CD4 T-cell Subsets Research Articles

Deep immune profiling of whole blood to identify early immune signatures that correlate to patient outcome after major trauma.

Major injury results in an early cascade of immunologic responses that increase susceptibility to infection and multiorgan dysfunction. Detailed immune profiling by mass cytometry has the potential to identify immune signatures that correspond to patient outcomes. Our objective was to determine the prognostic value of immune signatures early after major trauma injury. Trauma patients (n = 17) were prospectively enrolled between September 2018 and December 2019. Serial whole blood samples were obtained from trauma patients (mean Injury Severity Score, 26.2; standard error of the mean, 3.7) at Days 1 and 3 after injury, and from age- and sex-matched uninjured controls using a standardized protocol for fixation, storage, and labeling. Computational analyses including K-nearest neighbor automated clustering of immune cells and Spearman's correlation analysis were used to identify correlations between cell populations, clinical measures, and patient outcomes. Analysis revealed nine immune cell clusters that correlated with one or more clinical outcomes. On Days 1 and 3 postinjury, the abundance of immature neutrophil and classical monocytes exhibited a strong positive correlation with increased intensive care unit and hospital length of stay. Conversely, the abundance of CD4 T-cell subsets, namely Th17 cells, is associated with improved patient outcomes including decreased ventilator days (r = -0.76), hospital-acquired pneumonia (r = -0.69), and acute kidney injury (r = -0.73). Here, we provide a comprehensive multitime point immunophenotyping analysis of whole blood from patients soon after traumatic injury to determine immune correlates of adverse outcomes. Our findings indicate that alterations in myeloid-origin cell types may contribute to immune dysfunction after injury. Conversely, the presence of effector T cell populations corresponds with decreased hospital length of stay and organ dysfunction. Overall, these data identify novel immune signatures following traumatic injury that support the view that monitoring of immune (sub)-populations may provide clinical decision-making support for at-risk patients early in their hospital course. Prognostic/Epidemiologic, Level IV.

Dysregulated Host Response in Severe Acute Respiratory Syndrome Coronavirus 2-Induced Critical Illness.

BackgroundImpaired immune response has been reported to be the cause of the development of coronavirus disease 2019 (COVID-19)-related respiratory failure. Further studies are needed to understand the immunopathogenesis and to enable an improved stratification of patients who are at risk for critical illness.MethodsThirty-two severely ill patients hospitalized with COVID-19 were recruited in our center at the University Hospital Heidelberg. We performed a comprehensive analysis of immune phenotype, cytokine, and chemokine profiling and leukocyte transcripts in patients with severe COVID-19 and compared critically ill patients who required mechanical ventilation and high-flow oxygen therapy and noncritically ill patient who received low-flow oxygen therapy.ResultsCritically ill patients exhibited low levels of CD8 T cells and myeloid dendritic cells. We noted a pronounced CCR6+ TH17 phenotype in CD4 central memory cells and elevated circulating levels of interleukin-17 in the critical group. Gene expression of leukocytes derived from critically ill patients was characterized by an upregulation of proinflammatory cytokines and reduction of interferon (IFN)-responsive genes upon stimulation with Toll-like receptor 7/8 agonist. When correlating clinical improvement and immune kinetics, we found that CD8 T-cell subsets and myeloid dendritic cells significantly increased after disconnection from the ventilator.ConclusionCritical illness was characterized by a TH17-mediated response and dysfunctional IFN-associated response, indicating an impaired capacity to mount antiviral responses during severe acute respiratory syndrome coronavirus 2 severe infection.

Human Germinal Center Reaction: Assessing T Follicular Helper-B Cells Interaction In Vitro.

Antibody responses deeply rely on the interaction of antigen-primed B cells and CD4 helper T cells in the context of germinal center reactions, through signals provided by costimulatory molecules and cytokines. B-cell proliferation and differentiation in antibody-secreting plasma cells are processes that critically depend on the helper function of a specific CD4 T-cell subset, known as follicular helper T cells (Tfh). Here, we describe a method that mimics in vitro the cross talk between Tfh and B cells occurring in the germinal center. The procedure is based on setting up a coculture system with B cells and Tfh isolated from blood of healthy donors, or tonsils removed upon surgical intervention, in order to recapitulate in vitro the Tfh-dependent mechanisms leading to B cells' activation, proliferation, and differentiation.

Extensive longitudinal immune profiling reveals sustained innate immune activation in COVID-19 patients with unfavorable outcome

COVID-19 differs substantially between individuals, ranging from mild to severe or even fatal. Heterogeneity in the immune response against SARS-COV-2 likely contributes to this. Therefore, we explored the temporal dynamics of key cellular and soluble mediators of innate and adaptive immune activation in relation to COVID-19 severity and progression.Forty-four patients with a PCR-proven diagnosis of COVID-19 were included. Extensive cellular (leukocytes and T-lymphocyte subsets) and serological immune profiling (cytokines, soluble cell surface molecules, and SARS-CoV-2 antibodies) was performed at hospital admission and every 3–4 days during hospitalization. Measurements and disease outcome were compared between patients with an unfavorable (IC admission and/or death) and favorable (all others) outcome.Patients with an unfavorable outcome had higher leukocyte numbers at baseline, mostly due to increased neutrophils, whereas lymphocyte and monocyte numbers were reduced. CRP, IL-6, CCL2, CXCL10, and GM-CSF levels were higher at baseline in the unfavorable group, whereas IL-7 levels were lower. SARS-CoV-2 antibodies were more frequently absent in the unfavorable group. Longitudinal analysis revealed delayed kinetics of activated CD4 and CD8 T-lymphocyte subsets in the unfavorable group. Furthermore, whereas CRP, IL-6, CXCL10, and GM-CSF declined in the favorable group, these cytokines declined with delayed kinetics, remained increased, or even increased further in the unfavorable group.Our data indicate a state of increased innate immune activation in COVID19-patients with an unfavorable outcome at hospital admission, which remained over time, as compared with patients with a favorable outcome.

GB co-immunization with GP96 enhances pulmonary-resident CD8 T cells and exerts a long-term defence against MCMV pneumonitis.

Human cytomegalovirus (HCMV) infection in the respiratory tract leads to pneumonitis in immunocompromised hosts without available vaccine. Considering cytomegalovirus (CMV) mainly invades through the respiratory tract, CMV‐specific pulmonary mucosal vaccine development that provides a long‐lasting protection against CMV challenge gains our attention. In this study, N‐terminal domain of GP96 (GP96‐NT) was used as a mucosal adjuvant to enhance the induction of pulmonary‐resident CD8 T cells elicited by MCMV glycoprotein B (gB) vaccine. Mice were intranasally co‐immunized with 50 μg pgB and equal amount of pGP96‐NT vaccine 4 times at 2‐week intervals, and then i.n. challenged with MCMV at 16 weeks after the last immunization. Compared with pgB immunization alone, co‐immunization with pgB/pGP96‐NT enhanced a long‐lasting protection against MCMV pneumonitis by significantly improved pneumonitis pathology, enhanced bodyweight, reduced viral burdens and increased survival rate. Moreover, the increased CD8 T cells were observed in lung but not spleen from pgB/pGP96‐NT co‐immunized mice. The increments of pulmonary CD8 T cells might be mainly due to non‐circulating pulmonary‐resident CD8 T‐cell subset expansion but not circulating CD8 T‐cell populations that home to inflammation site upon MCMV challenge. Finally, the deterioration of MCMV pneumonitis by depletion of pulmonary site‐specific CD8 T cells in mice that were pgB/pGP96‐NT co‐immunization might be a clue to interpret the non‐circulating pulmonary‐resident CD8 T subset expansion. These data might uncover a promising long‐lasting prophylactic vaccine strategy against MCMV‐induced pneumonitis.

A Fully-Human Armored BCMA CAR Boosts Function of CD4+ CAR-T Cells and Resists TGF-β Suppression in Pre-Clinical Models of Multiple Myeloma

The B-cell maturation antigen (BCMA) is an immunotherapy target selectively expressed on multiple myeloma cells (MM). Despite recent success of experimental BCMA CAR-T cell therapy, clinical remissions in MM are often short, in part due to low persistence of the BCMA CAR-T cells. Additionally, immunosuppressive factors, notably TGF-β, are known to be elevated in the peripheral blood and tumor microenvironment in the bone marrow of MM patients, potentially contributing to the lack of durability of CAR-T cell therapy. We aimed to develop a fully-human BCMA CAR with long-term persistence and functional resistance to the suppressive effects of TGF-β. Initially, two fully human single chain variable fragments (scFv) specific for BCMA, derived by biopanning of a yeast display human scFv library, were characterized in a CAR format. Each of the two scFvs was cloned into a lentiviral vector CAR backbone, comprised of the CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain and CD3ζ signaling domain, and termed BCMA1 and BCMA2, respectively. BCMA2 and BCMA1 T cells, generated by lentiviral vector transduction of primary human T cells, exhibited high CAR expression at multiplicities of infection 10 to 40 (BCMA1: 64-81%; BCMA2: 84-94%), and consistently demonstrated potent cytotoxicity in an overnight co-culture with BCMA+ MM cell lines RPMI-8226 and MM1.S, but not the BCMA- 293T cells. BCMA2 CAR showed robust proliferative capacity in response to repeated, long-term exposure to MM1.S target cells, concordant with the sustained CD4 T-cell subset and high IL-2 production during the course of repeated exposure to target cells. This is in contrast to BCMA1, that showed precipitous decrease in CD4+ T-cell subset upon co-culture with MM cells, resulting in a CAR T-cell population dominated by CD8+ T-cells. Furthermore, the BCMA2 demonstrated prolonged potency in clearing tumor cells compared to BCMA1, even after continuous 20-day exposure to target cells. This was further confirmed in a mouse intradermal RPMI-8226 xenograft tumor model, in which infusion of BCMA2 T-cells resulted in the rapid and complete eradication of tumors, while BCMA1 showed a slower decline in tumor burden. To further improve the efficacy of the BCMA2, specifically within the TGF-β-rich immunosuppressive tumor microenvironment, we developed an armored BCMA2 CAR variant, which co-expresses the dominant negative TGF-β RII bicistronically via a 2A sequence (BCMA2-TbnegCAR). In a 10 day-long co-culture assay with MM1.S targets in the presence of spiked 10 ng/ml TGF-β, BCMA2-TbnegCAR retained high proliferative capacity and potent cytotoxicity, while the unarmored BCMA2 had diminished proliferation and substantially reduced cytokine and granzyme B production. Consistently, in the in vivo intradermal tumor model that utilizes RPMI-8226, which is a MM cell line that endogenously produces TGF-β, we observed that BCMA2-TbnegCAR treatment resulted in higher T-cell counts in the tumors and an earlier decrease of tumor burden compared to infusion with BCMA2, further demonstrating the increased efficacy and potency of the BCMA2-TbnegCAR. In conclusion, we have designed and characterized a new fully-human BCMA2-TbnegCAR, with a novel scFv that exhibits robust proliferative capacity and persistent cytotoxicity, and remains functionally resistant to the immunosuppressive effects of TGF-β. This novel BCMA CAR can potentially improve the effectiveness and durability of the current BCMA CAR-T cell therapy. Disclosures Alabanza: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Vu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Wu:Lentigen, a Miltenyi Biotec Company: Current Employment. Zhu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties.

Increased frequency of proinflammatory CD4 T cells and pathological levels of serum neurofilament light chain in adult drug-resistant epilepsy.

Adult drug-resistant epilepsy (DRE) is associated with significant morbidity. Infiltration of immune cells is observed in DRE epileptic foci; however, the relation between DRE and the peripheral immune cell compartment remains only partially understood. We aimed to investigate differences in immune cell populations, cytokines, and neurodegenerative biomarkers in the peripheral blood of subjects with epilepsy versus healthy controls, and in DRE compared to well-controlled epilepsy (WCE). Peripheral blood mononuclear cells and serum from >120 age- and sex-matched adults suffering from focal onset epilepsy and controls were analyzed by multipanel flow cytometry, multiplex immunoassays, and ultrasensitive single molecule array. Using a data-driven analytical approach, we identified that CD4 T cells in the peripheral blood are present in a higher proportion in DRE patients. Moreover, we observed that the frequency of CD4 T cells expressing proinflammatory cytokines interleukin (IL)-17A, IL-22, tumor necrosis factor, interferon-γ, and granulocyte-macrophage colony-stimulating factor, but not anti-inflammatory cytokines IL-10 and IL-4, is elevated in the peripheral blood of DRE subjects compared to WCE. In parallel, we found that Th17-related circulating proinflammatory cytokines are elevated, but Th2-related cytokine IL-4 is reduced, in the serum of epilepsy and DRE subjects. As Th17 cells can exert neurotoxicity, we measured levels of serum neurofilament light chain (sNfL), a marker of neuronal injury. We found significantly elevated levels of sNfL in DRE compared to controls, especially among older individuals. Our data support that DRE is associated with an expansion of the CD4 Tcell subset in the peripheral blood and with a shift toward a proinflammatory Th17/Th1 CD4 Tcell immune profile. Our results further show that pathological levels of sNfL are more frequent in DRE, supporting a potential neurodegenerative component in adult DRE. With this work, we provide evidence for novel potential inflammatory and degenerative biomarkers in DRE.

Area under Immunosurveillance: Dedicated Roles of Memory CD8 T-Cell Subsets.

Immunological memory, defined as the ability to respond in an enhanced manner upon secondary encounter with the same pathogen, can provide substantial protection against infectious disease. The improved protection is mediated in part by different populations of memory CD8 T cells that are retained after primary infection. Memory cells persist in the absence of pathogen-derived antigens and enable secondary CD8 T-cell responses with accelerated kinetics and of larger magnitude after reencounter with the same pathogen. At least three subsets of memory T cells have been defined that are referred to as central memory CD8 T cells (Tcm), effector memory CD8 T cells (Tem), and tissue-resident memory CD8 T cells (Trm). Tcm and Tem are circulating memory T cells that mediate bodywide immune surveillance in search of invading pathogens. In contrast, Trm permanently reside in peripheral barrier tissues, where they form a stationary defensive line of sentinels that alert the immune system upon pathogen reencounter. The characterization of these different subsets has been instrumental in our understanding of the strategies that memory T cells employ to counter invading pathogens. It is clear that memory T cells not only have a numerical advantage over naive T cells resulting in improved protection in secondary responses, but also acquire distinct sets of competencies that assist in pathogen clearance. Nevertheless, inherent challenges are associated with the allocation of memory T cells to a limited number of subsets. The classification of memory T cells into Tcm, Tem, and Trm may not take into account the full extent of the heterogeneity that is observed in the memory population. Therefore, in this review, we will revisit the current classification of memory subsets, elaborate on functional and migratory properties attributed to Tcm, Tem, and Trm, and discuss how potential heterogeneity within these populations arises and persists.

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.

Abstract 4476: PD-1 and TIGIT co-expression identifies a circulating CD8 T cell population predictive of response to anti-PD-1 therapy in melanoma and Merkel-cell carcinoma patients

Abstract Immunotherapies targeting the PD-1 pathway have profoundly transformed the clinical care of cancer patients for a growing variety of cancer types. However, most patients do not experience durable clinical benefit. The definition of robust and convenient biomarkers of PD-1 therapy efficacy to stratify patients beforehand or early after initiation of the therapy that could guide therapeutic management is still lacking while being a very active research field. Biomarkers described to date include tumor burden, neoantigen load, presence and number of PD-1+ CD8+ at the tumor margin, T-cell inflamed tumor microenvironment and PD-L1 expression by the tumor cells or other immune cells and composition of the gut microbiota. Most of these parameters are closely related/influenced by the presence, activation status and functional capacities of CD8+ T cells infiltrating the tumor site demonstrating their pivotal role for anti-PD-1 mediated anti-tumor efficacy. A population of PD-1high CD8 TILs was consequently described as predictive of PD-1 blockade in NSCLC. The exact contribution for clinical efficacy of TILs versus distinct CD8+ T cells from peripheral origins recirculating to the tumor site remains to be elucidated. Notably, immunological responses to PD-1 blockade at the periphery were described within the very first days following the first therapy dose. Therefore, describing circulating cellular population predictive of PD-1 inhibitor efficacy could represent a convenient, non-invasive and rapid method to assess anti-tumor benefits. Original findings reported in this study identified a circulating CD8 T cell population delineated by the co-expression of TIGIT and PD-1 inhibitory receptors as an early immune marker of anti-PD-1 efficacy in three independent cohorts of cancer patients (two melanoma patient's cohorts and one Merkel-cell carcinoma patient's cohort). The frequency of this double positive (DPOS) population even appeared predictive of PD-1 inhibitor therapy efficacy at baseline in the MCC cohort. Furthermore, to understand the mechanistical relevance of this subset for PD-1 blockade efficacy, we thoroughly described this DPOS T cell subset by flow cytometry, gene expression analysis, anti-tumor reactivity assay and TCR repertoire analysis, and compared it to its double negative (DNEG), PD-1 and TIGIT single positive counterparts. This DPOS subset was enriched in activated and proliferative T cells, retained expression of co-stimulatory molecules and was enriched for common features with Tfc. Moreover, this subpopulation exhibited a specific gene signature, strongly predictive of long-term survival in melanoma patients (TCGA analyses). We demonstrated that this subpopulation was enriched in tumor-specific T-cells (ELISPOT analysis against 11 antigen-derived peptides). Finally, clustering of TCR clonotypes revealed that the DPOS T cell population was significantly enriched in emerging clonotypes in responding patients, after 1 month of anti-PD-1 therapy echoing recent work from others. Our findings provide a compelling rationale to measure PD-1+TIGIT+ CD8 T-cell subset in the blood of cancer patients to monitor early anti-PD1 mediated clinical efficacy, and to use DPOS T cells as a window to study the dynamic changes that underly successful antitumor immunity. Citation Format: Sylvain Simon, Valentin Voillet, Virginie Vignard, Zhong Wu, Camille Dabrowski, Nicolas Jouand, Tiffany Beauvais, Amir Khammari, Cecile Braudeau, Regis Josien, Olivier Adotevi, Caroline Laheurte, Francçois Aubin, Charles Nardin, Samuel Rulli, Raphaël Gottardo, Nirasha Ramchurren, Cheever Martin, Steven P. Fling, Candice D. Church, Paul Nghiem, Brigitte Dreno, Stanley R. Riddell, Nathalie Labarriere. PD-1 and TIGIT co-expression identifies a circulating CD8 T cell population predictive of response to anti-PD-1 therapy in melanoma and Merkel-cell carcinoma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4476.

Disease-associated and patient-specific immune cell signatures in juvenile-onset systemic lupus erythematosus: patient stratification using a machine-learning approach.

SummaryBackgroundJuvenile-onset systemic lupus erythematosus (SLE) is a rare autoimmune rheumatic disease characterised by more severe disease manifestations, earlier damage accrual, and higher mortality than in adult-onset SLE. We aimed to use machine-learning approaches to characterise the immune cell profile of patients with juvenile-onset SLE and investigate links with the disease trajectory over time.MethodsThis study included patients who attended the University College London Hospital (London, UK) adolescent rheumatology service, had juvenile-onset SLE according to the 1997 American College of Rheumatology revised classification criteria for lupus or the 2012 Systemic Lupus International Collaborating Clinics criteria, and were diagnosed before 18 years of age. Blood donated by healthy age-matched and sex-matched volunteers who were taking part in educational events in the Centre for Adolescent Rheumatology Versus Arthritis at University College London (London, UK) was used as a control. Immunophenotyping profiles (28 immune cell subsets) of peripheral blood mononuclear cells from patients with juvenile-onset SLE and healthy controls were determined by flow cytometry. We used balanced random forest (BRF) and sparse partial least squares-discriminant analysis (sPLS-DA) to assess classification and parameter selection, and validation was by ten-fold cross-validation. We used logistic regression to test the association between immune phenotypes and k-means clustering to determine patient stratification. Retrospective longitudinal clinical data, including disease activity and medication, were related to the immunological features identified.FindingsBetween Sept 5, 2012, and March 7, 2018, peripheral blood was collected from 67 patients with juvenile-onset SLE and 39 healthy controls. The median age was 19 years (IQR 13–25) for patients with juvenile-onset SLE and 18 years (16–25) for healthy controls. The BRF model discriminated patients with juvenile-onset SLE from healthy controls with 90·9% prediction accuracy. The top-ranked immunological features from the BRF model were confirmed using sPLS-DA and logistic regression, and included total CD4, total CD8, CD8 effector memory, and CD8 naive T cells, Bm1, and unswitched memory B cells, total CD14 monocytes, and invariant natural killer T cells. Using these markers patients were clustered into four distinct groups. Notably, CD8 T-cell subsets were important in driving patient stratification, whereas B-cell markers were similarly expressed across the cohort of patients with juvenile-onset SLE. Patients with juvenile-onset SLE and elevated CD8 effector memory T-cell frequencies had more persistently active disease over time, as assessed by the SLE disease activity index 2000, and this was associated with increased treatment with mycophenolate mofetil and an increased prevalence of lupus nephritis. Finally, network analysis confirmed the strong association between immune phenotype and differential clinical features.InterpretationMachine-learning models can define potential disease-associated and patient-specific immune characteristics in rare disease patient populations. Immunological association studies are warranted to develop data-driven personalised medicine approaches for treatment of patients with juvenile-onset SLE.FundingLupus UK, The Rosetrees Trust, Versus Arthritis, and UK National Institute for Health Research University College London Hospital Biomedical Research Centre.

Inverse Association Between the Quantity of Human Peripheral Blood CXCR5+IFN-γ+CD8+ T Cells With De Novo DSA Production in the First Year After Kidney Transplant.

We recently reported that a novel CXCR5IFN-γCD8 T-cell subset significantly inhibits posttransplant alloantibody production in a murine transplant model. These findings prompted the current study to investigate the association of human CD8 T cells with the same phenotype with the development of de novo donor-specific antibody (DSA) after kidney transplantation. In the current studies, we prospectively and serially analyzed peripheral blood CD8 and CD4 T-cell subsets and monitored for the development of de novo DSA in kidney transplant recipients during the first-year posttransplant. We report results on 95 first-time human kidney transplant recipients with 1-year follow-up. Twenty-three recipients (24.2%) developed de novo DSA within 1-year posttransplant. Recipients who developed DSA had significantly lower quantities of peripheral CXCR5IFN-γCD8 T cells (P = 0.01) and significantly lower ratios of CXCR5IFN-γCD8 T cell to combined CD4 Th1/Th2 cell subsets (IFN-γCD4 and IL-4CD4 cells; P = 0.0001) compared to recipients who remained DSA-negative over the first-year posttransplant. Our data raise the possibility that human CXCR5IFN-γCD8 T cells are a homolog to murine CXCR5IFN-γCD8 T cells (termed antibody-suppressor CD8 T cells) and that the quantity of CXCR5IFN-γCD8 T cells (or the ratio of CXCR5IFN-γCD8 T cells to Th1/Th2 CD4 T cells) may identify recipients at risk for development of DSA.

Characterization of circulating and cultured Tfh-like cells in sickle cell disease in relation to red blood cell alloimmunization status

BackgroundPeople living with sickle cell disease (SCD) are prone to red blood cell (RBC) alloimmunization. We hypothesized that subjects with alloantibodies (responders) would have differences in circulating T-follicular helper (Tfh)-like cells compared to subjects without alloantibodies (non-responders). Materials and methodsPeripheral blood mononuclear cells were collected from 28 subjects, including those with SCD and controls. Circulating CD4 T-cell subsets were first evaluated at baseline. CD4 T-cell subsets were also evaluated after naïve CD4 T-cells were differentiated into Tfh-like cells following in vitro culture with CD3/CD28 beads, IL-7, IL-12, and Activin A. Transfusion and alloantibody histories were extracted from the electronic medical record. ResultsNon-responders had a lower percentage of CD45RA negative Tmemory cells than responders or controls (p<0.05). Notably, there were no differences in circulating Tfh-like cells between any group. However, naïve CD4 T-cells from subjects with SCD were more likely to express CXCR5 after in vitro culture than cells from controls. After culture, CXCR5 expressing cells from responders were more likely to express PD1 and ICOS (16.43 %, sd. 20.23) compared to non-responders (3.69 %, s.d. 3.09) or controls (2.78 %, s.d. 2.04). DiscussionThe tendency for naïve CD4 T-cells from responders to differentiate into Tfh-like cells after in vitro culture may suggest these cells are prepared to assist B-cells with antibody production regardless of antigen specificity. Further studies are needed, but it is possible that these results may explain why some responders form RBC alloantibodies with multiple specificities, in addition to RBC autoantibodies and HLA alloantibodies.

Apoptosis characterization in mononuclear blood leukocytes of HIV\xa0patients during dengue acute disease

Dengue virus (DENV) co-circulation in Brazil represents a challenge for treatment and vaccine development. Despite public health impact, the occurrence of coinfections with other viruses is a common event. Increased T cell activation and altered inflammatory response are found during DENV coinfection with Human Immunodeficiency Virus (HIV) impacting HIV-pathogenesis. Even with Antiretroviral therapy (ART), HIV- treated patients had chronic immune activation and lymphocyte apoptosis. However, apoptotic mechanisms have not been investigated during coinfection with DENV. Our attention was attracted to apoptotic cell markers expressions in PBMCs from DENV and DENV/HIV coinfected patients. We found CD4/CD8 ratio inversion in most coinfected patients. CD4 T and CD8 T-cell subsets from DENV and DENV/HIV groups expressed low levels of anti-apoptotic protein Bcl-2. Furthermore, CD8 CD95 double positive cells frequency expressing low levels of Bcl-2 were significantly higher in these patients. Additionally, the density of Bcl-2 on classical monocytes (CD14++CD16−) was significantly lower during DENV infection. Upregulation of pro-apoptotic proteins and anti-apoptotic proteins were found in DENV and DENV/HIV, while catalase, an antioxidant protein, was upregulated mainly in DENV/HIV coinfection. These findings provide evidence of apoptosis triggering during DENV/HIV coinfection, which may contribute to knowledge of immunological response during DENV acute infection in HIV-patients treated with ART.

Abstract IA12: Fueling T cells in inflammation and cancer

Abstract Lymphocyte activation leads to rapid proliferation and differentiation, and we have shown that CD4 T-cell subsets are metabolically distinct. These specific metabolic programs may allow new understanding and approaches to manipulate immunity. Using metabolic network analysis of metabolomics and proteomics, we defined several metabolic nodes differentially utilized by CD4 T-cell subsets, including glutamine metabolism. By genetically deleting the glucose transporter Glut1 or the glutaminolysis enzyme, glutaminase (GLS), we have shown that glycolysis and glutaminolysis are both used by activated T cells. All effector T cells require Glut1 and glycolysis. In contrast, we found that Th17 cells preferentially require GLS while Th1 cells are actively impaired by this enzyme. Thus, inhibition of GLS both reduces Th17 responses and promotes differentiation of Th1 cells and can lead to signs of T-cell exhaustion. We show that GLS deficiency can protect against Th17-mediated inflammatory models and also can augment effector function of Th1 CAR-T cells against B-cell targets. Understanding mechanisms that regulate T-cell metabolism may provide new tools to modulate immunity in the balance of T-cell effector populations to both suppress inflammation and promote effector function. Citation Format: Jeffrey C. Rathmell. Fueling T cells in inflammation and cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA12.

Abstract B61: High-dimensional analysis of tumor-resident CD4 and CD8 double-negative T-cell subset in multiple tumor types

Abstract Background: Checkpoint blockade therapies like PD-1 antibodies elicit durable long-lasting immunity in a subset of patients of certain tumor types. However, many patients across a range of tumor types are resistant to checkpoint blockade. Mechanisms that confer local response or resistance to checkpoint blockade are not well understood. Mounting evidence points to local suppression of T cell function as one of the most substantial barriers to effective antitumor immunity. Methods: The study involved multiparametric flow cytometry and genomic characterization of a subset of T cells in the tumors and lymphoid organs of syngeneic tumors, genetically engineered mouse model of cancers and human tumors. Results: A significant fraction (10-40%) of α/β TCR+ CD3+ T cells do not express CD4, CD8 or NKp46 in the tumors. The presence of double-negative T-cell subset (DN T cells) is restricted to tumor microenvironment and is relatively scarce in tumor draining lymph nodes and in spleen. To identify if DN T cells are antigen-specific, we performed AH-1 tetramer staining. While a substantial fraction of CD8 T cells are AH-1 tetramer positive, majority of DN T cells are AH-1 tetramer negative in CT26 tumors. Recently, it has been shown that higher frequency of CD39-negative bystander T cells correlated to lack of response to PD-1 therapy in non-small cell lung cancer patients. Mounting evidence also reveals presence of a specific subset of CD8 T cells with features of stem cell memory in tumors as defined by CXCR5 and TCF1 expression. Efforts are under way to understand if DN T cells exhibit bystander cell and/or memory stem-like phenotype. Majority of the DN T cells are proliferating (Ki67+) and express very low levels of PD-1 (in contrast to CD8 T cells) and Granzyme B. Treatment of mice bearing syngeneic tumors with PD-1 checkpoint blockade therapy does not impact the frequency of DN T-cell subset consistent with lower levels of PD-1 expression. Moreover, a substantial frequency (30-40%) of DN T cells in both syngeneic and KPC pancreatic GEMM tumors express CD103, a tissue resident marker. Furthermore, DN T cells are CD44+ CD127+ KLRG1- and may represent a memory precursor cell population. Upon ex vivo stimulation, these DN T cell subsets proliferate and express modest levels of PD-1. Preliminary analysis of single-cell RNA sequencing data from FACS sorted murine DN T cells reveals lack of CD4, CD8 expression and retention of CD2, CD44, CD69, CCR7 and CXCR5 expression. Flow cytometry analysis of human CD2+ TILs reveals presence of DN T cells in human tumors. Single-cell transcriptome analysis of CD2+ T cells isolated from human colorectal tumors revealed presence of DN T cells. Current efforts are focused on further characterization of this subset in both mouse and human tumors. Conclusions: In this study, we describe a tumor-resident CD4 and CD8 double-negative T-cell subset that may represent a bystander cell population with features of memory precursors and potential implications in modulation of response to checkpoint blockade therapy in cancer. Citation Format: Murali Gururajan, Ravi Kandasamy, Jessica Wong, Kalpit Shah, Shuoguo Wang, Adam Bata, Amy Truong, Sunil Kuppasani, Ching-Ping Ho, Robert Graziano, Michael Quigley, Anwar Murtaza, Jinqi Liu. High-dimensional analysis of tumor-resident CD4 and CD8 double-negative T-cell subset in multiple tumor types [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B61.

Abstract B64: CD4 help is required for the generation of a transcriptionally distinct cytolytic CD8 T-cell subset to control chronic infection and tumor

Abstract “CD4 help” is crucial to sustain the function of exhausted CD8 T cells, yet the detailed mechanisms by which CD4 help regulates CD8 T-cell differentiation and function during chronic viral infection remains elusive. Here, using single-cell RNA-sequencing (scRNA-seq) we show that CD8 T cells responding to chronic viral infection are more heterogeneous than previously appreciated, with three transcriptionally distinct subsets dominating the late phase of the antiviral response. Importantly, our findings uncover the formation of a previously unrecognized CD8 T-cell subset that exhibits potent cytolytic function. This subset is characterized by high expression of killer cell lectin-like receptors Klre1 and Klra9, the chemokine receptor CX3CR1, and the transcription factors T-bet and Zeb2. Notably, our data further demonstrate that the formation of this cytotoxic subset is critically dependent on CD4 help and that exploitation of this developmental pathway may be used therapeutically to enhance cytotoxic CD8 T-cell infiltration into the tumor. These findings uncover novel cellular and molecular mechanisms of how “CD4 help” regulates CD8 T-cell differentiation during persistent infection and have implications towards optimizing the generation of protective CD8 T cells in immunotherapy. Citation Format: Ryan Zander, David Schauder, Weiguo Cui. CD4 help is required for the generation of a transcriptionally distinct cytolytic CD8 T-cell subset to control chronic infection and tumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B64.

Blood and Lymph Node Dissemination of Clonal Genome-Intact Human Immunodeficiency Virus 1 DNA Sequences During Suppressive Antiretroviral Therapy.

The majority of cells with latent human immunodeficiency virus 1 infection are located in lymphoid tissues that are difficult to access. In the current study, we used single-genome near-full-length proviral sequencing to evaluate intact and defective proviruses in blood and lymph node CD4 T cells enriched for specific functional polarizations. We observed minor variations between the frequencies of proviral sequences within individual CD4 T-cell subsets and across tissue compartments. However, we noted multiple clonal clusters of identical intact or defective proviral sequences from distinct compartments and CD4 T-cell subpopulations, suggesting frequent interchanges between viral reservoir cells in blood and tissues.

Abstract A101: Single-cell analysis reveals the pivotal role of the innate immune compartment in aCTLA-4 antitumor response

Abstract Immunotherapies have revolutionized and continue to revolutionize cancer patient care; however, much of our understanding of the mechanism of action of these therapies is currently limited. The accumulation of regulatory T cells (Tregs) in the tumor hampers antitumor activity and correlates with bad prognosis in several human cancers. Anti-CTLA-4 monoclonal antibodies (mAbs) have been extensively studied, and their activity depends both on the blockade of the CTLA-4 coinhibitory molecule, as well as the intratumoral depletion of Tregs, increasing effector cell abundance, and favoring tumor rejection. Fc-gamma receptor (FcgR) coengagement has been proven to be important for the action of aCTLA-4, in addition to its Treg-depleting activity via antibody-dependent cellular cytotoxicity (ADCC); however, it is unclear what are the cellular changes involved. We therefore wished to dissect the differences between aCTLA-4 mIgG1, an antibody that only blocks the CTLA-4 receptor, and aCTLA-4 mIgG2a, which has a dual activity of both blocking the receptor and depleting Tregs. To address this, we performed single-cell RNA-seq of infiltrating leukocytes from tumors in mice treated with aCTLA-4 mIgG1, mIgG2a, or left untreated (UT). This high-resolution comparison revealed unique cellular profiles generated by each treatment. While the blocking-only mIgG1 was similar to UT, mIgG2a with ADCC effector function demonstrated major changes. Tumors in mIgG2a treated mice showed an immediate decline of immune suppressive macrophages, and the emergence of proinflammatory monocytes, as well as NK cells and naive CD8 T cells. In addition, mIgG2a-treated mice displayed a gradual increase of CD4 T-cell subsets throughout treatment. Finally, a vast tissue repair signature was observed in later time points of aCTLA-4 mIgG2a treatment, comprising MRC1+ macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs), while CD8 T-cell abundance declined. In summary, our findings provide an in-depth view of the differences in mechanisms of action between an aCTLA-4 blocking mAb and an optimized blocking-depleting mAb, underscoring how FcgRs coengagement leads to enhanced antitumor response via the innate immune compartment. Citation Format: Ido Yofe, Adam Jelinski, Isabelle Solomon, Tomer Landsberger, Marc Robert de Massy, Karl Peggs, Sergio Quezada, Ido Amit. Single-cell analysis reveals the pivotal role of the innate immune compartment in aCTLA-4 antitumor response [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A101.

Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube.

CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models—monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM), and common variable immunodeficiency (CVID). Overall, 145 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design–testing–evaluation–redesign, combined with in vitro functional studies, gene expression profiling, and multicentric evaluation of manual vs. automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0–89 years) were used to establish reference values, and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL (n = 8), SM (n = 7), and CVID (n = 10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T cells, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, Th2, and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4 tube allows fast, automated, and reproducible identification of ≥89 subsets of CD4+ blood T cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.