Memory Subsets Research Articles

IL-21 in Homeostasis of Resident Memory and Exhausted CD8 T Cells during Persistent Infection.

CD4 T cells guide the development of CD8 T cells into memory by elaborating mitogenic and differentiation factors and by licensing professional antigen-presenting cells. CD4 T cells also act to stave off CD8 T cell dysfunction during repetitive antigen stimulation in persistent infection and cancer by mitigating generation of exhausted T cells (TEX). CD4 T cell help is also required for establishing and maintaining tissue-resident memory T cells (TRM), the nonrecirculating memory T cell subset parked in nonlymphoid tissues to provide frontline defense against reinvading pathogens. Interleukin (IL)-21 is the signature cytokine secreted by follicular helper CD4 T cells (TFH) to drive B cell expansion and differentiation in germinal centers to mount high-affinity, isotype class-switched antibodies. In several infection models, IL-21 has been identified as the CD4 T help needed for formation and survival of TRM and TEX. In this review, we will explore the different memory subsets of CD8 T cells in persistent infections, the metabolic profiles associated with each, and evidence documenting the importance of CD4 T cell-derived IL-21 in regulating CD8 TRM and TEX development, homeostasis, and function.

Human-Animal Immune Simulations in Covid-19

The theme of Human-Animal immune simulations in covid-19 is of tripartite nature .First is the human, the second is the infecting virus and the third is the animal. The simulation criteria for human are; functional immune system ,evident harmony in the nervous-endocrine-immune axis, potent homeostatic mechanisms and good nutritional status. The infectious virus should be capable to; Find port of entry, did efficiently the receptor mediated entry to the target cell entrance ,production of outnumbering viral particles forming viral load, production of virulence factors and evading the host immune mechanisms. The virus first sound to be zoonotic in nature, but then it expresses anthropozoonotic and zooanthroponotic infection cycle. Human sars-cov-2 infection forms clustered in six infection forms. While in the mammalian experimental sars-cov-2 infection is mostly of mild respiratory form. The mammalian animal simulation criteria are; High percent similarity in structure and function to human immune system, similar harmony in the axis of nervous-endocrine-immune systems and evident similarity percent in the cellular events of the immune responses to that of man.Sars-cov-2 antigenic epitopes identified as spike protein epitope, nucleoprotein epitope and binding domain epitope, infection in man rise up specific antibodies as early in the immune response time curve as IgM, then class switched to IgA in acute course of the infection then lately switched to IgG in chronic state. Anti-spike antibodies are virus neutralizing parallel to recovery from the infection. The duration of antibody lasting in patient plasma around three months but their protective efficacy upon re- infection is a matter of debate. Neutralizing anti-spike protein antibodies prevents experimental reinfection in both monkeys and Syrian golden hamsters. Though the duration of such protection to experimental reinfection is rather unknown.CD4 T cells effector and memory subsets are of crucial protective role in human covid-19.CD8 T cells and NK cells are exhausted during the human acute infection form. The role of NK,CD4,CD8 T cells in covid-19 in experimental animals remained to be explored. Number of novel immune models concerns Sars-cov-2 virus in various facets of human infection remain to be performed. Keywords: Antibody ,Covid-19 ,epitope , harmony , homeostasis , infection , Sars-cov-2 ,virus DOI: 10.7176/JBAH/10-18-05 Publication date: September 30 th 2020

1947P Single cell analysis reveals that CD8+ T cell clone size determines response to immune checkpoint blockade

Immune checkpoint blockers (ICB) have markedly improved survival in patients with metastatic melanoma (MM). However, clinical response remains highly variable and peripheral biomarkers are lacking. CD8+ T cells are a key cellular subset mediating the effect of ICB and previous work has demonstrated that the presence of large CD8+ T cell clones in the peripheral blood (>0.5% of the total repertoire) is associated with positive clinical outcomes. We sought to further characterise and explore the clonal characteristics of the peripheral CD8+ T cell response to ICB at a single-cell level. Single cell transcriptome and V(D)J sequencing was performed on CD8+ T cells isolated from the peripheral blood of patients undergoing ICB treatment for MM (n=8) at baseline and 21 days post-treatment. Dimensionality reduction and trajectory inference were subsequently employed to identify and understand the effects of treatment on cellular subpopulations. We identified 7 subsets of CD8+ T cells, defined by distinctive phenotypes. ICB had a heterogenous effect across these subsets, with the greatest and most unique gene expression changes in the effector and effector memory subsets. Such changes were more marked in larger (>0.5% of repertoire) than smaller clones. By grouping cells based on clonal size, we identify that the effects of ICB vary as a function of clonal size, with clones between 0.5-1% of the repertoire having quantitatively fewer, but qualitatively more relevant, gene expression changes than either hyper-expanded (>2%) or small (<0.1%) clones. Further, ICB had a differential effect on clones that expand following treatment, than those that contract, with expanding clones having both a greater magnitude and qualitatively more relevant gene expression changes than contracting clones. Taken together, we describe a discrete subset of cells which uniquely respond to ICB. We identify that all cells are not equally affected by ICB, but a subset defined by clonal size and clonal behaviour demonstrate a marked and distinctive response, potentially representing a key cellular target of therapy. This work will aid the search for a prognostic biomarker and further advances understanding of the molecular mechanisms of ICB.

Rewiring Mitochondrial Metabolism for CD8+ T Cell Memory Formation and Effective Cancer Immunotherapy.

Memory T cells persist for long term to mediate robust recall response upon rechallenging with previous encountered pathogens. The memory T cell pool is highly heterogeneous based on distinct phenotypic, functional, and locational properties, and contains discrete subsets, which contribute to diverse immune responses. In this mini-review, we will briefly discuss the distinct subsets of memory T cells and then focus on mitochondria-related metabolic and epigenetic regulations of CD8+ T cell memory formation. In particular, we discuss many aspects of mitochondrial quality control systems (biogenesis, dynamics, etc.) in regulating CD8+ T cell fate decision and antitumor immunity. Importantly, targeting mitochondrial metabolism to boost T cell memory formation and metabolic fitness might represent an attractive strategy to improve cancer immunotherapy including CAR-T therapy.

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.

OMIP-067: 28-Color Flow Cytometry Panel to Evaluate Human T-Cell Phenotype and Function.

OMIP-067: 28-Color Flow Cytometry Panel to Evaluate Human T-Cell Phenotype and Function.

Fc receptor-like 4 and 5 define human atypical memory B cells.

Atypical memory B cells accumulate in chronic infections and autoimmune conditions, and commonly express FCRL4 and FCRL5, respective IgA and IgG receptors. We characterized memory cells from tonsils on the basis of both FCRL4 and FCRL5 expression, defining three subsets with distinct surface proteins and gene expression. Atypical FCRL4+FCRL5+ memory cells had the most discrete surface protein expression and were enriched in cell adhesion pathways, consistent with functioning as tissue-resident cells. Atypical FCRL4-FCRL5+ memory cells expressed transcription factors and immunoglobulin genes that suggest poised differentiation into plasma cells. Accordingly, the FCRL4-FCRL5+ memory subset was enriched in pathways responding to endoplasmic reticulum stress and IFN-γ. We reconstructed ongoing B-cell responses as lineage trees, providing crucial in vivo developmental context. Each memory subset typically maintained its lineage, denoting mechanisms enforcing their phenotypes. Classical FCRL4-FCRL5- memory cells were infrequently detected in lineage trees, suggesting the majority were in a quiescent state. FCRL4-FCRL5+ cells were the most represented memory subset in lineage trees, indicating robust participation in ongoing responses. Together, these differences suggest FCRL4 and FCRL5 are unlikely to be passive markers but rather active drivers of human memory B-cell development and function.

Single-cell TCR sequencing reveals phenotypically diverse clonally expanded cells harboring inducible HIV proviruses during ART

Clonal expansions occur in the persistent HIV reservoir as shown by the duplication of proviral integration sites. However, the source of the proliferation of HIV-infected cells remains unclear. Here, we analyze the TCR repertoire of single HIV-infected cells harboring translation-competent proviruses in longitudinal samples from eight individuals on antiretroviral therapy (ART). When compared to uninfected cells, the TCR repertoire of reservoir cells is heavily biased: expanded clonotypes are present in all individuals, account for the majority of reservoir cells and are often maintained over time on ART. Infected T cell clones are detected at low frequencies in the long-lived central memory compartment and overrepresented in the most differentiated memory subsets. Our results indicate that clonal expansions highly contribute to the persistence of the HIV reservoir and suggest that reservoir cells displaying a differentiated phenotype are the progeny of infected central memory cells undergoing antigen-driven clonal expansion during ART.

Abstract 6538: CMV-specific immuno-dysregulation in recurrent glioblastoma patients can be overcome with therapeutic vaccination which is associated with tumor response and overall survival benefits in a Phase I/IIA study

Abstract Numerous independent laboratories using several techniques have demonstrated cytomegalovirus (CMV) antigens in over 90% of glioblastoma (GBM) tumors. We initiated a phase I/IIA multicenter study designed to demonstrate safety and determine the most immunogenic dose of an enveloped virus-like particle (eVLP) vaccine expressing the CMV gB and pp65 antigens for treatment of recurrent GBM patients. In Part A of the trial, 6 patients in each of 3 different antigen dose cohorts were vaccinated monthly and followed until tumor progression, with PBMCs collected 2 weeks after each vaccination. Surprisingly, only 7/18 patients enrolled in Part A of the trial had baseline CMV-specific antibody (Ab) titers. This was unexpected given the documented high prevalence of CMV antigens in GBM tumor samples as well as epidemiological data demonstrating much higher CMV Ab seropositive rates in otherwise healthy individuals of a comparable age. In the low dose cohort, 4/6 patients had baseline CMV Ab titers. Of these 4 patients,vaccination boosted strong T cell responses in 2 patients (based on IFN-γ ELISPOT against CMV gB and pp65 antigens). Vaccination did not boost T-cell responses in the CMV Ab-negative patients. In the intermediate dose cohort, among 4 evaluable patients, T cell responses were robustly boosted in the only patient with a baseline CMV Ab titer. In marked contrast, the T-cell responses in the high dose cohort were boosted in 3/5 evaluable patients, all of which were CMV Ab seronegative at baseline. Moreover, the T-cell responses correlated with tumor responses (stable disease based on 2 or more stable MRI scans) as well as improved PFS and 6 month OS relative to vaccine non-responders. To further understand the apparent disconnect between baseline CMV Ab titers and vaccine-mediated impact on tumor and clinical responses, we developed a more sensitive flow cytometry-based assay which evaluated proliferating (Ki-67-positive) gB- and pp65-specific T cells within naïve, central memory (CD45RA-CCR7+) or effector memory (CD45RA-CCR7-) subsets. Among 2/3 tested vaccine responders evaluated thus far in the high dose cohort that were CMV Ab negative at baseline, high frequencies of effector memory cells (~2% total CD3+CD4+ T cells) against both the CMV pp65 and gB antigens were detected at baseline. Collectively, these data suggest that CMV-specific immune dysregulation exists in a substantial number of rGBM patients, which can be overcome with the appropriate dose of a CMV vaccine immunotherapeutic. Importantly, vaccine-mediated boosting of CMV immunity correlates with improved tumor response and clinical outcomes and warrants further confirmation. Citation Format: Tamara Berthoud, Felicia Deonarine, Spenser Ng Cheong Chung, Catalina Soare, Francisco Diaz-Mitoma, David E. Anderson. CMV-specific immuno-dysregulation in recurrent glioblastoma patients can be overcome with therapeutic vaccination which is associated with tumor response and overall survival benefits in a Phase I/IIA study [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 6538.

Abstract 6647: Sensitizing microsatellite stable colorectal cancer to immune checkpoint therapy utilizing Wnt pathway inhibition

Abstract Immunotherapies that target immune regulatory checkpoints such as CTLA-4 and PD-1 are widely used among many cancer types and have shown positive results in CRC with high microsatellite instability. However, in microsatellite stable (MSS) CRC there is a dismal response rate of 0%. The limited efficacy has shown to be partially due to the lack of T-cells in the tumor microenvironment and/or no activation/regulation of paramount cells in the immune system. The Wnt pathway is the most commonly altered pathway in CRC and is highly involved in driving tumor initiation and progression. Recent evidence also demonstrates the Wnt pathway is involved in T-lymphocyte development, maturation/activation of CD8+ effector T cells and recruitment of dendritic cells. Therefore, targeting the Wnt pathway utilizing a Porcupine (PORCN) inhibitor (ETC-159) in MSS CRC may be a promising strategy to sensitize tumors to immune checkpoint inhibition. Human Immune System BRGS (BALB/c, Rag2−/−, IL2RγC−/−, NODSIRPα) mice were engrafted with MSS CRC PDX (hPDX). The hPDX were randomized according to human chimerism into the following drug treatments groups: Vehicle, ETC-159, nivolumab, and the combination. Treatments began when tumors reached 100-300mm3 and tumors were measured twice weekly. At the end of study, sera, lymph nodes, spleen, and tumor tissue were collected for immunohistochemistry, single cell suspensions, and flow cytometry analysis. Combination therapy resulted in a significant decrease in tumor volume compared to both single agents and vehicle. Flow cytometric analysis demonstrated an increase in human immune cells, in particular human CD4 and CD8 cells in the combination compared to the vehicle and nivolumab treated groups. Additionally, these T-cells showed increased signs of activation and effector function, as indicated by increased CD69+ expression, effector memory subsets, and granzyme B+ cells in the TILs, with a further reduction in Treg populations, suggesting an overall increase in inflammation. An increase in MHC II expression on tumor cells was observed in the ETC-159 single agent with a statistically significant increase in the combination treated tumors demonstrating enhanced antigen presentation. Furthermore, PD-1 expression was upregulated on CD4+ T-cells in the ETC-159 single agent. Lastly, VECTRA analysis corroborates the flow cytometry data showing a changing tumor immune landscape through an increase in CD4+ and CD8+ T cells in the tumor and surrounding stroma. Our data demonstrates the combination treatment of ETC-159 + nivolumab in MSS CRC hPDX show increased tumor infiltration of human immune cells. Further preclinical data is compulsory but these results support further development of this combination in clinical trials. Citation Format: Stacey M. Bagby, Sarah J. Hartman, Natalie M. Navarro, Betelehem W. Yacob, Jeremy Shulman, Jessica Barkow, Christopher H. Lieu, S. Lindsey Davis, Alexis D. Leal, Wells A. Messersmith, Angela Minic, Kimberly R. Jordan, Julie Lang, Todd M. Pitts. Sensitizing microsatellite stable colorectal cancer to immune checkpoint therapy utilizing Wnt pathway inhibition [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 6647.

Acellular Wharton's Jelly, Potentials in T-Cell Subtypes Differentiation, Activation and Proliferation.

Purpose: Because of different potentials of T-cell subtypes in T-cell based cellular immunotherapy approaches such as CAR-T cell therapies; Regarding the high cost of the serum-free specific culture media, having distinct control on T-cell subset activation, expansion and differentiation seem crucial in T-cell expansion step of cell preparation methods. By the way, there was no clear data about the effect of acellular Wharton’s Jelly (AWJ) on T-cells expansion, activation or differentiation status. So, we have launched to study the effect of AWJ on T-cell’s immunobiological properties. Methods: CD3+ T-cells were isolated from healthy bone marrow allogeneic donors, sorted by FACS method and cultured on either routine phyto-hemagglutinin complemented and different concentrations of AWJ, lag phase and doubling time of the cells calculated from cell growth curve. After 3, 7 and 14-days T-cell subtypes cell markers and cell activity related genes expression rate have been evaluated by flow cytometry and real-time polymerase chain reaction (PCR) methods respectively. Results: AWJ in a 1:1 ratio compared with contemporary lymphocyte culture media showed significant activating and proliferative capacities. The introduced condition has not affected the frequency of CD4+ subpopulation of T-cells, but significantly increased even CD8+ cells and immune-activator genes in T-cells. The regulatory and memory subsets of T-cells in this study have not affected significantly. Conclusion: the study results revealed that AWJ can be utilized as a supportive substance to increase the memory properties of the T-cells, gives control to design a selective medium for expanding and differentiating memory T-cells, relatively.

Single-cell mass cytometry on peripheral blood identifies immune cell subsets associated with primary biliary cholangitis

The relationship between primary biliary cholangitis (PBC), a chronic cholestatic autoimmune liver disease, and the peripheral immune system remains to be fully understood. Herein, we performed the first mass cytometry (CyTOF)-based, immunophenotyping analysis of the peripheral immune system in PBC at single-cell resolution. CyTOF was performed on peripheral blood mononuclear cells (PBMCs) from PBC patients (n = 33) and age-/sex-matched healthy controls (n = 33) to obtain immune cell abundance and marker expression profiles. Hierarchical clustering methods were applied to identify immune cell types and subsets significantly associated with PBC. Subsets of gamma-delta T cells (CD3+TCRgd+), CD8+ T cells (CD3+CD8+CD161+PD1+), and memory B cells (CD3−CD19+CD20+CD24+CD27+) were found to have lower abundance in PBC than in control. In contrast, higher abundance of subsets of monocytes and naïve B cells were observed in PBC compared to control. Furthermore, several naïve B cell (CD3−CD19+CD20+CD24−CD27−) subsets were significantly higher in PBC patients with cirrhosis (indicative of late-stage disease) than in those without cirrhosis. Alternatively, subsets of memory B cells were lower in abundance in cirrhotic relative to non-cirrhotic PBC patients. Future immunophenotyping investigations could lead to better understanding of PBC pathogenesis and progression, and also to the discovery of novel biomarkers and treatment strategies.

Comparison of Lymphocyte Subset Populations in Children From South Africa, US and Europe.

Background: Typically, African healthcare providers use immunological reference intervals adopted from Europe and the United States (US). This may be inappropriate in a setting with many differences including exposure to different environmental stimuli and pathogens. We compared immunological reference intervals for children from Europe and the US with South African children to explore whether healthy children living in settings with high rates of infectious diseases have different baseline immunological parameters.Methodology: Blood was taken from 381 HIV-uninfected children aged between 2 weeks and 13 years of age from a Child Wellness Clinic in an informal settlement in Cape Town to establish local hematological and lymphocyte reference intervals for South African children. Flow-cytometry quantified percentage and absolute counts of the B-cells, NK-cells, and T-cells including activated, naïve, and memory subsets. These parameters were compared to three separate studies of healthy children in Europe and the US.Results: Increased activated T-cells, and natural killer cells were seen in the younger age-groups. The main finding across all age-groups was that the ratio of naïve/memory CD4 and CD8 T-cells reached a 1:1 ratio around the first decade of life in healthy South African children, far earlier than in resource-rich countries, where it occurs around the fourth decade of life.Conclusions: This is the largest data set to date describing healthy children from an African environment. These data have been used to create local reference intervals for South African children. The dramatic decline in the naïve/memory ratio of both CD4 and CD8 T-cells alongside increased activation markers may indicate that South African children are exposed to a wider range of environmental pathogens in early life than in resource-rich countries. These marked differences illustrate that reference intervals should be relevant to the population they serve. The implications for the developing pediatric immune system requires further investigation.

Single-cell repertoire tracing identifies rituximab-resistant B cells during myasthenia gravis relapses.

Rituximab, a B cell-depleting therapy, is indicated for treating a growing number of autoantibody-mediated autoimmune disorders. However, relapses can occur after treatment, and autoantibody-producing B cell subsets may be found during relapses. It is not understood whether these autoantibody-producing B cell subsets emerge from the failed depletion of preexisting B cells or are generated de novo. To further define the mechanisms that cause postrituximab relapse, we studied patients with autoantibody-mediated muscle-specific kinase (MuSK) myasthenia gravis (MG) who relapsed after treatment. We carried out single-cell transcriptional and B cell receptor profiling on longitudinal B cell samples. We identified clones present before therapy that persisted during relapse. Persistent B cell clones included both antibody-secreting cells and memory B cells characterized by gene expression signatures associated with B cell survival. A subset of persistent antibody-secreting cells and memory B cells were specific for the MuSK autoantigen. These results demonstrate that rituximab is not fully effective at eliminating autoantibody-producing B cells and provide a mechanistic understanding of postrituximab relapse in MuSK MG.

Transitional immature regulatory B cells and regulatory cytokines can discriminate chronic antibody-mediated rejection from stable graft function

BackgroundThe balance between inflammatory and anti-inflammatory responses of the immune system has been demonstrated to determine the fate of transplanted allografts. Here we analyzed CD19+CD24hiCD38hi immature transitional regulatory B (TRB) cells, as well as the gene and protein levels of interleukin (IL)-10 and transforming growth factor (TGF)-β in the three separate groups, include of stable transplanted subjects, chronic antibody-mediated rejection (cAMR) patients, and healthy individuals. MethodPeripheral blood mononuclear cells (PBMCs) from stable subjects (n = 36), cAMR patients (n = 36) and healthy controls (n = 18) were isolated. Flowcytometry was performed for CD19, CD24, and CD38 surface markers. ELISA and quantitative real-time PCR were performed for IL-10 and TGF-β cytokines. ResultThe percentages of immature TRB cells were significantly decrease in cAMR patients (0.98%) versus stable recipients (2.81%) and healthy subjects (4.03%) (P = 0.001 and P < 0.001, respectively). Total lymphocytes, circulating B cells, memory and mature subsets of B cells did not show any significant difference between the groups. TGF-β mRNA was 3-fold upregulated in the cAMR group compared to stable patients (P < 0.001.), but without significant alteration at the protein level. Also, long-term survival renal transplant recipients had a higher protein but not mRNA levels of IL-10 than short-term survival renal transplant recipients. ConclusionIt seems that immature TRB cell subpopulation might be a crucial regulator of immune system response and plays an important role in determining the transplantation outcome. Furthermore, immunosuppressive IL-10 and TGF-β cytokines might act as a double sword and can exhibit either pathogenic or protective effects against allograft.

Reappearance of effector T cells is associated with recovery from COVID-19.

BackgroundElucidating the role of T cell responses in COVID-19 is of utmost importance to understand the clearance of SARS-CoV-2 infection.Methods30 hospitalized COVID-19 patients and 60 age- and gender-matched healthy controls (HC) participated in this study. We used two comprehensive 11-colour flow cytometric panels conforming to Good Laboratory Practice and approved for clinical diagnostics.FindingsAbsolute numbers of lymphocyte subsets were differentially decreased in COVID-19 patients according to clinical severity. In severe disease (SD) patients, all lymphocyte subsets were reduced, whilst in mild disease (MD) NK, NKT and γδ T cells were at the level of HC. Additionally, we provide evidence of T cell activation in MD but not SD, when compared to HC. Follow up samples revealed a marked increase in effector T cells and memory subsets in convalescing but not in non-convalescing patients.InterpretationOur data suggest that activation and expansion of innate and adaptive lymphocytes play a major role in COVID-19. Additionally, recovery is associated with formation of T cell memory as suggested by the missing formation of effector and central memory T cells in SD but not in MD. Understanding T cell-responses in the context of clinical severity might serve as foundation to overcome the lack of effective anti-viral immune response in severely affected COVID-19 patients and can offer prognostic value as biomarker for disease outcome and control.FundingFunded by State of Lower Saxony grant 14–76,103–184CORONA-11/20 and German Research Foundation, Excellence Strategy – EXC2155“RESIST”–Project ID39087428, and DFG-SFB900/3–Project ID158989968, grants SFB900-B3, SFB900-B8.

TRM integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection

Tissue-resident memory CD8 T (TRM) cells are a unique immune memory subset that develops and remains in peripheral tissues at the site of infection, providing future host resistance upon reexposure to that pathogen. In the pulmonary system, TRM are identified through S1P antagonist CD69 and expression of integrins CD103/β7 and CD49a/CD29(β1). Contrary to the established role of CD69 on CD8 T cells, the functions of CD103 and CD49a on this population are not well defined. This study examines the expression patterns and functions of CD103 and CD49a with a specific focus on their impact on T cell motility during influenza virus infection. We show that the TRM cell surface phenotype develops by 2 wk postinfection, with the majority of the population expressing CD49a and a subset that is also positive for CD103. Despite a previously established role in retaining TRM in peripheral tissues, CD49a facilitates locomotion of virus-specific CD8 T cells, both in vitro and in vivo. These results demonstrate that CD49a may contribute to local surveillance mechanisms of the TRM population.

Modulation of inhibitory signals in CAR T cells leads to improved activity against glioblastoma.

3031 Background: Early clinical trials have demonstrated the safety of chimeric antigen receptor (CAR) T cells targeting glioblastoma (GBM), however, their efficacy remains limited by multiple obstacles including the immunosuppressive tumor microenvironment. Adoptively transferred CAR T cells remain susceptible to inhibition via the engagement of co-inhibitory receptors on their surface such as PD1, BTLA, CTLA4 and LAG3. The subsequent recruitment of Src homology region 2 containing protein tyrosine phosphatase 2 (SHP2) by these receptors to the immune synapse may represent a common mechanism of T cell inhibition, as SHP2 can de-phosphorylate key signaling molecules that mediate T cell activation (including CD28 and CD3ζ). We hypothesized that SHP2 deletion will simultaneously offset the effects of multiple co-inhibitory receptors, thereby improving the anti-tumor activity of CAR T cells. Methods: Electroporation of sgRNA/Cas9 ribonucleoprotein complexes into human T cells was used to knockout (KO) SHP2. Retroviral vector transduction was used to express a clinically-utilized second generation CAR (with a CD28 endodomain) targeting HER2. The phenotype of wild-type (WT) and SHP2KO CAR T cells was evaluated with mass cytometry and flow cytometry. Their anti-tumor function was tested in vitro using the xCELLigence assay (an impedance-based cytotoxicity assay), and in vivo, in an orthotopic xenograft mouse model of GBM. Results: Efficient and reproducible depletion of the SHP2 protein in human T cells was verified using western blotting. The Inference of CRISPR Efficiency (ICE) Assay confirmed efficient editing of the PTPN11 gene encoding SHP2. An anti-HER2 CAR was efficiently expressed in the SHP2KO T cells. SHP2 deletion did not significantly affect CAR T cell expansion, proliferation or baseline phenotype. However, following co-culture with HER2+ LN229-GBM cells, the CD8+ central memory (CCR7+ CD45RA-) and effector memory (CCR7- CD45RA-) subsets were enriched to a greater extent in the SHP2KO CAR T cells. The pattern of cytokine co-expression varied between donors in a single-cell analysis comparing SHP2KO to WT CAR T cells after encountering LN229 cells. Functionally, SHP2KO CAR T cells derived from the majority of healthy donor and patient peripheral blood eliminated LN229 cells more rapidly in vitro. In an orthotopic mouse model of GBM, SHP2KO CAR T cells showed better early control of established LN229 xenografts and improved survival in comparison to WT CAR T cells. Conclusions: SHP2 deletion in CD28ζ.CAR T cells improves their anti-tumor activity.

Characterization of Immune Phenotypes in Peripheral Blood of Adult Renal Transplant Recipients using Mass Cytometry (CyTOF)

Abstract Background Chronic immunosuppressive therapy in transplant patients can be associated with opportunistic infections and risks of cancer development. There is an important need to better measure the immune status of patients in a transplant setting to improve patient monitoring and guide development of appropriate immunosuppressive agents and regimen. Results In this study, CyTOF was performed to comprehensively characterize the immune differences between 10 renal transplant recipients and 11 age-matched healthy donors, longitudinally over 6 months. Overall, the immune cells of transplant patients showed signs of increased CD8+ T cell activation/differentiation. Higher levels of CD57 on CD8+ T cells were observed. CD8+ T cells and their memory subsets also showed increased production of MIP-1β and IFNγ. Further, CD107a and Granzyme B expression were also increased in CD8+ T cells and CD56hi NK cells, while Tregs had decreased IL-10 production. These changes are consistent with a more stimulated immune system, despite the administration of immune suppressants to the patients. However, there were also some signs of immune suppression in the transplant patients consistent with the treatment regimen. γδT cells showed decreased TNFα and IFNγ expression, while MIP-1β expression was reduced in NKT, CD56hi NK and γδT cells. CD8+ T memory cell subsets also showed decreased expression of IL-2, IL-17 and GM-CSF. Conclusions This mosaic pattern of functional changes highlights the importance of comprehensive immune monitoring of these patients. Such information will aid in optimizing individual treatment strategies and development of improved immunosuppressants that specifically target overly activated populations.

Generating protective immunity to severe influenza disease in Indigenous Australians

Abstract The 2009 influenza pandemic caused generally mild infections due, at least partially, to broadly cross-reactive pre-existing CD8+ T cell immunity. In contrast, severe disease was observed in Indigenous populations worldwide, as shown by disproportionate hospitalisation rates. Human leukocyte antigen-I (HLA-I) molecules present viral peptides to CD8+ T cells, eliciting anti-viral responses that contribute to accelerated viral clearance. We identified several HLA-I alleles which are highly prevalent in Indigenous Australians (HLA-A*11:01, A*24:02, A*34:01, B*13:01 and B*15:21) but largely understudied. Using specific HLA-allomorph expressing C1R cell lines, we identified influenza A and B immunopeptidomes by mass spectrometry. A total of 653 influenza peptides derived from various viral proteins were presented during infection by different HLA-Is. Using virus-specific PBMC expansions, we screened these peptides for their potential to reactivate influenza-specific memory CD8+ T cells. We identified on average 4.6 (2–8) immunogenic epitopes per HLA-I for influenza A and B. We defined epitope-specific CD8+ T cells ex vivo across different human tissues and determined their memory subsets, phenotype and activation in healthy donors and influenza-infected patients. Overall, our approach is effective in detection of immunogenic epitopes and understanding CD8+ T cell pools in Indigenous populations, thus providing potential vaccine targets to protect Indigenous populations globally, from severe influenza disease. This is the first study to identify hallmarks of immunogenic responses and reveal key targets of an effective CD8+ T cell vaccine in Indigenous people.