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In recent years, Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past, the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study, we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally, we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications.

Background: T lymphocytes in tumor microenvironment play a pivotal role in the anti-tumor immunity, and the memory of T cells contributes to the long-term protection against tumor antigens. Compared to solid tumors, studies focusing on the T-cell differentiation in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment remain limited., Patients and Methods: Fresh BM specimens collected from 103 adult AML patients at diagnosis and 12 healthy donors (HDs) were tested T-cell differentiation subsets by multi-parameter flow cytometry., Results: CD4 and CD8 T-cell compartments had different constituted profiles of T-cell differentiated subsets, which was similar between AML patients and HDs. Compared to HDs, AML patients as a whole had a significantly higher proportion of CD8 effector T cells (Teff, P = 0.048). Moreover, the T-cell compartment of AML patients with no DNMT3A mutations skewed toward terminal differentiation at the expense of memory T cells (CD4 Teff: P = 0.034; CD8 Teff: P = 0.030; CD8 memory T: P = 0.017), whereas those with mutated DNMT3A had a decrease in CD8 naïve T (Tn) and CD4 effector memory T cells (Tem) as well as an increase in CD4 central memory T cells (Tcm) ( P = 0.037, 0.053 and 0.053). Adverse ELN genetic risk correlated with a lower proportion of CD8 Tn. In addition, the low proportions of CD4 Tem and CD8 Tn independently predicted poorer relapse-free survival (RFS, HR [95%CI]: 5.7 (1.4-22.2), P = 0.017 and 4.8 [1.3-17.4], P = 0.013) and event-free survival (EFS, HR [95% CI]: 3.3 (1.1-9.5), P = 0.029; 4.0 (1.4-11.5), P = 0.010), respectively., Conclusions: AML patients had abnormal profiles of BM T-cell differentiation subsets at diagnosis, which was related to DNMT3A mutations. The low proportions of CD4 Tem and CD8 Tn predicted poor outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Shi, Wang, Xie, Liu, Jiang, Jiang, Huang and Qin.)

Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin + cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Historically, the immune system was believed to develop along a linear axis of maturity from fetal life to adulthood. Now, it is clear that distinct layers of immune cells are generated from unique waves of hematopoietic progenitors during different windows of development. This model, known as the layered immune model, has provided a useful framework for understanding why distinct lineages of B cells and γδ T cells arise in succession and display unique functions in adulthood. However, the layered immune model has not been applied to CD8 + T cells, which are still often viewed as a uniform population of cells belonging to the same lineage, with functional differences between cells arising from environmental factors encountered during infection. Recent studies have challenged this idea, demonstrating that not all CD8 + T cells are created equally and that the functions of individual CD8 + T cells in adults are linked to when they were created in the host. In this review, we discuss the accumulating evidence suggesting there are distinct ontogenetic subpopulations of CD8 + T cells and propose that the layered immune model be extended to the CD8 + T cell compartment., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

After exposure to an antigen, CD8 T cells reach a decision point about their fate: to become either short-lived effector cells (SLECs) or memory progenitor effector cells (MPECs). SLECs are specialized in providing an immediate effector function but have a shorter lifespan and lower proliferative capacity compared to MPECs. Upon encountering the cognate antigen during an infection, CD8 T cells rapidly expand and then contract to a level that is maintained for the memory phase after the peak of the response. Studies have shown that the contraction phase is mediated by TGFβ and selectively targets SLECs, while sparing MPECs. The aim of this study is to investigate how the CD8 T cell precursor stage determines TGFβ sensitivity. Our results demonstrate that MPECs and SLECs have differential responses to TGFβ, with SLECs being more sensitive to TGFβ than MPECs. This difference in sensitivity is associated with the levels of TGFβRI and RGS3, and the SLEC-related transcriptional activator T-bet binding to the TGFβRI promoter may provide a molecular basis for increased TGFβ sensitivity in SLECs.

Increased demand for novel, highly effective vaccination strategies necessitates a better understanding of long-lived memory CD8 T cell differentiation. To achieve this understanding, we used the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. We reexamined classical memory CD8 T cell subsets and performed in-depth, longitudinal analysis of their phenotype, transcriptional programming, and anatomic location within the spleen. All analyses were performed at multiple time points from 8 days to 1 year postinfection. Memory subsets are conventionally defined by their expression of KLRG1 and IL-7Rα, as follows: KLRG1 + IL-7Rα - terminal effectors (TEs) and KLRG1 - IL-7Rα + memory precursors (MPs). But we also characterized a third KLRG1 + IL-7Rα + subset which we refer to as KLRG1 + MPs. In these analyses, we defined a comprehensive memory phenotype that is associated with higher levels of CD28 expression. We also demonstrated that MPs, KLRG1 + MPs, and TEs have distinct localization programs within the spleen. We found that MPs became preferentially enriched in the white pulp as early as 1 to 2 weeks postinfection, and their predominance in the white pulp was maintained throughout the course of a year. On the other hand, KLRG1 + MPs and TEs localized to the red pulp just as early, and they consistently localized to the red pulp thereafter. These findings indicate that location may be crucial for memory formation and that white pulp-derived signals may contribute to long-term memory survival. Achieving robust memory responses following vaccination may require more deliberate consideration of which memory phenotypes are induced, as well as where they traffic, as these factors could impact their longevity. IMPORTANCE CD8 T cells play a critical role in viral immunity and it is important to understand how memory cells are formed and what processes lead to their long-term maintenance. Here, we use a mouse model of acute infection to perform an in-depth, longitudinal analysis of memory CD8 T cell differentiation, examining the phenotype and location of memory cells out to 1 year postinfection.

Post-Covid pulmonary fibrosis is evident following severe COVID-19. There is an urgent need to identify the cellular and pathophysiological characteristics of chronic lung squeals of Covid-19 for the development of future preventive and/or therapeutic interventions. Tissue-resident memory T (T RM ) cells can mediate local immune protection against infections and cancer. Less beneficially, lung T RM cells cause chronic airway inflammation and fibrosis by stimulating pathologic inflammation. The effects of Janus kinase (JAK), an inducer pathway of cytokine storm, inhibition on acute Covid-19 cases have been previously evaluated. Here, we propose that Tofacitinib by targeting the CD8 + T RM cells could be a potential candidate for the treatment of chronic lung diseases induced by acute SARS-CoV-2 infection., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

T cell receptor (TCR) signal strength is a key determinant of T cell responses. We developed a cancer mouse model in which tumor-specific CD8 T cells (TST cells) encounter tumor antigens with varying TCR signal strength. High-signal-strength interactions caused TST cells to up-regulate inhibitory receptors (IRs), lose effector function, and establish a dysfunction-associated molecular program. TST cells undergoing low-signal-strength interactions also up-regulated IRs, including PD1, but retained a cell-intrinsic functional state. Surprisingly, neither high- nor low-signal-strength interactions led to tumor control in vivo, revealing two distinct mechanisms by which PD1hi TST cells permit tumor escape; high signal strength drives dysfunction, while low signal strength results in functional inertness, where the signal strength is too low to mediate effective cancer cell killing by functional TST cells. CRISPR-Cas9-mediated fine-tuning of signal strength to an intermediate range improved anti-tumor activity in vivo. Our study defines the role of TCR signal strength in TST cell function, with important implications for T cell-based cancer immunotherapies., Competing Interests: Disclosures: N.A. Defranoux reports personal fees from Alector outside the submitted work. M.D. Hellman receives institutional research funding from Bristol-Myers Squibb; has been a compensated consultant for Merck, Bristol-Myers Squibb, AstraZeneca, Genentech/Roche, Nektar, Syndax, Mirati, Shattuck Labs, Immunai, Blueprint Medicines, Achilles, and Arcus; received travel support/honoraria from AstraZeneca, Eli Lilly, and Bristol-Myers Squibb; has options from Factorial, Shattuck Labs, Immunai, and Arcus; reported personal fees from Adicet, DaVolterra, Genzyme/Sanofi, Janssen, Immunai, Instil Bio, Mana Therapeutics, Natera, Pact Pharms, Chattuck Labs, and Regenron out side the submitted work; and has a patent filed by his institution related to the use of tumor mutation burden to predict response to immunotherapy (PCT/US2015/062208), which has received licensing fees from PGDx. D.K. Wells is a founder, equity holder, and receives advisory fees from Immunai. No other disclosures were reported., (© 2021 Shakiba et al.)

Classic T cell subsets are defined by a small set of cell surface markers, while single-cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq clustered populations (scCPops) and cell surface marker-defined classic T cell subsets remains unclear. In this article, we integrated six bead-enriched T cell subsets with 62,235 single-cell transcriptomes from human PBMCs and clustered them into nine scCPops. Bead-enriched CD4 + /CD45RA + /CD25 - naive T and CD8 + /CD45RA + naive T cells were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops, including mucosal-associated invariant T cells and NKT cells. The multiple T cell subsets forming one scCPop exhibit similar expression patterns, but not vice versa, indicating scCPop is a more homogeneous cell population with similar cell states. Interestingly, we discovered and named IFN signaling-associated gene (ISAG) high T (ISAG hi T) cells, a T cell subpopulation that highly expressed ISAGs. We further enriched ISAG hi T cells from human PBMCs by FACS of BST2 for scRNA-seq analyses. The ISAG hi T cell cluster disappeared on t -distributed stochastic neighbor embedding plot after removing ISAGs, whereas the ISAG hi T cell cluster showed up by analysis of ISAGs alone, indicating ISAGs are the major contributor of the ISAG hi T cell cluster. BST2 + and BST2 - T cells showing different efficiencies of T cell activation indicate that a high level of ISAGs may contribute to quick immune responses., (Copyright © 2022 by The American Association of Immunologists, Inc.)

According to the WHO 2009 classification, dengue with warning signs is at the risk of developing severe form of dengue disease. One of the most important warning signs is plasma leakage, which can be a serious complication associated with higher morbidity and mortality. We report that the frequency of CD4 + CD8 + double-positive (DP) T cells is significantly increased in patients at risk of developing plasma leakage. Transcriptomic analysis demonstrated that CD4 + CD8 + DP cells were distinct from CD4 + Single Positive (SP) T cells but co-clustered with CD8 + SP cells, indicating a largely similar transcriptional profile. Twenty significant differentially expressed (DE) genes were identified between CD4 + CD8 + DP and CD8 + SP cells. These genes encode OX40 and CCR4 proteins as well as other molecules associated with cell signaling on the cell surface ( NT5E , MXRA8 , and PTPRK ). While comparing the profile of gene expression in CD4 + CD8 + DP cells from patients with and without warning signs of plasma leakage, similar expression profile was observed, implying a role of CD4 + CD8 + DP cells in plasma leakage through a quantitative increase rather than functional alteration. This study provided novel insight into the host immune response during the acute febrile phase of DENV infection and the role of CD4 + CD8 + DP T cells in the pathogenesis of plasma leakage.

Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS "CD8+ TOXPHOS cells." We validated that higher levels of OXPHOS in tumor- and peripheral blood-derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients., Competing Interests: Disclosures: P. Funchain reported grants from Pfizer and Bristol Myers Squibb and personal fees from Eisai outside the submitted work. A. Rodriguez reported non-financial support from Lipid Genomics during the conduct of the study and non-financial support from Lipid Genomics outside the submitted work; in addition, A. Rodriguez had a patent to LAG3 issued (Lipid Genomics). No other disclosures were reported., (© 2021 Li et al.)

T cells play a central role in cancer immunotherapy, but we lack systematic comparison of the heterogeneity and dynamics of tumor-infiltrating T cells across cancer types. We built a single-cell RNA-sequencing pan-cancer atlas of T cells for 316 donors across 21 cancer types and revealed distinct T cell composition patterns. We found multiple state-transition paths in the exhaustion of CD8 + T cells and the preference of those paths among different tumor types. Certain T cell populations showed specific correlation with patient properties such as mutation burden, shedding light on the possible determinants of the tumor microenvironment. T cell compositions within tumors alone could classify cancer patients into groups with clinical trait specificity, providing new insights into T cell immunity and precision immunotherapy targeting T cells.

T cell exhaustion limits anti-tumor immunity and responses to immunotherapy. Here, we explored the microenvironmental signals regulating T cell exhaustion using a model of chronic lymphocytic leukemia (CLL). Single-cell analyses identified a subset of PD-1 hi , functionally impaired CD8 + T cells that accumulated in secondary lymphoid organs during disease progression and a functionally competent PD-1 int subset. Frequencies of PD-1 int TCF-1 + CD8 + T cells decreased upon Il10rb or Stat3 deletion, leading to accumulation of PD-1 hi cells and accelerated tumor progression. Mechanistically, inhibition of IL-10R signaling altered chromatin accessibility and disrupted cooperativity between the transcription factors NFAT and AP-1, promoting a distinct NFAT-associated program. Low IL10 expression or loss of IL-10R-STAT3 signaling correlated with increased frequencies of exhausted CD8 + T cells and poor survival in CLL and in breast cancer patients. Thus, balance between PD-1 hi , exhausted CD8 + T cells and functional PD-1 int TCF-1 + CD8 + T cells is regulated by cell-intrinsic IL-10R signaling, with implications for immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Programmed cell death-1 (PD-1) blockade has a profound effect on the ability of the immune system to eliminate tumors, but many questions remain about the cell types involved and the underlying mechanisms of immune activation. To shed some light on this, the cellular and molecular events following inhibition of PD-1 signaling was investigated in the MC-38 colon carcinoma model using constitutive (PD-1 KO) and conditional (PD1cKO) mice and in wild-type mice treated with PD-1 antibody. The impact on both tumor growth and the development of tumor immunity was assessed. In the PD-1cKO mice, a complete deletion of Pdcd1 in tumor-infiltrating T cells (TILs) after tamoxifen treatment led to the inhibition of tumor growth of both small and large tumors. Extensive immune phenotypic analysis of the TILs by flow and mass cytometry identified 20-different T cell subsets of which specifically 5-CD8 positive ones expanded in all three models after PD-1 blockade. All five subsets expressed granzyme B and interferon gamma (IFNγ). Gene expression analysis of the tumor further supported the phenotypic analysis in both PD-1cKO- and PD-1 Ab-treated mice and showed an upregulation of pathways related to CD4 and CD8 T-cell activation, enhanced signaling through costimulatory molecules and IFNγ, and non-T-cell processes. Altogether, using PD-1cKO mice, we define the intrinsic nature of PD-1 suppression of CD8 T-cell responses in tumor immunity., Competing Interests: Authors SR, NT-S, AS, MS, EP and AC were employed by Merck & Co., Inc., Kenilworth, NJ, USA. CK and RW were employed by Merck & Co., Inc., Kenilworth, NJ, USA at the time of the study and are currently employed by Synthekine. WB, EM, and TR are currently employed by Merck & Co., Inc., Kenilworth, NJ, USA., (Copyright © 2021 Raghavan, Tovbis-Shifrin, Kochel, Sawant, Mello, Sathe, Blumenschein, Muise, Chackerian, Pinheiro, Rosahl, Luche and de Waal Malefyt.)

The infiltration of tumor-reactive T cells in the tumor site is associated with better survival and immunotherapy response. However, tumor-reactive T cells were often represented by the infiltration of total CD8+ T cells, which was confounded by the presence of bystander T cells. To identify tumor-reactive T cells at the cancer lesion, we performed integration analyses of three scRNA-seq data sets of T cells in melanoma. Extensive heterogeneous functional states of T cells were revealed in the tumor microenvironment. Among these states, we identified a subset of tumor-reactive T cells which specifically expressed tumor-reactive markers and T cell activation signature, and were strongly enriched for larger T cell receptor (TCR) clones. We further identified and validated a tumor-reactive T cell signature (TRS) to evaluate the tumor reactivity of T cells in tumor patients. Patients with high TRS scores have strong immune activity and high mutation burden in the TCGA-SKCM cohort. We also demonstrated a significant association of the TRS with the clinical outcomes of melanoma patients, with higher TRS scores representing better survival, which was validated in four external independent cohorts. Furthermore, the TRS scores exhibited greater performance on prognosis prediction than infiltration levels of CD8+ T cells and previously published prognosis-related signatures. Finally, we observed the capability of TRS to predict immunotherapy response in melanoma. Together, based on integrated analysis of single-cell RNA-sequencing, we developed and validated a tumor-reactive-related signature that demonstrated significant association with clinical outcomes and response to immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yan, Hu, Ping, Xu, Liao, Jiang, Pang, Sun, Zhang, Xiao and Li.)

Background: In infants admitted to an ICU with respiratory failure, there is an association between the ratio of CD8 + to CD4 + T cells within the upper respiratory tract and disease severity. Whether this ratio is associated with respiratory disease severity within children presenting to a pediatric emergency department is not known., Methods: We studied a convenience sample of 63 children presenting to a pediatric emergency department with respiratory symptoms. T cell subsets in the nasal mucosa were analyzed by flow cytometry. We compared CD4 + and CD8 + T cells subsets in these samples and analyzed the proportion of these subsets that expressed markers associated with tissue residency., Results: We were able to identify major subsets of CD8 and CD4 T cells within the nasal mucosa using flocked swabs. We found no difference in the ratio CD8 + to CD4 + T cells in children with upper or lower respiratory illness. A positive association between tissue-resident memory T cell frequency and patient age was identified., Conclusions: In our patient populations, the CD8 + :CD4 + ratio was not associated with disease severity. The majority of T cells collected on nasal swabs are antigen experienced, and there is an association between the frequency of tissue-resident T cells and age., Impact: Immune cell populations from the nasal mucosa can be captured using flocked nasal swabs and analyzed by flow cytometry. Nasal CD8 + :CD4 + ratio does not predict respiratory illness severity in children presenting to the emergency department. The frequency of CD8 + and CD4 + resident memory T cells within the nasal mucosa increases with age., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

CD8+ T cells play a key role in mediating protective immunity after immune challenges such as infection or vaccination. Several subsets of differentiated CD8+ T cells have been identified, however, a deeper understanding of the molecular mechanism that underlies T-cell differentiation is lacking. Conventional approaches to the study of immune responses are typically limited to the analysis of bulk groups of cells that mask the cells' heterogeneity (RNA-seq, microarray) and to the assessment of a relatively limited number of biomarkers that can be evaluated simultaneously at the population level (flow and mass cytometry). Single-cell analysis, on the other hand, represents a possible alternative that enables a deeper characterization of the underlying cellular heterogeneity. In this study, a murine model was used to characterize immunodominant hemagglutinin (HA 533-541 )-specific CD8+ T-cell responses to nucleic- and protein-based influenza vaccine candidates, using single-cell sorting followed by transcriptomic analysis. Investigation of single-cell gene expression profiles enabled the discovery of unique subsets of CD8+ T cells that co-expressed cytotoxic genes after vaccination. Moreover, this method enabled the characterization of antigen specific CD8+ T cells that were previously undetected. Single-cell transcriptome profiling has the potential to allow for qualitative discrimination of cells, which could lead to novel insights on biological pathways involved in cellular responses. This approach could be further validated and allow for more informed decision making in preclinical and clinical settings., Competing Interests: This work was undertaken at the request of and sponsored by GlaxoSmithKline Biologicals SA. GSK directed all aspects of this study. CS, ST, SN, RK, ES and SB are all employees of the GSK group of companies. SN, DMe, ES, SB hold GSK shares. MF59 is a trademark of Novartis AG., (Copyright © 2021 Meldgaard, Blengio, Maffione, Sammicheli, Tavarini, Nuti, Kratzer, Medini, Siena and Bertholet.)

Exhaustion of HIV-1-specific CD8 + T cells prevents optimal control of HIV-1 infection. Identifying unconventional CD8 + T cell subsets to effectively control HIV-1 replication is vital. In this study, the role of CD11c + CD8 + T cells during HIV-1 infection was evaluated. The frequencies of CD11c + CD8 + T cells significantly increased and were negatively correlated with viral load in HIV-1-infected treatment-naïve patients. HIV-1-specific cells were enriched more in CD11c + CD8 + T cells than in CD11c - CD8 + T cells, which could be induced by HIV-1-derived overlapping peptides, marking an HIV-1-specific CD8 + T cell population. This subset expressed higher levels of activating markers (CD38 and HLA-DR), cytotoxic markers (granzyme B, perforin, and CD107a), and cytokines (IL-2 and TNF-α), with lower levels of PD-1 compared to the CD11c - CD8 + T cell subset. In vitro analysis verified that CD11c + CD8 + T cells displayed a stronger HIV-1-specific killing capacity than the CD11c - counterparts. These findings indicate that CD11c + CD8 + T cells have potent immunotherapeutic efficacy in controlling HIV-1 infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Guo, Zhao, Gao, Huang, Zhang, Zhang, Song, Xu, Fan, Shi, Jiao and Wang.)

Most persons living with HIV (PLWH) experience a significant restoration of their immunity associated with successful inhibition of viral replication after antiretroviral therapy (ART) initiation. Nevertheless, with the robust quantitative and qualitative restoration of CD4 + T-lymphocytes, a fraction of patients co-infected with tuberculosis develop immune reconstitution inflammatory syndrome (TB-IRIS), a dysregulated inflammatory response that can be associated with significant tissue damage. Several studies underscored the role of adaptive immune cells in IRIS pathogenesis, but to what degree T lymphocyte activation contributes to TB-IRIS development remains largely elusive. Here, we sought to dissect the phenotypic landscape of T lymphocyte activation in PLWH coinfected with TB inititating ART, focusing on characterization of the profiles linked to development of TB-IRIS. We confirmed previous observations demonstrating that TB-IRIS individuals display pronounced CD4 + lymphopenia prior to ART initiation. Additionally, we found an ART-induced increase in T lymphocyte activation, proliferation and cytotoxicity among TB-IRIS patients. Importantly, we demonstrate that TB-IRIS subjects display higher frequencies of cytotoxic CD8 + T lymphocytes which is not affected by ART. Moreover, These patients exhibit higher levels of activated (HLA-DR + ) and profilerative (Ki-67 + ) CD4 + T cells after ART commencenment than their Non-IRIS counterparts. Our network analysis reveal significant negative correlations between Total CD4 + T cells counts and the frequencies of Cytotoxic CD8 + T cells in our study population which could suggest the existance of compensatory mechanisms for Mtb-infected cells elimination in the face of severe CD4 + T cell lymphopenia. We also investigated the correlation between T lymphocyte activation profiles and the abundance of several inflammatory molecules in plasma. We applied unsupervised machine learning techniques to predict and diagnose TB-IRIS before and during ART. Our analyses suggest that CD4 + T cell activation markers are good TB-IRIS predictors, whereas the combination of CD4 + and CD8 + T cells markers are better at diagnosing TB-IRIS patients during IRIS events Overall, our findings contribute to a more refined understanding of immunological mechanisms in TB-IRIS pathogenesis that may assist in new diagnostic tools and more targeted patient management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tibúrcio, Barreto-Duarte, Naredren, Queiroz, Anbalagan, Nayak, Ravichandran, Subramani, Antonelli, Satagopan, Anbalagan, Porter, Sher, Swaminathan, Sereti and Andrade.)

Background: Tuberculosis still threatens human health. We aimed to investigate the T cell immune status and the role of multifunctional T cells in pulmonary tuberculosis patients., Methods: Thirty active pulmonary tuberculosis (APTB) patients, 30 latent tuberculosis infection (LTBI) patients, 25 cured pulmonary tuberculosis (CPTB) patients and 25 healthy controls (HCs) enrolled in this study. Flow cytometer for detecting T cell phenotype and function. CBA Flex Set was used to measure chemokine., Results: Compared with HCs and LTBI patients, APTB patients had fewer CD4 + T and CD8 + T cells, but the expression of granzyme A, granzyme B and perforin on CD8 + T cells increased. Compared to LTBI and CPTB patients, Mycobacterium tuberculosis-specific CD8 + T cells in APTB patients appeared to be more differentiated CD45RA - CCR7 - cells, and there were more multifunctional CD4 + T and CD8 + T cells. Importantly, the frequency of multifunctional CD4 + T cells in the pleural fluid of APTB patients was higher than that of peripheral blood. And the proportion of multifunctional CD4 + T cells expressing the migration receptor CXCR3 in the peripheral blood of APTB patients decreased, while the concentrations of its ligands, chemokine MIG, IP-10 and I-TAC increased significantly in plasma, especially in pleural fluid., Conclusions: Decreased T lymphocytes in APTB patients may cause compensatory activation of CD8 + T cells. Multifunctional CD4 + T cells in peripheral blood could migrate to the lungs under the action of CXCR3 and associated chemokine. Multifunctional CD4 + T cells and Multifunctional CD8 + T cells were of great significance in monitoring disease treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sepsis reduces the number and function of memory CD8 T cells within the host, contributing to the long-lasting state of immunoparalysis. Interestingly, the relative susceptibility of memory CD8 T cell subsets to quantitative/qualitative changes differ after cecal ligation and puncture (CLP)-induced sepsis. Compared with circulatory memory CD8 T cells (T CIRCM ), moderate sepsis (0-10% mortality) does not result in numerical decline of CD8 tissue-resident memory T cells (T RM ), which retain their "sensing and alarm" IFN-γ-mediated effector function. To interrogate this biologically important dichotomy, vaccinia virus-immune C57BL/6 (B6) mice containing CD8 T CIRCM and skin T RM underwent moderate or severe (∼50% mortality) sepsis. Severe sepsis led to increased morbidity and mortality characterized by increased inflammation compared with moderate CLP or sham controls. Severe CLP mice also displayed increased vascular permeability in the ears. Interestingly, skin CD103 + CD8 T RM , detected by i.v. exclusion or two-photon microscopy, underwent apoptosis and subsequent numerical loss following severe sepsis, which was not observed in mice that experienced moderate CLP or sham surgeries. Consequently, severe septic mice showed diminished CD8 T cell-mediated protection to localized skin reinfection. Finally, the relationship between severity of sepsis and demise in circulatory versus tissue-embedded memory CD8 T cell populations was confirmed by examining tumor-infiltrating and nonspecific CD8 T cells in B16 melanoma tumors. Thus, sepsis can differentially affect the presence and function of Ag-specific CD8 T cells that reside inside tissues/tumors depending on the severity of the insult, a notion with direct relevance to sepsis survivors and their ability to mount protective memory CD8 T cell-dependent responses to localized Ag re-encounter., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The T cell immune responses in filarial infections are primarily mediated by CD4 + T cells and type 2-associated cytokines. Emerging evidence indicates that CD8 + T cell responses are important for anti-filarial immunity, however, could be suppressed in co-infections. This review summarizes what we know so far about the activities of CD8 + T cell responses in filarial infections, co-infections, and the associations with the development of filarial pathologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kwarteng, Asiedu, Koranteng and Asiedu.)

Cytomegalovirus (CMV) latent infection and aging contribute to alterations in the function and phenotype of the T-cell pool. We have demonstrated that CMV-seropositivity is associated with the expansion of polyfunctional CD57+ T-cells in young and middle-aged individuals in response to different stimuli. Here, we expand our results on the effects of age and CMV infection on T-cell functionality in a cohort of healthy middle-aged and older individuals stratified by CMV serostatus. Specifically, we studied the polyfunctional responses (degranulation, IFN-γ and TNF-α production) of CD4+, CD8+, CD8+CD56+ (NKT-like), and CD4-CD8- (DN) T-cells according to CD57 expression in response to Staphylococcal Enterotoxin B (SEB). Our results show that CD57 expression by T-cells is not only a hallmark of CMV infection in young individuals but also at older ages. CD57+ T-cells are more polyfunctional than CD57- T-cells regardless of age. CMV-seronegative individuals have no or a very low percentages of cytotoxic CD4+ T-cells (CD1017a+) and CD4+CD57+ T-cells, supporting the notion that the expansion of these T-cells only occurs in the context of CMV infection. There was a functional shift in T-cells associated with CMV seropositivity, except in the NKT-like subset. Here, we show that the effect of CMV infection and age differ among T-cell subsets and that CMV is the major driving force for the expansion of highly polyfunctional CD57+ T-cells, emphasizing the necessity of considering CMV serology in any study of immunosenescence.

During chronic viral infection, CD8 + T cells develop into three major phenotypically and functionally distinct subsets: Ly108 + TCF-1 + progenitors, Ly108 - CX 3 CR1 - terminally exhausted cells and the recently identified CX 3 CR1 + cytotoxic effector cells. Nevertheless, how CX 3 CR1 + effector cell differentiation is transcriptionally and epigenetically regulated remains elusive. Here, we identify distinct gene regulatory networks and epigenetic landscapes underpinning the formation of these subsets. Notably, our data demonstrate that CX 3 CR1 + effector cells bear a striking similarity to short-lived effector cells during acute infection. Genetic deletion of Tbx21 significantly diminished formation of the CX 3 CR1 + subset. Importantly, we further identify a previously unappreciated role for the transcription factor BATF in maintaining a permissive chromatin structure that allows the transition from TCF-1 + progenitors to CX 3 CR1 + effector cells. BATF directly bound to regulatory regions near Tbx21 and Klf2, modulating their enhancer accessibility to facilitate the transition. These mechanistic insights can potentially be harnessed to overcome T cell exhaustion during chronic infection and cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Ageing dramatically affects number and function of both innate and adaptive arms of immune system, particularly T cell subsets, contributing to reduced vaccination efficacy, decreased resistance to infections and increased prevalence of cancer in older people. In the present paper, we analysed the age-related changes in the absolute number of lymphocytes in 214 Sicilian subjects, and in the percentages of T and natural killer (NK) cells in a subcohort of donors. We compared these results with the immunophenotype of the oldest living Italian supercentenarian (aged 111 years). The results were also sorted by gender. The correlation between number/percentage of cells and age in all individuals. and separately in males and females, was examined using a simple linear regression analysis. We did not record the increase in the rate of inversion of the CD4/CD8 ratio, frequently reported as being associated with ageing in literature. Our observation was the direct consequence of a flat average trend of CD4 + and CD8 + T cell percentages in ageing donors, even when gender differences were included. Our results also suggest that CD4 + and CD8 + subsets are not affected equally by age comparing females with males, and we speculated that gender may affect the response to cytomegalovirus (CMV) infection. The supercentenarian showed a unique immunophenotypic signature regarding the relative percentages of her T cell subsets, with CD4 + and CD8 + T cell percentages and CD4 + naive T cell values in line with those recorded for the octogenarian subjects. This suggests that the supercentenarian has a naive 'younger' T cell profile comparable to that of a >80-year-old female., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Introduction: Gaps still exist regarding knowledge on regulatory cells in transplant recipients. We studied the phenotypic patterns of CD4+, CD8+CD28- Tregs, and CD19+IL-10+ Bregs in the blood of healthy controls (HC), end-stage kidney disease patients (ESKD), early and late stable renal transplant recipients (Tx), and transplant recipients with steroid-treated acute cellular rejection 1 week-3 months after successful treatment. We also investigated the relationship between immunosuppressive drugs and the aforementioned regulatory cells in transplant recipients., Methods: We recruited 32 HC, 83 ESKD, 51 early Tx, 95 late Tx, and 9 transplant patients with a recent steroid-treated acute cellular rejection. Besides CD19+IL-10+ Bregs, we analyzed absolute and relative frequencies of CD4+CD25+CD127-Foxp3+ Tregs and CD8+CD28- Tregs and their expression of IL-10, TGF-ß, IFN-g, and Helios., Results: We found a negative correlation between absolute CD4+CD25+CD127-Foxp3+ Treg and relative CD19+IL-10+ Breg frequencies in early Tx recipients (r=-0.433, p =0.015, n=31). In that group, absolute CD4+CD25+CD127-Foxp3+ Tregs were negatively associated with steroid dose and tacrolimus trough levels (r=-0.377, p = 0.021, n=37; r=-0.43, p =0.033, n=25, respectively), opposite to IL-10+ Bregs, whose frequency apparently was not negatively affected by potent immunosuppression early posttransplant. We found also lower CD4+CD25+CD127-Foxp3+ Tregs in patients treated with basiliximab or rATG as compared with ESKD patients ( p =0.001 and p < 0.001, respectively). No difference in absolute IL-10+ Bregs could be detected among these 3 patient groups. Early Tx recipients showed lower CD4+CD25+CD127-Foxp3+ Tregs within 3 months of antibody induction than after 3 months ( p = 0.034), whereas IL-10+ Bregs showed higher relative counts during the first 3 months post antibody induction than after 3 months ( p = 0.022). Our findings suggest that IL-10+ Bregs decrease with time posttransplantation independent of the effect of antibody induction and dose of other immunosuppressive drugs., Conclusion: These findings suggest that CD19+IL-10+ Bregs and CD4+CD25+CD127-Foxp3+ Tregs behave in opposite ways during the early posttransplant period, possibly due to a predominant negative impact of high doses of immunosuppressants on Tregs. CD19+IL-10+Bregs do not seem to be suppressed by antibody induction and early potent immunosuppression with chemical drugs., Competing Interests: CM and GO together with the University of Heidelberg, are cofounders of TolerogenixX GmbH, a biotechnology company that holds licenses for modified immune cell (MIC) treatment. GO holds a patent for MIC treatment (“Immunosuppressive blood cells and methods of producing the same.” Patent no. WO 2010/000730, EP 2318020). CM and VD filed a patent application for MIC treatment (“MIC therapy for specific immunosuppression in transplantation” Patent no. PCT/EP2019/062857). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aly, Ibrahim, Karakizlis, Weimer, Opelz, Morath, Zeier, Ekpoom and Daniel.)

Herein we measured CD4+ T-cell responses against common cold coronaviruses (CCC) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC-reactive T cells in SARS-CoV-2-seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 T-cell reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses is consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC T-cell reactivity was decreased in SARS-CoV-2-infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego. CD4+ T-cell responses against common cold coronaviruses (CCC) are elevated in SARS-CoV-2 seronegative high-risk health care workers (HCW) compared to COVID-19 convalescent HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses and/or cross-reactivity associated with a protective effect., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Objective: To compare immune cell phenotype and function in psoriatic arthritis (PsA) versus psoriasis in order to better understand the pathogenesis of PsA., Methods: In-depth immunophenotyping of different T cell and dendritic cell subsets was performed in patients with PsA, psoriasis, or axial spondyloarthritis and healthy controls. Subsequently, we analyzed cells from peripheral blood, synovial fluid (SF), and skin biopsy specimens using flow cytometry, along with high-throughput transcriptome analyses and functional assays on the specific cell populations that appeared to differentiate PsA from psoriasis., Results: Compared to healthy controls, the peripheral blood of patients with PsA was characterized by an increase in regulatory CD4+ T cells and interleukin-17A (IL-17A) and IL-22 coproducing CD8+ T cells. One population specifically differentiated PsA from psoriasis: i.e., CD8+CCR10+ T cells were enriched in PsA. CD8+CCR10+ T cells expressed high levels of DNAX accessory molecule 1 and were effector memory cells that coexpressed skin-homing receptors CCR4 and cutaneous lymphocyte antigen. CD8+CCR10+ T cells were detected under inflammatory and homeostatic conditions in skin, but were not enriched in SF. Gene profiling further revealed that CD8+CCR10+ T cells expressed GATA3, FOXP3, and core transcriptional signature of tissue-resident memory T cells, including CD103. Specific genes, including RORC, IFNAR1, and ERAP1, were up-regulated in PsA compared to psoriasis. CD8+CCR10+ T cells were endowed with a Tc2/22-like cytokine profile, lacked cytotoxic potential, and displayed overall regulatory function., Conclusion: Tissue-resident memory CD8+ T cells derived from the skin are enhanced in the circulation of patients with PsA compared to patients with psoriasis alone. This may indicate that aberrances in cutaneous tissue homeostasis contribute to arthritis development., (© 2021 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Background/aim: The role of immune cells and PD-L1 in cutaneous squamous carcinogenesis is unclear. This study examines T-cell populations, Langerhans cells (LCs) and PD-L1 in invasive squamous cell carcinoma (inSCC), adjacent precursors and normal skin (NS) to investigate their participation in tumorigenesis., Materials and Methods: Cases of cutaneous inSCC with adjacent precursors (n=125) were selected. In situ SCC (isSCC) and actinic keratosis (AK) were observed in 53 and 123 cases, respectively, whereas NS was present in 123 lesions. Immunohistochemistry was performed for CD3, CD8, Foxp3, CD1a and PD-L1., Results: T-cells, LCs and PD-L1 gradually increase during the evolution from AK to isSCC and inSCC, with statistical significance between all lesions, except for CD3+ and CD8+ cells between isSCC and inSCC. Epithelial PD-L1 expression correlates with tumor diameter and thickness., Conclusion: The progressive increase of T-cells, LCs and PD-L1 in cutaneous squamous carcinogenesis provides rationale for immunotherapy and identification of predictive biomarkers., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

IL-26 is a newly discovered IL-10 cytokine family member mainly secreted by Th17 cells. However, the relationship between IL-26 and lung cancer remains unclear. The present study reported that IL-26 is involved in the production and promotion of malignant pleural effusion (MPE) for the first time. The concentrations of IL-26 and several Th17-related cytokines in MPE and peripheral blood (PB) from MPE patients were measured. IL-26, IL-10, and IL-6 were elevated in MPE compared to PB. The cell resource of IL-26 was primary Th17 cells measured by flow cytometry, whereas Tc17 cells and macrophages could also contribute to higher concentration of IL-26 in MPE. Abundant IL-6 and IL-23 in MPE could promote the frequency of IL-26 expressed by CD4 + T cells through phosphorylating STAT3 signaling pathway and promoting the expression of a specific Th17 lineage marker RORγt subsequently. IL-26 could selectively increase Th22 proportion through up-regulating the percentage of Ki-67 expressed by CD4 + T cells and the expression of IL-22 secreted by memory CD4 + T cells. In addition, IL-26 could decrease secretion of granzyme B. The tumor-killing activity of CD8 + T cells were inhibited as well when cocultured with malignant cells. Furthermore, the accumulation of IL-26 protein in MPE predicted poor patient survival. In summary, our results indicated that IL-26 was involved in the pathogenesis of MPE by exerting its impacts on both CD4 + T cells and CD8 + T cells., (©2021 Society for Leukocyte Biology.)

CD8 + T cells have conventionally been studied in relationship to pathogen or tumor clearance. Recent reports have identified novel functions of CXCR5 + CD8 + T cells that can home to lymphoid follicles, a key site of antibody production. In this review we provide an in-depth analysis of conflicting reports regarding the impact of CXCR5 + CD8 + T cells on antibody production and examine the data supporting a role for antibody-enhancement (B cell "helper") and antibody-downregulation (antibody-suppressor) by CXCR5 + CD8 + T cell subsets. CXCR5 + CD8 + T cell molecular phenotypes are associated with CD8-mediated effector functions including distinct subsets that regulate antibody responses. Co-inhibitory molecule PD-1, among others, distinguish CXCR5 + CD8 + T cell subsets. We also provide the first in-depth review of human CXCR5 + CD8 + T cells in the context of clinical outcomes and discuss the potential utility of monitoring the quantity of peripheral blood or tissue infiltrating CXCR5 + CD8 + T cells as a prognostic tool in multiple disease states., Competing Interests: Disclosure: The authors have no financial conflicts of interest.

Although CD8 + T cell tolerance to tissue-specific antigen (TSA) is essential for host homeostasis, the mechanisms underlying peripheral cross-tolerance and whether they may differ between tissue sites remain to be fully elucidated. Here, we demonstrate that peripheral cross-tolerance to intestinal epithelial cell (IEC)-derived antigen involves the generation and suppressive function of FoxP3 + CD8 + T cells. FoxP3 + CD8 + T reg generation was dependent on intestinal cDC1, whose absence led to a break of tolerance and epithelial destruction. Mechanistically, intestinal cDC1-derived PD-L1, TGFβ, and retinoic acid contributed to the generation of gut-tropic CCR9 + CD103 + FoxP3 + CD8 + T regs Last, CD103-deficient CD8 + T cells lacked tolerogenic activity in vivo, indicating a role for CD103 in FoxP3 + CD8 + T reg function. Our results describe a role for FoxP3 + CD8 + T regs in cross-tolerance in the intestine for which development requires intestinal cDC1., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Some long-term non-progressors (LTNPs) have decreasing CD4 + T cell counts and progress to AIDS. Exploring which subsets of CD4 + T cell decreasing and the determinants associated with the decay in these patients will improve disease progression surveillance and provide further understanding of HIV pathogenesis., Methods: Twenty-five LTNPs infected with HIV by blood products were classified as decreased (DG) if their CD4 + cell count dropped to < 400 cells/μL during follow-up or as non-decreased (non-DG) if their CD4 + cell count was ≥400 cells/μL. Laboratory and clinical assessments were conducted at 6 consecutive visits to identify DG characteristics., Results: The LTNPs were infected with HIV for 12 (IQR: 11.5-14) years, and 23 were classified as the B' subtype. Six individuals lost LTNP status 14.5 (IQR: 12.5-17.5) years after infection (DG), and the CD4 + T cell count decreased to 237 (IQR: 213-320) cells/μL at the latest visit. The naïve CD4 + T cell count decrease was greater than that of memory CD4 + T cells [- 128 (IQR: - 196, - 107) vs - 64 (IQR: - 182, - 25) cells/μL)]. Nineteen individuals retained LTNP status (non-DG). At enrolment, the viral load (VL) level (p = 0.03) and CD8 + CD38 + percentage (p = 0.03) were higher in DG than non-DG individuals. During follow-up, viral load and CD8 + CD38 + percentage were significantly increased and negatively associated with CD4 + cell count [(r = - 0.529, p = 0.008), (r = - 0.476, p = 0.019), respectively]. However, the CD8 + CD28 + percentage and B cell count dropped in DG and were positively correlated with CD4 + T cell count [(r = 0.448, p = 0.028), (r = 0.785, p < 0.001)]., Conclusion: Immunological progression was mainly characterized by the decrease of naïve CD4 + T cell in LTNPs infected with HIV by blood products and it may be associated with high HIV RNA levels.

Background: Psoriasis is an inflammatory, IL-17-driven skin disease in which autoantigen-induced CD8 + T cells have been identified as pathogenic drivers., Objective: Our study focused on comprehensively characterizing the phenotypic variation of CD8 + T cells in psoriatic lesions., Methods: We used single-cell RNA sequencing to compare CD8 + T-cell transcriptomic heterogeneity between psoriatic and healthy skin., Results: We identified 11 transcriptionally diverse CD8 + T-cell subsets in psoriatic and healthy skin. Among several inflammatory subsets enriched in psoriatic skin, we observed 2 Tc17 cell subsets that were metabolically divergent, were developmentally related, and expressed CXCL13, which we found to be a biomarker of psoriasis severity and which achieved comparable or greater accuracy than IL17A in a support vector machine classifier of psoriasis and healthy transcriptomes. Despite high coinhibitory receptor expression in the Tc17 cell clusters, a comparison of these cells with melanoma-infiltrating CD8 + T cells revealed upregulated cytokine, cytolytic, and metabolic transcriptional activity in the psoriatic cells that differed from an exhaustion program., Conclusion: Using high-resolution single-cell profiling in tissue, we have uncovered the diverse landscape of CD8 + T cells in psoriatic and healthy skin, including 2 nonexhausted Tc17 cell subsets associated with disease severity., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Previously, we have found that IL-22 could be not only secreted outside of cells, but also highly expressed on the T cells membrane in HIV-1 negative patients with tuberculosis (TB). However, the study on membrane-bound IL-22+ cells of HIV-1 infected patients is rare. Therefore, we investigated antigen-specific membrane-bound IL-22+ T cell subsets in Mycobacterium tuberculosis (M.tb) coinfection of HIV-1 infected individuals., Methods: A case-control study that enrolled 74 HIV-1 infected participants was carried out, including HIV-1 monoinfection (HIV+TB-, n = 43), HIV-1 infected patients with latent TB (HIV+LTB, n = 18) and HIV-1 coinfected patients with active TB (HIV+TB+, n = 13). We made use of an IFN-γ release assay (IGRA) to screen LTB individuals. Purified protein derivative (PPD) and phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) were used as specific-stimulators to detect the levels of peripheral blood membrane-bound IL-22+ T cell subsets via cell surface staining and flow cytometry among three groups., Results: An approximate rate of 24.3% (n = 18 out of 74) of latent M.tb infection among HIV-1 positive population in Eastern China. Interestingly, HMBPP-specific CD3+Vγ2+ T cells were impaired in HIV+TB+patients compared with HIV+LTB patients (P < 0.05). Furthermore, increases of PPD-specific and HMBPP-specific membrane-bound IL-22+ T cell subsets including CD3+, CD3+CD4+ and CD3+Vγ2+ T cells were observed in HIV+TB+group rather than HIV+LTB groups (all P < 0.05)., Conclusion: Antigen-specific membrane-bound IL-22+ T cells were highly expressed in M.tb coinfection of HIV-1 infected individuals, and may play an important role in anti-TB immune response during coinfection with HIV-1., Competing Interests: Declaration of Competing Interest All authors declare that they have no competing interests regarding this manuscript., (Copyright © 2020. Published by Elsevier B.V.)

The association of immune markers and clinicopathologic features and patient outcome has not been extensively studied in Merkel cell carcinoma (MCC). We correlated tumoral PD-L1 and IDO1 expression, and intratumoral CD8+ and FoxP3+ lymphocytes count with clinicopathologic variables, Merkel cell polyomavirus (MCPyV) status, and patient outcomes in a series of 132 MCC. By univariate analyses, tumoral PD-L1 expression >1% and combined tumoral PD-L1 >1% and high intratumoral FoxP3+ lymphocyte count correlated with improved overall survival (OS) ( p = 0.016, 0.0072), MCC-specific survival (MSS) ( p = 0.019, 0.017), and progression-free survival (PFS) ( p = 0.043, 0.004, respectively). High intratumoral CD8+ and FoxP3+ lymphocyte count correlated with longer MSS ( p = 0.036) and improved PFS ( p = 0.047), respectively. Ulceration correlated with worse OS and worse MSS. Age, male gender, and higher stage (3 and 4) significantly correlated with worse survival. MCPyV positivity correlated with immune response. By multivariate analyses, only ulceration and age remained as independent predictors of worse OS; gender and stage remained for shorter PFS. Tumoral PD-L1 expression and increased density of intratumoral CD8+ lymphocytes and FoxP+ lymphocytes may represent favorable prognosticators in a subset of MCCs. Tumoral PD-L1 expression correlated with intratumoral CD8+ and FoxP3+ lymphocytes, which is supportive of an adaptive immune response.

Myeloid-derived suppressor cells (MDSCs) play an important role in tumor progression through both immunologic and non-immunologic mechanisms. This study was conducted to evaluate the expression of S100A8, a well-known MDSC marker, and the significance of its expression in pre-invasive and invasive breast cancers. S100A8 expression in tumor cells (TCs) and immune cells (ICs) was assessed by immunohistochemistry, and its association with clinicopathologic features and infiltration of other IC subsets including CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocytes (TILs) and PD-L1+ ICs was evaluated. S100A8 expression in TCs and ICs showed a positive correlation in pre-invasive carcinoma and invasive carcinoma. S100A8+ ICs, but not S100A8+ TCs, were significantly higher in number in invasive carcinoma than in pre-invasive carcinoma. Infiltration of S100A8+ ICs was revealed as a poor prognostic indicator in pre-invasive and invasive carcinomas, especially in hormone receptor-positive subgroup. Infiltration of CD4+, CD8+, and FOXP3+ TIL subsets and PD-L1+ ICs was significantly higher in S100A8+ IC (+) group than in S100A8+ IC (-) group. Combined analyses of IC subset infiltration revealed that infiltration of S100A8+ ICs was associated with poor clinical outcome in the PD-L1+ IC (-), CD8+ TIL-low, and FOXP3+ TIL-low subgroups. In conclusion, S100A8+ ICs seem to undergo a dynamic change during breast cancer progression in association with other IC subset infiltration. The prognostic impact of S100A8+ IC infiltration was greater in less immunogenic tumors.

Tissue-resident memory T (T RM ) cells are well known to play critical roles in peripheral tissues during virus infection and tumor immunology. Our previous studies indicated that CD69 + CD4 + and CD69 + CD8 + T cells in tuberculous pleural effusion (TPE) were antigen-specific memory T cells. However, the phenotypical and functional characteristics of CD8 + T RM cells in tuberculosis remain unknown. We found that CD103 + CD8 + T cells were the predominant subset of CD103 + lymphocytes in TPE; both CD103 and CD69 expressed on memory CD8 + T cells from TPE were significantly increased compared with those from paired peripheral blood. Phenotypically, CD103 + CD69 + and CD103 + CD69 - CD8 + T cells expressed higher levels of CD45RO than CD103 - CD69 + CD8 + T cells did; CD103 + CD69 - CD8 + T cells highly expressed CD27, CD127, and CD62L and some chemokine receptors. We further compared the functional differences among the four distinct CD45RO + CD8 + T subsets identified by CD103 and CD69 expression. In consist with our published results, CD69 + CD8 + T cells, but not CD103 + CD8 + , produced high levels of IFN- γ after treatment with BCG in the presence of BFA. Nevertheless, CD103 - CD69 + and CD103 + CD69 + memory CD8 + T cells expressed higher levels of Granzyme B, while CD103 + CD69 - memory CD8 + T cells were characterized as a possibly immunosuppressive subset by highly expressing CTLA-4, CD25, and FoxP3. Furthermore, TGF- β extremely increased CD103 expression but not CD69 in vitro . Together, CD103 + CD8 + T cells form the predominant subset of CD103 + lymphocytes in TPE; CD103 and CD69 expression defines distinct CD8 + T RM -like subsets exhibiting phenotypical and functional heterogeneity. Our findings provide an important theoretical basis to optimize and evaluate new tuberculosis vaccines., Competing Interests: The authors have declared that no conflict of interest exists., (Copyright © 2021 Sifei Yu et al.)

Birdshot Retinochoroiditis (BSRC) is a progressive non-infectious intraocular inflammation that affects choroid and retina. Inflammatory processes have adverse effects on vision by affecting photoreceptor-bearing cells that do not regenerate. This study aimed at characterizing inflammatory CD4 + and CD8 + T cell subsets in the peripheral blood of active and inactive BSRCs. Furthermore, we correlated phenotypical and functional immunological analyses with clinical data. We observed a slight increase of terminally differentiated effector memory CD8 + T cells expressing CD45RA (T EMRA ) in blood of inactive, compared to active BSRCs. Moreover, we identified a trend for a decreased population of T H 2 cells and increased T H 1 frequencies in active BSRCs, a typical sign of ongoing autoimmune processes. Functional assays demonstrated severe and overall impairment of effector function of both, CD4 + and CD8 + inflammatory T cells, which might reflect T cell exhaustion. Although the eye is the main site of inflammation in BSRC, we observed altered T cell subset compositions in the peripheral blood, dependent on the disease status. Our results indicate that T cells may play a major role in BSRC pathology, although our cohort size is too limited for definitve conclusions. Future studies with larger BSRCs have to be performed.

Crohn's disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30 + γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated T H 17 but decreased CD8 + T, γδT, T FH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8 + , as well as reduced CD4 + T cells with an elevated T H 17 over Treg/T FH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.

Background: The aim of this study is to investigate the changes of peripheral blood lymphocyte subsets before and after treatment with pembrolizumab for non-small cell lung cancer and its clinical significance., Methods: A total of 32 patients with non-small cell lung cancer who received pembrolizumab treatment in The Affiliated Hospital of Qingdao University and Weifang People's Hospital of Shandong Province from January 2015 to December 2020 were selected as the observation group, and 30 healthy people during the same period were selected as the control group. Before treatment and in cycles 1, 2 and 4 after treatment, fluid cytometry was used to detect changes in the levels of lymphocyte subsets in the peripheral blood of patients., Results: The CD3⁺, CD4⁺, CD4⁺/CD8⁺ indexes of patients with non-small cell lung cancer before the treatment were significantly lower than those in the control group (P<0.05), and the CD8⁺ level was significantly increased (P<0.05); After 1 cycle of pembrolizumab treatment, there was no significant difference in the changes of lymphocyte subsets compared with before immunotherapy; After 2 cycles of the treatment, the CD3⁺, CD4⁺, CD4⁺/CD8⁺ values were higher than before the treatment (P>0.05), and the CD8⁺ index was slightly lower than before the treatment (P<0.05); After the fourth cycle of treatment, the CD3⁺, CD4⁺, CD4⁺/CD8⁺ values were significantly improved compared to before the treatment (P<0.05), and the CD8⁺ index was significantly lower than before the treatment (P<0.05); In the treatment process of patients with stable disease (SD)/partial response (PR), the CD3⁺, CD4⁺, CD4⁺/CD8⁺ values of the fourth cycles were higher than before the treatment (P<0.05), and the CD8⁺ index was lower than before the treatment (P<0.05); During the treatment of progressive disease (PD) patients, the changes of lymphocyte subsets in the fourth cycles were not significantly different from those before the treatment (P>0.05). At the same time, this article shows through analysis that the expression of programmed cell death ligand 1 (PD-L1) and pathological types have no obvious influence on the effect of immunotherapy. Multi-factor analysis shows that it is more meaningful to observe the changes of CD3⁺, CD4⁺ and CD8⁺ at the same time to predict the effect of immunotherapy., Conclusions: Pembrolizumab can regulate the changes of T lymphocyte subsets in patients with non-small cell lung cancer, improve the immune status of the patients, and there is no obvious adverse reaction. At the same time, monitoring the changes of lymphocyte subsets during treatment can predict the effect of immunotherapy.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. We have previously demonstrated that CNS-specific CD8 T cells possess a disease-suppressive function in MS and variations of its animal model, experimental autoimmune encephalomyelitis (EAE), including the highly clinically relevant relapsing-remitting EAE disease course. Regulatory CD8 T cell subsets have been identified in EAE and other autoimmune diseases, but studies vary in defining phenotypic properties of these cells. In relapsing-remitting EAE, PLP 178-191 CD8 T cells suppress disease, whereas PLP 139-151 CD8 T cells lack this function. In this study, we used this model to delineate the unique phenotypic properties of CNS-specific regulatory PLP 178-191 CD8 T cells versus nonregulatory PLP 139-151 or OVA 323-339 CD8 T cells. Using multiparametric flow cytometric analyses of phenotypic marker expression, we identified a CXCR3 + subpopulation among activated regulatory CD8 T cells, relative to nonregulatory counterparts. This subset exhibited increased degranulation and IFN-γ and IL-10 coproduction. A similar subset was also identified in C57BL/6 mice within autoregulatory PLP 178-191 CD8 T cells but not within nonregulatory OVA 323-339 CD8 T cells. This disease-suppressing CD8 T cell subpopulation provides better insights into functional regulatory mechanisms, and targeted enhancement of this subset could represent a novel immunotherapeutic approach for MS., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The immune system plays a major role in recognizing and eliminating malignant cells, and this has been exploited in the development of immunotherapies aimed at either activating or reactivating the anti-tumor activity of a patient's immune system. A wide range of therapeutic approaches involving T lymphocytes, such as programmed cell death protein ligand-1 (PDL-1) inhibitors, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) blockers, and CD19-targeted T-cell therapy through chimeric antigen receptor (CAR)-T cells or CD19/CD3 bi-specific T-cell engagers, have been introduced to the field of oncology, leading to significant improvements in overall survival of adult cancer patients. During the past few years, the availability and approval of T-cell based immunotherapies have become a reality also for the treatment of childhood cancers. However, the distribution, ratio of regulatory to effector cells and the quality of T-cell responses early in life are distinct from those during adolescence and adulthood, raising the possibility that these differences impact the efficacy of immunotherapy. Herein we provide a brief overview of the properties of conventional T cell subsets during early life. Focusing on the most common cancer type during childhood, acute lymphoblastic leukemia (ALL), we describe how current conventional therapies used against ALL influence the T-cell compartment of small children. We describe early life T-cell responses in relation to immunotherapies engaging T-cell anticancer reactivity and present our opinion that it is not only immaturity of the adaptive immune system, but also the impact of an immunosuppressive environment that may prove disadvantageous in the setting of immunotherapies targeting pediatric cancer cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saghafian-Hedengren, Sverremark-Ekström and Nilsson.)

To date, pregnancy is an immunological paradox. The semi-allogenic fetus must be accepted by the maternal immune system, while defense against pathogens and immune surveillance cannot be compromised. Gamma/delta T cells are believed to play an important role in this immunological puzzle. In this study, we analyzed peripheral blood CD56+ γδT cells from pregnant women (1 st , 2 nd , and 3 rd trimester) and non-pregnant women by multicolor flow cytometry. Interestingly, γδT cells represent almost half of CD3+/CD56+ cells. Among γδT cells, the CD56+ population expands in the 2 nd and 3 rd trimester. CD56+ γδT cells maintained a predominantly CD4-/CD8- or CD8+ phenotype, while CD56- γδT cells were in similar rates CD4-/CD8- or CD4+ during pregnancy. Investigation of the lysosomal degranulation marker CD107a revealed a preserved elevated rate of potentially cytotoxic CD56+ γδT cells in pregnancy, while their cytotoxic strength was reduced. Furthermore, CD56+ γδT cells continuously showed a higher prevalence of PD-1 expression. CD56+ γδT cells' rate of PD-1 increased in the 1 st trimester and decreased hereafter back to normal level. We correlated the cytotoxic potential and the expression of the inhibitory immune checkpoint PD-1 and were able to demonstrate that highly cytotoxic cells within this CD56+ γδT population tend to express PD-1, which might allow the inhibition of these cells after binding its ligand in the placenta. These findings should support the understanding of the complex processes, which ensure the maintenance of pregnancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nörenberg, Jaksó and Barakonyi.)

Decreasing graft rejection and increasing graft and patient survival are great challenges facing liver transplantation (LT). Different T cell subsets participate in the acute cellular rejection (ACR) of the allograft. Cell-mediated immunity markers of the recipient could help to understand the mechanisms underlying acute rejection. This study aimed to analyse different surface antigens on T cells in a cohort of adult liver patients undergoing LT to determine the influence on ACR using multi-parametric flow cytometry functional assay. Thirty patients were monitored at baseline and during 1 year post-transplant. Two groups were established, with (ACR) and without (NACR) acute cellular rejection. Leukocyte, total lymphocyte, percentages of CD4 + CD154 + and CD8 + CD154 + T cells, human leukocyte antigen (HLA) mismatch between recipient-donor and their relation with ACR as well as the acute rejection frequencies were analysed. T cells were stimulated with concanavalin A (Con-A) and surface antigens were analysed by fluorescence activated cell sorter (FACS) analysis. A high percentage of CD4 + CD154 + T cells (P = 0·001) and a low percentage of CD8 + CD154 + T cells (P = 0·002) at baseline were statistically significant in ACR. A receiver operating characteristic analysis determined the cut-off values capable to stratify patients at high risk of ACR with high sensitivity and specificity for CD4 + CD154 + (P = 0·001) and CD8 + CD154 + T cells (P = 0·002). In logistic regression analysis, CD4 + CD154 + , CD8 + CD154 + and HLA mismatch were confirmed as independent risk factors to ACR. Post-transplant percentages of both T cell subsets were significantly higher in ACR, despite variations compared to pretransplant. These findings support the selection of candidates for LT based on the pretransplant percentages of CD4 + CD154 + and CD8 + CD154 + T cells in parallel with other transplant factors., (© 2020 British Society for Immunology.)

It has become increasingly clear that a subset of T cells which persist at diverse infection sites, known as tissue-resident memory T cells (T RM ), can mediate efficacious protective immunity against many types of viral infections. Recent studies have elucidated the mechanisms by which T RM coordinate enhanced viral clearance in different sites through rapid production of effector cytokines and cytolytic mediators, in situ expansion, differentiation to circulating effector cells, and immune cell recruitment. This tissue-localized response also includes enhancement at the local lymphoid sites which contribute to fortifying T RM -mediated protection. Understanding how these responses occur in a tissue-wide context will provide key insights for development of vaccines and therapeutics., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Persisting stimulation can skew CD8 T cells towards a hypofunctional state commonly referred to as T cell exhaustion. This functional attenuation likely constitutes a mechanism which evolved to balance T cell mediated viral control versus overwhelming immunopathology. Here, we highlight the recent progress in defining the genetic mechanisms and factors shaping the differentiation of exhausted CD8 T cells. We review how the transcription factor Tox imposes an exhausted phenotype in the Tcf1+ progenitors and how CD4 help fine-tunes the effector subsets that emerge from this progenitor population. Both processes critically shape the spectrum of effector function performed by CD8 T cells and the level of resulting virus control. Finally, we discuss how these insights can be exploited to boost the immune response in chronic infection and cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Nocardia rubra cell wall skeleton (Nr-CWS) for external use is an immune enhancer, which has been widely used in human cervix diseases such as cervical erosion, but the mechanism of Nr-CWS enhancing immunity is still unclear. The purpose of this study was to explore the effect and mechanism of Nr-CWS on the local immune status of cervical tissue in patients with high-risk human papillomavirus (HR-HPV) infection and cervical precancerous lesion, cervical intraepithelial neoplasia (CIN). The recruited patients with HR-HPV infection and CIN were treated with Nr-CWS. The specimens were taken from these patients before and after local application of Nr-CWS respectively. The normal control specimens were tested simultaneously. Serial section analysis of immunohistochemistry and co-expression analysis were performed to characterize populations of T cells and the expressions of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1). The levels of cytokines in local cervical tissue were also detected. Nr-CWS significantly increased T cells including CD4 + , CD8 + T cells, and reduced the expression of PD-L1 in the patients' local cervical tissues. Co-expression analyses showed that the proportions of PD-1 + CD4 + cells in CD4 + T cells and PD-1 + CD8 + cells in CD8 + T cells decreased after Nr-CWS application. Furthermore, the increase in the number of immune cells was accompanied by increased pro-inflammatory cytokines interleukin-12 (IL-12), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and decreased suppressive cytokine IL-10. The results indicate that Nr-CWS, as an immunotherapeutic agent for HR-HPV infection and CIN, plays an immune promoting role related to the upregulation of T cell subsets and the inhibition of PD-1/PD-L1 pathway., Competing Interests: YiZ was employed by the company Weihai Greatest Pharmaceutical Research Institute Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Zhang, Zhao, Shao, Zhang, Li, Bai, Guo, Liu, Tang and Zhang.)

Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B-cell lymphoma or acute lymphoblastic leukemia. Impaired T-cell fitness of CLL patients may be involved in treatment failure. Less-differentiated naïve-like T cells play an important role in CART expansion and long-term persistence in vivo. These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B-cell receptor inhibitor ibrutinib targeting Bruton's tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin-2-inducible T-cell kinase (ITK) which is involved in T-cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells from nine healthy donors and eight CLL patients were used to generate CART cells. T-cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient-derived CART cells. Furthermore, ibrutinib enriched CART cells with less-differentiated naïve-like phenotype and decreased expression of exhaustion markers including PD-1, TIM-3 and LAG-3. In addition, ibrutinib increased the cytokine release capacity of CLL patient-derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient-derived CART cell products., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of Union for International Cancer Control.)