T cells may provide significant clinical benefits to patients with cancer. Our approach is to reduce chronic TCR stimulation by inhibiting IL2-inducible T-cell kinase (ITK), a pivotal component of TCR signaling. In our study, we found that intermittent ITK inhibitor treatment significantly improved ICB response in three immune checkpoint blockade (ICB) -resistant solid tumors (melanoma, mesothelioma or pancreatic cancer) in vivo. In vitro, we demonstrated that specific ITK inhibitor and ibrutinib, an inhibitor of Bruton’s tyrosine kinase (BTK) and ITK, directly reversed the exhaustion-related phenotypes in exhausted CTLs. The changes included enhanced cytokine production, decreased inhibitory receptor expression and changes in transcription factors. Subsequently, RNA-seq data revealed that exhaustion-related transcriptional profile of in vitro CTLs is reduced by ibrutinib treatment. Ibrutinib mediated reversal of CTL exhaustion was BTK independent. To conclude, our study demonstrates that ITK inhibition can be used to directly ameliorate CTLs exhaustion and overcomes immune checkpoint blockade resistance in solid tumors. This work was supported by the department of Immunology, KWF Grant 128371 (PDK), in part from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779295 (PDK), and by the China Scholarship Council for funding PhD fellowships (No. 201506160120 for MZ and 201906210055 for LL).

Abstract 3340: Bladder field cancerization impacts tumor development and T-cell exhaustion, and is reflected in urinary tumor DNA

Abstract Introduction: Patients with high-risk non-muscle invasive bladder cancer (NMIBC) are treated with Bacillus Calmette-Guérin (BCG), which targets altered normal-appearing urothelium (field cancerization). Field cancerization may affect treatment response and outcome. High urinary tumor DNA (utDNA) levels in patients with NMIBC have been associated with worse clinical outcomes, and utDNA may be used for real-time assessment of residual disease. The prognostic and predictive values of field cancerization and utDNA need further investigation. Materials and methods: We analyzed samples procured from 134 patients with NMIBC - including 210 tumors, 751 selected site biopsies (SSBs) and 187 urine samples. The median follow-up time was 7.8 years. Samples were collected before and after BCG-treatment. Sixty-four patients (48%) progressed to muscle-invasive bladder cancer or experienced high-grade NMIBC recurrence within two years after BCG treatment, indicating BCG-failure. Tumor samples were analyzed using whole-exome and RNA-sequencing. DNA from SSBs and urine samples was subjected to tumor-informed deep-targeted sequencing. SSB DNA was sequenced to a mean coverage of 1,357X, and urinary DNA to a mean coverage of 2,153X. Unique molecular identifiers were applied and the deepSNV pipeline was used for error-robust mutation calling. The level of field cancerization in each patient was estimated by the highest number of called mutations adjusted by the number of mutations on the sequencing panel. Results: We detected mutations in 458 out of 751 analyzed SSBs. Mutations in normal-appearing SSBs were detected at a mean frequency of 0.04 (range: 0.002-0.39). The level of field cancerization increased with age (p=0.0027). utDNA levels reflected the bladder disease status, increasing with higher tumor stage and multiplicity and was shed from both bladder field cancerization and tumors. High utDNA levels after BCG-treatment were associated with high recurrence rates (p<0.001), high risk of progression (p=0.0014) and worse high-grade recurrence-free survival (p=0.047). Field cancerization level was not associated with response to BCG treatment. High levels of field cancerization and utDNA were associated with high tumor-associated CD8 T-cell exhaustion. Additionally, a high field cancerization level correlated with a high tumor neoantigen load (p=0.029), indicating a possible link to the observed CD8 T-cell exhaustion. Conclusion: A high level of field cancerization was associated with an immune exhausted phenotype of the tumor. Measurements of utDNA reflected the overall disease status of the bladder, including field cancerization. Collectively, these results suggest that field cancerization may impact the tumor microenvironment and utDNA levels may be used for assessing clinical risk in NMIBC and for guiding surveillance and early treatment interventions. Citation Format: Trine Strandgaard, Iver Nordentoft, Karin Birkenkamp-Demtröder, Liina Salminen, Frederik Prip, Emil Christensen, Philippe Lamy, Tine Ginnerup Andreasen, Sia Viborg Lindskrog, Michael Knudsen, Julie Rasmussen, Torben Steiniche, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Bladder field cancerization impacts tumor development and T-cell exhaustion, and is reflected in urinary tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3340.

THE PRECIOUS STUDY: A PROSPECTIVE, OBSERVATIONAL STUDY OF CD8+ BIOMARKER BASED PROGNOSTIC TEST IN NORTH AMERICAN IBD PATIENTS

The clinical course of IBD varies considerably among patients1. One barrier to personalized medicine in IBD is the lack of a reliable prognostic assay that can be used at diagnosis to guide effective therapy for each patient, to optimize clinical outcomes and minimize complications. To address this need, a whole-blood qPCR-based prognostic assay was developed to divide IBD patients into two subgroups2. This transcriptional signature yields CD8 T-cell exhaustion level data that correlated with disease outcomes among IBD patients in UK2. Here, we aimed to determine if this prognostic assay could stratify an ethnically diverse North American cohort of IBD patients at diagnosis into low and high-risk subgroups.

THE PRECIOUS STUDY: A PROSPECTIVE, OBSERVATIONAL STUDY OF CD8+ BIOMARKER BASED PROGNOSTIC TEST IN NORTH AMERICAN IBD PATIENTS

Abstract BACKGROUND The clinical course of IBD varies considerably among patients1. One barrier to personalized medicine in IBD is the lack of a reliable prognostic assay that can be used at diagnosis to guide effective therapy for each patient, to optimize clinical outcomes and minimize complications. To address this need, a whole-blood qPCR-based prognostic assay was developed to divide IBD patients into two subgroups2. This transcriptional signature yields CD8 T-cell exhaustion level data that correlated with disease outcomes among IBD patients in UK2. Here, we aimed to determine if this prognostic assay could stratify an ethnically diverse North American cohort of IBD patients at diagnosis into low and high-risk subgroups. METHODS PRECIOUS (Predicting Crohn’s & Colitis Outcomes in the United States) is a multi-centre, observational, double-blinded study of patients with active IBD, who are not receiving systemic steroids, immunomodulator, advanced small molecules or biologics. The study is recruiting up to 200 patients between 16-80 years of age, from 14 centres across the US and Canada. Patients are prospectively followed for >12 months after enrolment to monitor disease course, including the requirement for IBD-related surgery or treatment escalations. Patients and clinicians are blinded to the biomarker results and patients are treated according to local practice. RESULTS Of 120 patients recruited to date, 86 have reached the 12-month follow-up timepoint (Table 1). Of the 102 patients with complete race and ethnicity data (Table 1), 63.5% are non-Hispanic Whites, 13.7% identified as Hispanic/Latino, 11.8% Asian and 9.8% Black/African American. Biomarker results were obtained for 80 (88.8%) of the 90 samples tested to date – 10 (11.1%) of samples failed quality control. Of these 80 results, 38 patients (47.5%) were identified as high risk for disease progression and 42 (52.5%) as low risk – a similar breakdown to that seen in the UK cohort2. DISCUSSION This is the first trial of a prognostic biomarker for patients with CD and UC in North America. This patient population was significantly more diverse than the UK cohort which had ~98% White. This diversity is not generally reflected in clinical trials performed in the United States in which approximately 90% of participants for all conditions are White whereas non-Hispanic Whites represent approximately 60% of the U.S. population. 4,5. Our enrolment of only 63.5% non-Hispanic White subjects speaks to our targeted efforts to recruit a representative level of diversity into our study population to that represents the diversity of patients with IBD that are being diagnosed globally6.

Impact of CD4 T cells on intratumoral CD8 T-cell exhaustion and responsiveness to PD-1 blockade therapy in mouse brain tumors

BackgroundGlioblastoma is a fatal disease despite aggressive multimodal therapy. PD-1 blockade, a therapy that reinvigorates hypofunctional exhausted CD8 T cells (Tex) in many malignancies, has not shown efficacy in glioblastoma....

SARS-CoV2 Infection During Pregnancy Causes Persistent Immune Abnormalities in Women Without Affecting the Newborns.

SARS-CoV2 infection in pregnancy and exposed newborns is poorly known. We performed a longitudinal analysis of immune system and determined soluble cytokine levels in pregnant women infected with SARS-CoV2 and in their newborns. Women with confirmed SARS-CoV2 infection and their exposed uninfected newborns were recruited from Hospital General Universitario Gregorio Marañón. Peripheral blood mononuclear cells (PBMCs), cord cells and plasma were collected at birth and 6 months later. Immunophenotyping of natural killer (NK), monocytes and CD4/CD8 T-cells were studied in cryopreserved PBMCs and cord cells by multiparametric flow cytometry. Up to 4 soluble pro/anti-inflammatory cytokines were assessed in plasma/cord plasma by ELISA assay. SARS-CoV2-infected mothers and their newborns were compared to matched healthy non-SARS-CoV2-infected mothers and their newborns. The TNFα and IL-10 levels of infected mothers were higher at baseline than those of healthy controls. Infected mothers showed increased NK cells activation and reduced expression of maturation markers that reverted after 6 months. They also had high levels of Central Memory and low Effector Memory CD4-T cell subsets. Additionally, the increased CD4- and CD8-T cell activation (CD154 and CD38) and exhaustion (TIM3/TIGIT) levels at baseline compared to controls remained elevated after 6 months. Regarding Treg cells, the levels were lower at infected mothers at baseline but reverted after 6 months. No newborn was infected at birth. The lower levels of monocytes, NK and CD4-T cells observed at SARS-CoV2-exposed newborns compared to unexposed controls significantly increased 6 months later. In conclusion, SARS-CoV2 infection during pregnancy shows differences in immunological components that could lead newborns to future clinical implications after birth. However, SARS-CoV2 exposed 6-months-old newborns showed no immune misbalance, whereas the infected mothers maintain increased activation and exhaustion levels in T-cells after 6 months.

Selected Articles from This Issue

Interleukin-15 (IL-15) regulates the generation, persistence, and differentiation of natural killer (NK) and CD8 T cells. To expand its role in cancer immunotherapy, Chen and colleagues designed a multitumor model treated with the combination of intratumoral anti-CD40, parenteral IL-15 and anti-PD-1. Anti-CD40 given intratumorally not only reduced dosing requirements and toxicity, but it also stimulated the antitumor activity of the distal tumors, suggestive of an abscopal effect. IL-15 with intratumoral anti-CD40 was additive/synergistic, and the inclusion of the anti-PD-1 was additive to the combination. Thus, this treatment shows promise for patients with solid tumors or resistance to anti-PD-1 therapy.Cancer-associated fibroblasts (CAF) are a heterogenous cell population with a complex immunoregulatory role, which has not been well defined in head and neck squamous cell carcinoma (HNSCC). Leveraging protein activity inference tools to analyze scRNA-sequencing of HNSCC patients, pre- and post-nivolumab, Obradovic and colleagues identified distinct subpopulations of head and neck CAF (HNCAF). Enrichment of a T-cell stimulating population (tsCAF) was found to associate with treatment response and ex vivo assays confirmed this CAF subset reduces CD8 T-cell exhaustion. Analysis of an independent HNSCC cohort treated with pembrolizumab validated this tsCAF subset as a potential biomarker of response.Approximately 30% of MSI-H/dMMR tumors do not respond to immune checkpoint inhibitors (ICI), highlighting the importance of identifying predictive biomarkers or molecular profiling correlated with resistance to these agents. Here, Chida and colleagues performed transcriptomic profiling of MSI-H/dMMR gastrointestinal tumors treated with PD-1 blockade. The study revealed that nonresponders had the enrichment of epithelial mesenchymal transition, angiogenesis, hypoxia, mTORC1, KRAS, Wnt/β-catenin, TGF-β, and various metabolism-related signaling pathways, which have been associated with an immunosuppressive tumor microenvironment. Meanwhile, the IFN-γ pathway was upregulated in responders. High expression of VEGF-A might predict a negative response to ICI in this population.Acral melanoma is a rare subtype of melanoma that arises on non-sun exposed skin of the hands, feet, and nailbeds. Patients with acral melanoma have lower response rates to immunotherapy than those with nonacral cutaneous melanoma, suggesting unique microenvironmental features. In their study, Li and colleagues performed comprehensive single cell analysis of a cohort of acral melanoma specimens and found them to harbor fewer effector CD8 T cells and NK cells and a complete absence of γδ T cells compared to non-acral cutaneous melanoma. VISTA, TIGIT, and adenosine signaling were identified as targetable immune checkpoints in acral melanoma.

High CD169 Monocyte/Lymphocyte Ratio Reflects Immunophenotype Disruption and Oxygen Need in COVID-19 Patients.

Background: Sialoadhesin (CD169) has been found to be overexpressed in the blood of COVID-19 patients and identified as a biomarker in early disease. We analyzed CD169 in the blood cells of COVID-19 patients to assess its role as a predictive marker of disease progression and clinical outcomes. Methods: The ratio of the median fluorescence intensity of CD169 between monocytes and lymphocytes (CD169 RMFI) was analyzed by flow cytometry in blood samples of COVID-19 patients (COV) and healthy donors (HDs) and correlated with immunophenotyping, inflammatory markers, cytokine mRNA expression, pulmonary involvement, and disease progression. Results: CD169 RMFI was high in COV but not in HDs, and it correlated with CD8 T-cell senescence and exhaustion markers, as well as with B-cell maturation and differentiation in COV. CD169 RMFI correlated with blood cytokine mRNA levels, inflammatory markers, and pneumonia severity in patients who were untreated at sampling, and was associated with the respiratory outcome throughout hospitalization. Finally, we also report the first evidence of the specific ability of the spike protein of SARS-CoV-2 to trigger CD169 RMFI in a dose-dependent manner in parallel with IL-6 and IL-10 gene transcription in HD PBMCs stimulated in vitro. Conclusion: CD169 is induced by the spike protein and should be considered as an early biomarker for evaluating immune dysfunction and respiratory outcomes in COVID-19 patients.

Inverted CD8 T-Cell Exhaustion and Co-Stimulation Marker Balance Differentiate Aviremic HIV-2-Infected From Seronegative Individuals.

HIV-2 is less pathogenic compared to HIV-1. Still, disease progression may develop in aviremic HIV-2 infection, but the driving forces and mechanisms behind such development are unclear. Here, we aimed to reveal the immunophenotypic pattern associated with CD8 T-cell pathology in HIV-2 infection, in relation to viremia and markers of disease progression. The relationships between pathological differences of the CD8 T-cell memory population and viremia were analyzed in blood samples obtained from an occupational cohort in Guinea-Bissau, including HIV-2 viremic and aviremic individuals. For comparison, samples from HIV-1- or dually HIV-1/2-infected and seronegative individuals were obtained from the same cohort. CD8 T-cell exhaustion was evaluated by the combined expression patterns of activation, stimulatory and inhibitory immune checkpoint markers analyzed using multicolor flow cytometry and advanced bioinformatics. Unsupervised multidimensional clustering analysis identified a cluster of late differentiated CD8 T-cells expressing activation (CD38+, HLA-DRint/high), co-stimulatory (CD226+/-), and immune inhibitory (2B4+, PD-1high, TIGIThigh) markers that distinguished aviremic from viremic HIV-2, and treated from untreated HIV-1-infected individuals. This CD8 T-cell population displayed close correlations to CD4%, viremia, and plasma levels of IP-10, sCD14 and beta-2 microglobulin in HIV-2 infection. Detailed analysis revealed that aviremic HIV-2-infected individuals had higher frequencies of exhausted TIGIT+ CD8 T-cell populations lacking CD226, while reduced percentage of stimulation-receptive TIGIT-CD226+ CD8 T-cells, compared to seronegative individuals. Our results suggest that HIV-2 infection, independent of viremia, skews CD8 T-cells towards exhaustion and reduced co-stimulation readiness. Further knowledge on CD8 T-cell phenotypes might provide help in therapy monitoring and identification of immunotherapy targets.

CD4 Tcell help prevents CD8 Tcell exhaustion and promotes control of Mycobacterium tuberculosis infection.

SUMMARYCD4 T cells are essential for immunity to tuberculosis because they produce cytokines, including interferon-γ. Whether CD4 T cells act as “helper” cells to promote optimal CD8 T cell responses during Mycobacterium tuberculosis is unknown. Using two independent models, we show that CD4 T cell help enhances CD8 effector functions and prevents CD8 T cell exhaustion. We demonstrate synergy between CD4 and CD8 T cells in promoting the survival of infected mice. Purified helped, but not helpless, CD8 T cells efficiently restrict intracellular bacterial growth in vitro. Thus, CD4 T cell help plays an essential role in generating protective CD8 T cell responses against M. tuberculosis infection in vitro and in vivo. We infer vaccines that elicit both CD4 and CD8 T cells are more likely to be successful than vaccines that elicit only CD4 or CD8 T cells.

CD4 T-Cell Exhaustion: Does It Exist and What Are Its Roles in Cancer?

In chronic infections and in cancer, persistent antigen stimulation under suboptimal conditions can lead to the induction of T-cell exhaustion. Exhausted T cells are characterized by an increased expression of inhibitory markers and a progressive and hierarchical loss of function. Although cancer-induced exhaustion in CD8 T cells has been well-characterized and identified as a therapeutic target (i.e., via checkpoint inhibition), in-depth analyses of exhaustion in other immune cell types, including CD4 T cells, is wanting. While perhaps attributable to the contextual discovery of exhaustion amidst chronic viral infection, the lack of thorough inquiry into CD4 T-cell exhaustion is particularly surprising given their important role in orchestrating immune responses through T-helper and direct cytotoxic functions. Current work suggests that CD4 T-cell exhaustion may indeed be prevalent, and as CD4 T cells have been implicated in various disease pathologies, such exhaustion is likely to be clinically relevant. Defining phenotypic exhaustion in the various CD4 T-cell subsets and how it influences immune responses and disease severity will be crucial to understanding collective immune dysfunction in a variety of pathologies. In this review, we will discuss mechanistic and clinical evidence for CD4 T-cell exhaustion in cancer. Further insight into the derivation and manifestation of exhaustive processes in CD4 T cells could reveal novel therapeutic targets to abrogate CD4 T-cell exhaustion in cancer and induce a robust antitumor immune response.

Exploring Markers of Exhausted CD8 T Cells to Predict Response to Immune Checkpoint Inhibitor Therapy for Hepatocellular Carcinoma

Background: Reversal of CD8 T-cell exhaustion was considered a major antitumor mechanism of anti-programmed cell death-1 (PD-1)/ anti-programmed death ligand-1 (PD-L1)-based immune checkpoint inhibitor (ICI) therapy. Objectives: The aim of this study was to identify markers of T-cell exhaustion that is best associated with ICI treatment efficacy for advanced hepatocellular carcinoma (HCC). Methods: Immune cell composition of archival tumor samples was analyzed by transcriptomic analysis and multiplex immunofluorescence staining. Results: HCC patients with objective response after anti-PD-1/anti-PD-L1-based ICI therapy (n = 42) had higher expression of genes related to T-cell exhaustion. A 9-gene signature (LAG3, CD244, CCL5, CXCL9, CXCL13, MSR1, CSF3R, CYBB, and KLRK1) was defined, whose expression was higher in patients with response to ICI therapy, correlated with density of CD8⁺LAG3⁺ cells in tumor microenvironment, and independently predicted better progression-free and overall survival. This 9-gene signature had similar predictive values for patients who received single-agent or combination ICI therapy and was not associated with prognosis in HCC patients who received surgery, suggesting that it may outperform other T-cell signatures for predicting efficacy of ICI therapy for HCC. For HCC patients who underwent surgery for both the primary liver and metastatic lung tumors (n = 31), lung metastatic HCC was associated with a higher exhausted CD8 T-cell signature, consistent with prior observation that patients with lung metastatic HCC may have higher probability of response to ICI therapy. Conclusions: CD8 T-cell exhaustion in tumor microenvironment may predict better efficacy of ICI therapy for HCC.

Profiling of immune dysfunction in COVID-19 patients allows early prediction of disease progression.

With a rising incidence of COVID-19-associated morbidity and mortality worldwide, it is critical to elucidate the innate and adaptive immune responses that drive disease severity. We performed longitudinal immune profiling of peripheral blood mononuclear cells from 45 patients and healthy donors. We observed a dynamic immune landscape of innate and adaptive immune cells in disease progression and absolute changes of lymphocyte and myeloid cells in severe versus mild cases or healthy controls. Intubation and death were coupled with selected natural killer cell KIR receptor usage and IgM+ B cells and associated with profound CD4 and CD8 T-cell exhaustion. Pseudo-temporal reconstruction of the hierarchy of disease progression revealed dynamic time changes in the global population recapitulating individual patients and the development of an eight-marker classifier of disease severity. Estimating the effect of clinical progression on the immune response and early assessment of disease progression risks may allow implementation of tailored therapies.

Organized immune cell interactions within tumors sustain a productive T-cell response.

Tumor-infiltrating CD8 T cells are associated with improved patient survival and response to immunotherapy in various cancers. Persistent antigen leads to CD8 T-cell exhaustion, where proliferation/self-renewal and killing are divided within distinct subsets of CD8 T cells in the tumor. CD8 T-cell responses in chronic antigen settings must be maintained for long periods of time, suggesting that mechanisms that regulate chronic CD8 T-cell responses may differ from those in acute settings. Currently, factors that regulate the maintenance of stem-like CD8 T cells in the tumor or their differentiation into terminally differentiated cells are unknown. In this review, we discuss the role of dendritic cells in the activation and differentiation of CD8 T-cell subsets within secondary lymphoid tissue and tumors. In addition, we examine changes in CD4 T-cell differentiation in response to chronic antigens and consider how subset-specific mechanisms could assist the stem-like and terminally differentiated CD8 T-cell subsets. Finally, we highlight how tumor-infiltrating CD4 T cells and dendritic cells interact with CD8 T cells within organized lymphoid-like areas in the tumor and propose a CD8 T-cell differentiation model that requires the collaboration of CD4 T cells and dendritic cells. These organized interactions coordinate the anti-tumor response and control disease progression by mechanisms that regulate CD8 T-cell differentiation, which permit the maintenance of an effective balance of stem-like and terminally differentiated CD8 T cells.