Immunological memory is thought to depend upon a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T-cell population that displays enhanced self-renewal and multipotent capacity to derive central memory, effector memory and effector T cells. These cells, specific for multiple viral and self-tumor antigens, were found within a CD45RO−, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7Rα+ T-cell compartment characteristic of naïve T cells. However, they expressed increased levels of CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibited numerous functional attributes distinctive of memory cells. Compared to known memory populations, these lymphocytes displayed increased proliferative capacity, more efficiently reconstituted immunodeficient hosts and mediated superior anti-tumor responses in a humanized mouse model. The identification of a human stem cell-like memory T-cell population is of direct relevance to the design of vaccines and T-cell therapies.

• Antimicrobial CD8 ϩ MAIT cells are activated, exhausted, and progressively and persistently depleted during chronic HIV-1 infection.• This decline in MAIT cell level and function may seriously impair the ability to mount immune responses to bacterial and fungal pathogens. IntroductionHIV-1 infection is associated with a range of pathologic changes to the immune system, including systemic immune activation, CD4 T-cell loss and CD8 T-cell expansion. The state of broad and persistent immune activation develops early during infection, 1,2 contributes to the rapid aging of the immune system seen during chronic progressive HIV-1 disease, and persists despite effective long-term virologic suppression by combination antiretroviral therapy (cART; reviewed in by Deeks, 3 Appay et al, 4 and Desai and Landay 5 ). These pathologic processes lead to the progressive destruction of lymphoid organs and loss of CD4 helper T cells. 6,7 Already during primary infection, HIV-1 depletes intestinal CD4 T cells and disrupts the structure and function of the intestinal immune system. [8][9][10][11][12][13] One consequence of this is increased permeability of the intestinal epithelium with translocation of microbial products into the local tissue, the portal circulation, the liver and eventually into systemic circulation. 14 This process may continue despite effective long-term cART. 15,16 Disruption of immune homeostasis and barrier function at the mucosa is a considerable challenge for the host immune system because the microbial proteins, carbohydrates, and lipids form a range of antigens that will engage innate as well as adaptive immune mechanisms (reviewed by Brenchley and Douek 17 ). Despite considerable advances in the treatment and management of HIV-1 disease, certain infections still present a significant clinical challenge particularly among HIV-infected individuals who are diagnosed at advanced stages, those who lack access to antiretroviral therapy, and those who cannot maintain adherence to therapy and clinical care. [18][19][20] This includes an increased risk of developing bacterial pneumonia even in HIV-1-infected patients with relatively normal CD4 counts, 21 indicating that impaired CD4 T-cell independent control of certain infections still exists even in the context of treated HIV-1 disease. Mucosal-associated invariant T (MAIT) cells are a relatively recently discovered subset of unconventional, innate-like T cells that are highly abundant in mucosal tissues, liver, and peripheral blood. [22][23][24][25] Human MAIT cells express an invariant T-cell receptor (TCR) carrying the V␣7.2 ␣-chain segment, a restricted V␤ repertoire (V␤2 or V␤13), and recognize antigens in complex with the evolutionarily conserved MHC-Ib-related protein (MR1). 24,25 In addition to the V␣7.2 TCR segment, MAIT cells are defined by Submitted July 27, 2012; accepted November 26, 2012. Prepublished online as Blood First Edition paper, December 13, 2012; DOI 10.1182 DOI 10. /blood-2012 The online version of this article contains a data suppleme...

Mucosa-associated invariant T (MAIT) cells represent a large innate-like evolutionarily conserved antimicrobial T-cell subset in humans. MAIT cells recognize microbial riboflavin metabolites from a range of microbes presented by MR1 molecules. MAIT cells are impaired in several chronic diseases including HIV-1 infection, where they show signs of exhaustion and decline numerically. Here, we examined the broader effector functions of MAIT cells in this context and strategies to rescue their functions. Residual MAIT cells from HIV-infected patients displayed aberrant baseline levels of cytolytic proteins, and failed to mobilize cytolytic molecules in response to bacterial antigen. In particular, the induction of granzyme B (GrzB) expression was profoundly defective. The functionally impaired MAIT cell population exhibited abnormal T-bet and Eomes expression patterns that correlated with the deficiency in cytotoxic capacity and cytokine production. Effective antiretroviral therapy (ART) did not fully restore these aberrations. Interestingly, IL-7 was capable of arming resting MAIT cells from healthy donors into cytotoxic GrzB+ effector T cells capable of killing bacteria-infected cells and producing high levels of pro-inflammatory cytokines in an MR1-dependent fashion. Furthermore, IL-7 treatment enhanced the sensitivity of MAIT cells to detect low levels of bacteria. In HIV-infected patients, plasma IL-7 levels were positively correlated with MAIT cell numbers and function, and IL-7 treatment in vitro significantly restored MAIT cell effector functions even in the absence of ART. These results indicate that the cytolytic capacity in MAIT cells is severely defective in HIV-1 infected patients, and that the broad-based functional defect in these cells is associated with deficiency in critical transcription factors. Furthermore, IL-7 induces the arming of effector functions and enhances the sensitivity of MAIT cells, and may be considered in immunotherapeutic approaches to restore MAIT cells.

The human naive T cell repertoire is the repository of a vast array of TCRs. However, the factors that shape their hierarchical distribution and relationship with the memory repertoire remain poorly understood. In this study, we used polychromatic flow cytometry to isolate highly pure memory and naive CD8+ T cells, stringently defined with multiple phenotypic markers, and used deep sequencing to characterize corresponding portions of their respective TCR repertoires from four individuals. The extent of interindividual TCR sharing and the overlap between the memory and naive compartments within individuals were determined by TCR clonotype frequencies, such that higher-frequency clonotypes were more commonly shared between compartments and individuals. TCR clonotype frequencies were, in turn, predicted by the efficiency of their production during V(D)J recombination. Thus, convergent recombination shapes the TCR repertoire of the memory and naive T cell pools, as well as their interrelationship within and between individuals.

Naive and central memory CD8 T cells use CCR7 to recirculate through T cell zones of secondary lymphoid organs where they can encounter antigen. Here we describe a subset of human CD8 T cells expressing CXCR5 which enables homing in response to CXCL13 produced within B cell follicles. CXCR5 + CD8 T cells were found in tonsil B cell follicles, and isolated cells migrated towards CXCL13 in vitro. They expressed CD27, CD28, CD45RO, CD69, and were CD7 low , and produced IFN-c and granzyme A but lacked perforin, a functional profile suggesting that these cells are early effector memory cells in the context of contemporary T cell differentiation models. Receptors important in the interaction with B cells, including CD70, OX40 and ICOS, were induced upon activation, and CXCR5 + CD8 T cells could to some extent support survival and IgG production in tonsil B cells. Furthermore, CXCR5 + CD8 T cells expressed CCR5 but no CCR7, suggesting a migration pattern distinct from that of follicular CD4 T cells. The finding that a subset of early effector memory CD8 T cells use CXCR5 to locate to B cell follicles indicates that MHC class I-restricted CD8 T cells are part of the follicular T cell population. IntroductionCD8 T cells are lymphocytes of the adaptive immune system which recognize viral and bacterial peptides presented by MHC class I molecules and are indispensable in the control of many intracellular infections [1]. When appropriately stimulated by antigen presented by dendritic cells (DC) in secondary lymphoid tissues, they proliferate vigorously, become effector cells, and upon resolution of infection a small population of antigen-specific CD8 T cells are maintained as long-lived memory cells [2][3][4]. A primary effector mechanism of CD8 T cells is the killing of infected cells via the targeted release of the lytic effector molecules perforin and granzymes, and via expression of death receptors. However, there is significant functional heterogeneity in CD8 T cells and they are also efficient in producing anti-viral and pro-inflammatory cytokines [5][6][7][8]. The effector mechanisms used may depend on the pathogen and on the type of cell or tissue that is infected [8].CD8 T cell responses involve the differentiation of naive T cells into distinct types of effector and memory cells [2, 3,9]. One approach to analyse a T cell response is to examine the expression of receptors that mediate homing of T cells, such as CD62L, which binds to glycosylation-dependent cell adhesion molecule 1 on [4,20,21]. CXCR5 is a chemokine receptor which is expressed on all B cells and on specialized subsets of DC and T cells [22]. It has only one known ligand, the chemokine CXCL13, mainly produced by stromal cells and follicular DC within B cell follicles [23][24][25]. CXCR5 + CD4 T cells, known as follicular homing T helper cells, are found in B cell follicles and contribute to the generation of effective humoral immune responses by supporting antibody production and isotype class switching [26,27]. However, some of the processes and mec...

Adaptive T-cell immunity relies on the recruitment of antigenspecific clonotypes, each defined by the expression of a distinct T-cell receptor (TCR), from an array of naïve T-cell precursors. Despite the enormous clonotypic diversity that resides within the naïve T-cell pool, interindividual sharing of TCR sequences has been observed within mobilized T-cell responses specific for certain peptide-major histocompatibility complex (pMHC) antigens. The mechanisms that underlie this phenomenon have not been fully elucidated, however. A mechanism of convergent recombination has been proposed to account for the occurrence of shared, or "public," TCRs in specific memory T-cell populations. According to this model, TCR sharing between individuals is directly related to TCR production frequency; this, in turn, is determined on a probabilistic basis by the relative generation efficiency of particular nucleotide and amino acid sequences during the recombination process. Here, we tested the key predictions of convergent recombination in a comprehensive evaluation of the naïve CD8 + TCRβ repertoire in mice. Within defined segments of the naïve CD8 + T-cell repertoire, TCRβ sequences with convergent features were (i) present at higher copy numbers within individual mice and (ii) shared between individual mice. Thus, the naïve CD8 + T-cell repertoire is not flat, but comprises a hierarchy of recurrence rates for individual clonotypes that is determined by relative production frequencies. These findings provide a framework for understanding the early mobilization of public CD8 + T-cell clonotypes, which can exert profound biological effects during acute infectious processes.

Adoptive transfer of viral antigen-specific memory T cells can reconstitute antiviral immunity, but in a recent report a majority of virus-specific cytotoxic T-lymphocyte (CTL) lines showed in vitro cross-reactivity against allo-human leukocyte antigen (HLA) molecules as measured by interferon-␥ secretion. We therefore reviewed our clinical experience with adoptive transfer of allogeneic hematopoietic stem cell transplantation donor-derived virus-specific CTLs in 153 recipients, including 73 instances where there was an HLA mismatch. There was no de novo acute graft-versus-host disease after infusion, and incidence of graft-versus-host disease reactivation was low and not significantly different in recipients of matched or mismatched CTL. However, we found that IntroductionAfter stem cell transplantation, there are high morbidity and mortality from viral disease. 1 Such complications are commonest where the donor and recipient are partially human leukocyte antigen (HLA)-mismatched or the donor graft has been depleted of mature T lymphocytes to prevent alloreactivity and graft-versushost disease (GVHD). As a consequence, several investigators have administered donor-derived virus-specific T cells to transplantation recipients to reduce the incidence and severity of posttransplantation viral disease with apparent clinical benefit. 2-9 A recent study by Amir et al, however, suggests that transfer of HLA-mismatched virus-specific cytotoxic T-lymphocytes (CTLs) might risk graftversus-host alloreactions. 10 In that study, T-cell lines reactive against Epstein-Barr virus (EBV), cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells and target cells transduced with single HLA molecules. 10 Remarkably, 80% of virus-specific T-cell lines and 45% of virus-specific T-cell clones derived therefrom were crossreactive against allo-HLA molecules, as measured by ␥-interferon secretion. 10 This cross-reactivity was observed in both CD8 ϩ and CD4 ϩ T-cell clones, being directed primarily against HLA class I and II antigens, respectively. These observations raise the concern that virus-specific T cells might mediate graft rejection or GVHD when administered to HLA class I or II mismatched recipients. 10 Notwithstanding the apparently high level of cross-reactivity in the in vitro assays reported by Amir et al,10 there are no data to suggest that cross-reactivity of virus-specific T cells with HLA specificities leads to clinical complications. [3][4][5][6][7][8][9] None of these studies, however, formally dissected responses in recipients who had received HLA partially mismatched virus-specific CTLs, or examined whether the observed lack of any GVHD was simply the result of fortuitous absence of alloreactivity in the administered lines.We now report that, in 73 recipients of virus-specific CTLs from an HLA-mismatched donor, we have not observed GVHD associated with the cell infusion. In 4 patients, the alloreactivity of infused lines was characterized in an in vitro assay ...

BackgroundIt remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies.MethodsWe employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods.ResultsWe demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors.ConclusionsSingle-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.Electronic supplementary materialThe online version of this article (10.1186/s40425-017-0293-7) contains supplementary material, which is available to authorized users.