The CD8+ T cell response to Epstein-Barr virus (EBV) is well characterized. Much less is known about the evolution of the CD4+ T cell response. Here we show that EBV stimulates a primary burst of effector CD4+ T cells and this is followed by a period of down-regulation. A small population of EBV-specific effector CD4+ T cells survives during the lifelong persistent phase of infection. The EBV-specific effector CD4+ T cells accumulate within a CD27+ CD28+ differentiation compartment during primary infection and remain enriched within this compartment throughout the persistent phase of infection. Analysis of CD4+ T cell responses to individual epitopes from EBV latent and lytic cycle proteins confirms the observation that the majority of the effector cells express both CD27 and CD28, although CD4+ T cells specific for lytic cycle antigens have a greater tendency to express CD45RA than those specific for the latent antigens. In clear contrast, effector CD4+ T cells specific for cytomegalovirus (CMV) accumulate within the CD27− CD28+ and CD27− CD28− compartments. There are striking parallels in terms of the differentiation of CD8+ T cells specific for EBV and CMV. The results challenge current ideas on the definition of memory subsets.

Background. Changes in the phenotype and function of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4+ and CD8+ T-cell subsets in response to stage of infection may allow discrimination between active tuberculosis and latent tuberculosis infection.Methods. A prospective comparison of M. tuberculosis-specific cellular immunity in subjects with active tuberculosis and latent tuberculosis infection, with and without human immunodeficiency virus (HIV) coinfection. Polychromatic flow cytometry was used to measure CD4+ and CD8+ T-cell subset phenotype and secretion of interferon γ (IFN-γ), interleukin 2 (IL-2), and tumor necrosis factor α (TNF-α).Results. Frequencies of CD4+ and CD8+ cells secreting IFN-γ-only, TNF-α-only and dual IFN-γ/TNF-α were greater in active tuberculosis vs latent tuberculosis infection. All M. tuberculosis-specific CD4+ subsets, with the exception of IL-2-only cells, switched from central to effector memory phenotype in active tuberculosis vs latent tuberculosis infection, accompanied by a reduction in IL-7 receptor α (CD127) expression. The frequency of PPD-specific CD4+ TNF-α-only-secreting T cells with an effector phenotype accurately distinguished active tuberculosis from latent tuberculosis infection with an area under the curve of 0.99, substantially more discriminatory than measurement of function alone.Conclusions. Combined measurement of T-cell phenotype and function defines a highly discriminatory biomarker of tuberculosis disease activity. Unlocking the diagnostic and monitoring potential of this combined approach now requires validation in large-scale prospective studies.

Current tuberculosis (TB) vaccine strategies are largely aimed at activating conventional T cell responses to mycobacterial protein antigens. However, the lipid-rich cell wall of Mycobacterium tuberculosis (M. tuberculosis) is essential for pathogenicity and provides targets for unconventional T cell recognition. Group 1 CD1-restricted T cells recognize mycobacterial lipids, but their function in human TB is unclear and their ability to establish memory is unknown. Here, we characterized T cells specific for mycolic acid (MA), the predominant mycobacterial cell wall lipid and key virulence factor, in patients with active TB infection. MA-specific T cells were predominant in TB patients at diagnosis, but were absent in uninfected bacillus Calmette-Guérin-vaccinated (BCG-vaccinated) controls. These T cells were CD1b restricted, detectable in blood and disease sites, produced both IFN-γ and IL-2, and exhibited effector and central memory phenotypes. MA-specific responses contracted markedly with declining pathogen burden and, in patients followed longitudinally, exhibited recall expansion upon antigen reencounter in vitro long after successful treatment, indicative of lipid-specific immunological memory. T cell recognition of MA is therefore a significant component of the acute adaptive and memory immune response in TB, suggesting that mycobacterial lipids may be promising targets for improved TB vaccines.

BackgroundIFN-γ and IL-2 cytokine-profiles define three functional T-cell subsets which may correlate with pathogen load in chronic intracellular infections. We therefore investigated the feasibility of the immunospot platform to rapidly enumerate T-cell subsets by single-cell IFN-γ/IL-2 cytokine-profiling and establish whether immunospot-based T-cell signatures distinguish different clinical stages of human tuberculosis infection.MethodsWe used fluorophore-labelled anti-IFN-γ and anti-IL-2 antibodies with digital overlay of spatially-mapped colour-filtered images to enumerate dual and single cytokine-secreting M. tuberculosis antigen-specific T-cells in tuberculosis patients and in latent tuberculosis infection (LTBI). We validated results against established measures of cytokine-secreting T-cells.ResultsFluorescence-immunospot correlated closely with single-cytokine enzyme-linked-immunospot for IFN-γ-secreting T-cells and IL-2-secreting T-cells and flow-cytometry-based detection of dual IFN-γ/IL-2-secreting T-cells. The untreated tuberculosis signature was dominated by IFN-γ-only-secreting T-cells which shifted consistently in longitudinally-followed patients during treatment to a signature dominated by dual IFN-γ/IL-2-secreting T-cells in treated patients. The LTBI signature differed from active tuberculosis, with higher proportions of IL-2-only and IFN-γ/IL-2-secreting T-cells and lower proportions of IFN-γ-only-secreting T-cells.ConclusionsFluorescence-immunospot is a quantitative, accurate measure of functional T-cell subsets; identification of cytokine-signatures of pathogen burden, distinct clinical stages of M. tuberculosis infection and long-term immune containment suggests application for treatment monitoring and vaccine evaluation.

Antigen-primed cytotoxic T lymphocytes (CTL) may express leukocyte immunoglobulin-like receptors (LILRs) and natural killer receptors (NKRs). Published work suggests that expression of some of these receptors confers survival advantage, leading to the idea that cells expressing such receptors may accumulate as an antigen-specific response evolves. Here we tested this hypothesis by analyzing expression of CD85j (also known as LILRB1 or ILT2), KIRs, CD94, and CD161 by Epstein- Barr virus (EBV)-specific CTL during the primary and persistent phases of EBV infection in humans. During primary infection, few EBV-specific CTL expressed these receptors and this proportion was equally low in early persistent infection. Thus, expression of these molecules does not influence capacity to survive downregulation of the primary response. However, in donors persistently infected with EBV for many years, a significantly higher proportion of EBV-specific CTL expressed CD85j and NKRs, suggesting that cells expressing these receptors can accumulate with time. Using FACS analysis, we confirmed, at a single cell level, that expression of CD85j, defined by staining with the antibody VMP55, was associated with reduced capacity of EBV-specific CD8+ T cells to respond to antigen. Thus, in the later stages of persistent infection, protective immunity to EBV may be reduced due to the preferential accumulation of hyporesponsive EBV-specific CD8+ T cells.

Leukocyte immunoglobulin-like receptor-1 (LIR-1) is an inhibitory receptor that negatively regulates T cell effector functions after interaction with host class I major histocompatibility complex molecules and, additionally, binds to UL18, a human cytomegalovirus (HCMV)-encoded class I homologue. Here, we demonstrate that virus-specific cytotoxic T lymphocytes (CTLs) differentially express LIR-1, with high frequencies of expression on HCMV-specific CD8 + T cells and intermediate and low frequencies of expression on influenza virus-specific and Epstein-Barr virus (EBV)-specific CTLs, respectively. Expression of LIR-1 was dependent on CTL-antigen specificity and was associated with a differentiated effector memory phenotype, as demonstrated by decreased expression of CD28 and increased expression of CD57. During primary HCMV and EBV infections, expression of LIR-1 on virus-specific CTLs was low and increased slowly. These results indicate that expression of LIR-1 increases during differentiation of virus-specific CD8+ effector T cells. Furthermore, they suggest that a potential immunoregulatory function of UL18 may be to preferentially target highly differentiated HCMV-specific effector memory T cells during persistent infection.The leukocyte immunoglobulin-like receptor (LIR) family (LIRs, immunoglobulin-like transcripts [ILTs], monocyte/macrophage immunoglobulin-related receptors, LILRs, and CD85) comprises a set of related immunoreceptors with inhibitory (LIR-1, -2, -3, -5, and -8), activatory , or soluble (LIR-4) functions encoded within the leukocyte receptor cluster on chromosome 19 [1,2]. LIR-1, the most broadly expressed member, is a type I transmembrane protein that is present on monocytes,

HIV co-infection is an important risk factor for tuberculosis (TB) providing a powerful model in which to dissect out defective, protective and dysfunctional Mycobacterium tuberculosis (MTB)-specific immune responses. To identify the changes induced by HIV co-infection we compared MTB-specific CD4+ responses in subjects with active TB and latent TB infection (LTBI), with and without HIV co-infection. CD4+ T-cell subsets producing interferon-gamma (IFN-γ), interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) and expressing CD279 (PD-1) were measured using polychromatic flow-cytometry. HIV-TB co-infection was consistently and independently associated with a reduced frequency of CD4+ IFN-γ and IL-2-dual secreting T-cells and the proportion correlated inversely with HIV viral load (VL). The impact of HIV co-infection on this key MTB-specific T-cell subset identifies them as a potential correlate of mycobacterial immune containment. The percentage of MTB-specific IFN-γ-secreting T-cell subsets that expressed PD-1 was increased in active TB with HIV co-infection and correlated with VL. This identifies a novel correlate of dysregulated immunity to MTB, which may in part explain the paucity of inflammatory response in the face of mycobacterial dissemination that characterizes active TB with HIV co-infection.

Activation induced deaminase (AID) initiates somatic hypermutation and class switch recombination of the Ig genes in antigen-activated B cells, underpinning antibody affinity maturation and isotype switching. AID can also be pathogenic by contributing to autoimmune diseases and oncogenic mutations. Moreover, AID can exert non-canonical functions when aberrantly expressed in epithelial cells. The lack of specific inhibitors prevents therapeutic applications to modulate AID functions. Here, we have exploited our previous finding that the HSP90 molecular chaperoning pathway stabilizes AID in B cells, to test whether HSP90 inhibitors could target AID in vivo. We demonstrate that chronic administration of HSP90 inhibitors decreases AID protein levels and isotype switching in immunized mice. HSP90 inhibitors also reduce disease severity in a mouse model of acute B-cell lymphoblastic leukemia in which AID accelerates disease progression. We further show that human AID protein levels are sensitive to HSP90 inhibition in normal and leukemic B cells, and that HSP90 inhibition prevents AID-dependent epithelial to mesenchymal transition in a human breast cancer cell line in vitro. Thus, we provide proof-of-concept that HSP90 inhibitors indirectly target AID in vivo and that endogenous human AID is widely sensitive to them, which could have therapeutic applications.