Circulating gluten-specific FOXP3 + CD39 + regulatory T cells have impaired suppressive function in patients with celiac disease

Background Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. Objective We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3)+ Treg cells. Methods Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4+ T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4+ T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). Results Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. Conclusion This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten-specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten-specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis

Mucosal-associated invariant T (MAIT) cells are activated in the gastrointestinal tissue of patients with combination ipilimumab and nivolumab therapy-related colitis in a pathology distinct from ulcerative colitis

The aim of this study was to investigate the pathogenesis of combination ipilimumab and nivolumab-associated colitis (IN-COL) by measuring gut-derived and peripheral blood mononuclear cell (GMNC; PBMC) profiles. We studied GMNC and PBMC from patients with IN-COL, IN-treated with no adverse-events (IN-NAE), ulcerative colitis (UC) and healthy volunteers using flow cytometry. In the gastrointestinal-derived cells we found high levels of activated CD8+ T cells and mucosal-associated invariant T (MAIT) cells in IN-COL, changes that were not evident in IN-NAE or UC. UC, but not IN-C, was associated with a high proportion of regulatory T cells (Treg). We sought to determine if local tissue responses could be measured in peripheral blood. Peripherally, checkpoint inhibition instigated a rise in activated memory CD4+ and CD8+ T cells, regardless of colitis. Low circulating MAIT cells at baseline was associated with IN-COL patients compared with IN-NAE in one of two cohorts. UC, but not INCOL, was associated with high levels of circulating plasmablasts. In summary, the alterations in T cell subsets measured in IN-COL-affected tissue, characterized by high levels of activated CD8+ T cells and MAIT cells and a low proportion of Treg, reflected a pathology distinct from UC. These tissue changes differed from the periphery, where T cell activation was a widespread on-treatment effect, and circulating MAIT cell count was low but not reliably predictive of colitis

Tfh Cells in Health and Immunity: Potential Targets for Systems Biology Approaches to Vaccination

T follicular helper (Tfh) cells are a specialised subset of CD4+ T cells that play a significant role in the adaptive immune response, providing critical help to B cells within the germinal centres (GC) of secondary lymphoid organs. The B cell receptors of GC B cells undergo multiple rounds of somatic hypermutation and affinity maturation within the GC response, a process dependent on cognate interactions with Tfh cells. B cells that receive sufficient help from Tfh cells form antibody-producing long-lived plasma and memory B cells that provide the basis of decades of effective and efficient protection and are considered the gold standard in correlates of protection post-vaccination. However, the T cell response to vaccination has been understudied, and over the last 10 years, exponential improvements in the technological underpinnings of sampling techniques, experimental and analytical tools have allowed multidisciplinary characterisation of the role of T cells and the immune system as a whole. Of particular interest to the field of vaccinology are GCs and Tfh cells, representing a unique target for improving immunisation strategies. Here, we discuss recent insights into the unique journey of Tfh cells from thymus to lymph node during differentiation and their role in the production of high-quality antibody responses as well as their journey back to the periphery as a population of memory cells. Further, we explore their function in health and disease and the power of next-generation sequencing techniques to uncover their potential as modulators of vaccine-induced immunity

Uncovering the primary CD4+ T cell immune response to HIV-1 and Vaccinia virus.

Ex-vivo T cell responses to vaccines and viral antigens are not always detectable by conventional functional T cell assays such as lymphoproliferation, IFN-γ ELIspot and intracellular cytokine staining (ICC) assays. A culture amplified multiparametric intracellular cytokine assay (CAMP-ICC) was developed to detect antigen-specific T cells that may be present at low frequencies, typical of those induced by DNA vaccines in humans. The assay combined methodologies of proliferation detected by carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution and cytokine expression by ICC, amplified by interleukin (IL)-15 and IL-21. The CAMP-ICC provides additional information about the functionality of CD4+ and CD8+ T cells with improved sensitivity, without loss of specificity. The CAMP-ICC was utilised to uncover priming responses to a candidate prophylactic HIV-1 DNA vaccine. No differences were detected between placebo and active vaccine recipients either pre- or post-vaccination, strongly suggesting this vaccine was non-immunogenic. In an attempt to uncover mechanisms of immune control of HIV-1, CAMP-ICC was utilised to compare proliferative and cytokine expression profiles to Gag between HIV-1 controllers and non-controllers. Despite performing extensive analyses few comparisons were statistically significant. These were consistent with the current literature. To understand the developmental stages of CD4+ T cell responses to viral infection and their differentiation into long-term memory cells, individuals immunised with vaccinia virus (VV) were studied. At the peak of primary effector cell responses to VV, day 13 post-vaccination, activated effector (CD45RO+CD38+++) and naïve (CD45RO-CD38dim) CD4+ T cells were purified, mRNA extracted and microarray analysis conducted. Surprisingly, in activated effector CD4+ compared to naïve CD4+ T cells there was a strong up-regulation of cytotoxic T-lymphocyte (CTL) associated genes, of which Granzyme K was most highly expressed. This finding was confirmed at the mRNA and protein level. Generation of anti-viral human CD4+ T cell memory during primary immune responses is critical but poorly understood. The role of CTL associated genes in this process has not been explored. Understanding their role in the generation of effective memory may lead us closer to the development of a more effective HIV vaccine or enhance the modestly efficacious vaccines currently available for prevention of HIV-1 infection

Altered Immune Reconstitution in Allogeneic Stem Cell Transplant Recipients with Human Immunodeficiency Virus (HIV)

Background: Persons living with human immunodeficiency virus (HIV) are at elevated risk of developing the malignant diseases that require allogeneic stem cell transplantation (ASCT). Recent data suggest that these individuals are also at an elevated risk of certain complications post-ASCT. This risk may result from preexisting HIV-related factors affecting dynamics of immune reconstitution post-ASCT. However, to date, there has been little work describing the dynamics of immune reconstitution post-ASCT in persons with HIV and none comparing these data to controls without HIV. Methods: We assessed T-cell reconstitution in 6 ASCT with HIV recipients (HIV+ASCT) compared to a control population of 21 ASCT without HIV recipients. In a subset of HIV+ASCT recipients we performed additional flow cytometry profiling of CD8+ T-cell subsets and antigen specificity of reconstituting CD4+ and CD8+ T cells. Results: We observe no difference in post-ASCT CD4+ T cells between HIV+ASCT and HIV-negative ASCT recipients, despite much lower pre-ASCT CD4+ T-cell counts in the HIV+ASCT group. In contrast, we observed significantly higher CD8+ T-cell numbers in the HIV+ASCT group post-ASCT. The reconstituting CD8+ T-cells were predominantly CD45RO+, whereas homing markers and antigen specificity of these cells varied between participants. Conclusion: This study represents the most extensive characterization of immune-reconstitution post-ASCT in persons with HIV, and the first to our knowledge to compare these data to ASCT controls without HIV. The results indicate that immune reconstitution in this group can be affected by preexisting HIV infection and post-ASCT antigen exposure

The Role of ZEB2 in Human CD8 T Lymphocytes: Clinical and Cellular Immune Profiling in Mowat–Wilson Syndrome

The Zeb2 gene encodes a transcription factor (ZEB2) that acts as an important immune mediator in mice, where it is expressed in early-activated effector CD8 T cells, and limits effector differentiation. Zeb2 homozygous knockout mice have deficits in CD8 T cells and NK cells. Mowat–Wilson syndrome (MWS) is a rare genetic disease resulting from heterozygous mutations in ZEB2 causing disease by haploinsufficiency. Whether ZEB2 exhibits similar expression patterns in human CD8 T cells is unknown, and MWS patients have not been comprehensively studied to identify changes in CD8 lymphocytes and NK cells, or manifestations of immunodeficiency. By using transcriptomic assessment, we demonstrated that ZEB2 is expressed in early-activated effector CD8 T cells of healthy human volunteers following vaccinia inoculation and found evidence of a role for TGFß-1/SMAD signaling in these cells. A broad immunological assessment of six genetically diagnosed MWS patients identified two patients with a history of recurrent sinopulmonary infections, one of whom had recurrent oral candidiasis, one with lymphopenia, two with thrombocytopenia and three with detectable anti-nuclear antibodies. Immunoglobulin levels, including functional antibody responses to protein and polysaccharide vaccination, were normal. The MWS patients had a significantly lower CD8 T cell subset as % of lymphocytes, compared to healthy controls (median 16.4% vs. 25%, p = 0.0048), and resulting increased CD4:CD8 ratio (2.6 vs. 1.8; p = 0.038). CD8 T cells responded normally to mitogen stimulation in vitro and memory CD8 T cells exhibited normal proportions of subsets with important tissue-specific homing markers and cytotoxic effector molecules. There was a trend towards a decrease in the CD8 T effector memory subset (3.3% vs. 5.9%; p = 0.19). NK cell subsets were normal. This is the first evidence that ZEB2 is expressed in early-activated human effector CD8 T cells, and that haploinsufficiency of ZEB2 in MWS patients had a slight effect on immune function, skewing T cells away from CD8 differentiation. To date there is insufficient evidence to support an immunodeficiency occurring in MWS patients

CD127(+)CCR5(+)CD38(+++) CD4(+) Th1 Effector Cells Are an Early Component of the Primary Immune Response to Vaccinia Virus and Precede Development of Interleukin-2(+) Memory CD4(+) T Cells

The stages of development of human antigen-specific CD4(+) T cells responding to viral infection and their differentiation into long-term memory cells are not well understood. The inoculation of healthy adults with vaccinia virus presents an opportunity to study these events intensively. Between days 11 and 14 postinoculation, there was a peak of proliferating CCR5(+)CD38(+++) CD4(+) effector cells which contained the cytotoxic granule marker T-cell intracellular antigen 1 and included gamma interferon (IFN-γ)-producing vaccinia virus-specific CD4(+) T cells. The majority of these initial vaccinia virus-specific CD4(+) T cells were CD127(+) and produced interleukin-2 (IL-2) but not CTLA-4 in response to restimulation in vitro. Between days 14 and 21, there was a switch from IFN-γ and IL-2 coexpression to IL-2 production only, coinciding with a resting phenotype and an increased in vitro proliferation response. The early CCR5(+)CD38(+++) vaccinia virus-specific CD4(+) T cells were similar to our previous observations of human immunodeficiency virus (HIV)-specific CD4(+) T cells in primary HIV type 1 (HIV-1) infection, but the vaccinia virus-specific cells expressed much more CD127 and IL-2 than we previously found in their HIV-specific counterparts. The current study provides important information on the differentiation of IL-2(+) vaccinia virus-specific memory cells, allowing further study of antiviral effector CD4(+) T cells in healthy adults and their dysfunction in HIV-1 infection

Persistent survival of prevalent clonotypes within an immunodominant HIV gag-specific CD8+ T cell response

CD8+ T cells play a significant role in the control of HIV replication, yet the associated qualitative and quantitative factors that determine the outcome of infection remain obscure. In this study, we examined Ag-specific CD8+ TCR repertoires longitudinally in a cohort of HLA-B*2705+ long-term nonprogressors with chronic HIV-1 infection using a combination of molecular clonotype analysis and polychromatic flow cytometry. In each case, CD8+ T cell populations specific for the immunodominant p24 Gag epitope KRWIILGLNK (KK10; residues 263–272) and naturally occurring variants thereof, restricted by HLA-B*2705, were studied at multiple time points; in addition, comparative data were collected for CD8+ T cell populations specific for the CMV pp65 epitope NLVPMVATV (NV9; residues 495–503), restricted by HLA-A*0201. Dominant KK10-specific clonotypes persisted for several years and exhibited greater stability than their contemporaneous NV9-specific counterparts. Furthermore, these dominant KK10-specific clonotypes exhibited cross-reactivity with antigenic variants and expressed significantly higher levels of CD127 (IL-7Rα) and Bcl-2. Of note, we also found evidence that promiscuous TCR α-chain pairing associated with alterations in fine specificity for KK10 variants could contribute to TCR β-chain prevalence. Taken together, these data suggest that an antiapoptotic phenotype and the ability to cross-recognize variant epitopes contribute to clonotype longevity and selection within the peripheral memory T cell pool in the presence of persistent infection with a genetically unstable virus