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The worldwide rollout of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccinations in the last 2 years has produced a multitude of studies investigating T-cell responses in the peripheral blood and a limited number in secondary lymphoid tissues. As a key component to an effective immune response, vaccine-specific T follicular helper (Tfh) cells are localized in the draining lymph node (LN) and assist in the selection of highly specific B-cell clones for the production of neutralizing antibodies. While these cells have been noted in the blood as circulating Tfh (cTfh) cells, they are not often taken into consideration when examining effective CD4 + T-cell responses, particularly in immunocompromised groups. Furthermore, site-specific analyses in locations such as the LN have recently become an attractive area of investigation. This is mainly a result of improved sampling methods via ultrasound-guided fine-needle biopsy (FNB)/fine-needle aspiration (FNA), which are less invasive than LN excision and able to be performed longitudinally. While these studies have been undertaken in healthy individuals, data from immunocompromised groups are lacking. This review will focus on both Tfh and cTfh responses after SARS-CoV-2 vaccination in healthy and immunocompromised individuals. This area of investigation could identify key characteristics of a successful LN response required for the prevention of infection and viral clearance. This furthermore may highlight responses that could be fine-tuned to improve vaccine efficacy within immunocompromised groups that are at a risk of more severe disease., (© 2023 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Activation induced marker (AIM) assays are being used increasingly to measure antigen-specific T-cell responses, but this activation can alter cell lineage defining phenotypic markers. We aimed to extend the utility of AIM assays to enable pre-activation defined cell populations to be tracked and quantified within T-cell memory responses. We sorted three ex vivo CD4 + T-cell populations prior to any activation using well defined ex vivo lineage surface marker combinations. These populations were memory non-Tregs, CD39 + Tregs and CD39 neg Tregs, although any three memory CD4 + T-cell populations able to be isolated by cell surface markers could potentially be tracked. These cells were labeled with three distinct fluorescent cell proliferation dyes (CFSE, CellTrace Violet and Cell Proliferation Dye eF670) and then all autologous PBMCs were reconstituted maintaining ex vivo cell ratios and CD25/OX40 AIM assays performed with CMV and HSV antigens. This approach enabled tracking of pre-defined cell populations within antigen stimulated responses using both activation marker and cell proliferation readouts. We confirmed that although CD39 + Tregs comprise a substantial proportion of AIM assay responses, they do not make substantial contributions to the proliferative response. This extends the utility of AIM assays to enable parallel analysis of the relative contribution of several CD4 + memory T-cell subsets to recall responses., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia Ltd, on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Recent studies of protein and gene expression at the single-cell level have revealed that the memory T-cell compartment is more heterogeneous than previously acknowledged. Identifying different T helper subsets involved in memory responses at the single-cell level is thus necessary to understand the level of heterogeneity within this population. Antigen-specific CD4 + T cells were measured using the CD25/OX40 assay together with a qualitative multiplex single-cell RT-PCR assay. Transcription profiles and subset proportions within the antigen-specific CD4 + T-cell population were dissected. Cytomegalovirus (CMV)-specific CD4 + T-cell responses skewed toward a Th1 response, whereas Tetanus toxoid responses skewed toward a Th2 type response. Fluctuations in CD4 + T-cell subsets were observed within the HIV-Gag-specific response during ongoing antiretroviral therapy. Strong effector responses (Th1) were observed in early treatment, however with ongoing therapy this effector response significantly decreased in combination with an increase in Tregs and circulating Tfh-like BCL-6 + memory cells. The apparent increase in Tcm in peripheral blood after a several weeks of antiretroviral therapy may be due to Tfh-like cell egress from germinal centers into the periphery.

Mucosal-associated invariant T (MAIT) cells home to mucosal sites and exert antimicrobial activity against bacteria and other microorganisms. HIV infection leads to early depletion of gut T cells and translocation of bacterial products. There are reports that MAIT cells, defined by coexpression of Vα7.2 and CD161, are depleted during HIV infection and residual MAIT cells are functionally impaired. However, one study suggested that MAIT cells might remain after HIV infection but evade detection through CD161 downregulation. Thus, the impact of HIV infection on MAIT cells is unclear. We studied longitudinal blood samples from 31 HIV-infected subjects for MAIT cell numbers, phenotype and function using both standard Vα7.2/CD161 surface markers and an MR1 tetramer. We found that MAIT cells were depleted early during HIV infection, and although there was a concomitant rise in Vα7.2(+)CD161(-) cells, these were MR1 tetramer negative, indicating that these are unlikely to be altered MAIT cells. Antigen-mediated activation of residual MAIT cells showed that they remained functional out to 2 years following HIV infection. Although MAIT cells are depleted in HIV infection, residual and functionally active MAIT cells persist and may still be able to assist in controlling bacterial translocation during HIV infection.

Antibody-dependent phagocytosis (ADP) is a potentially important immune mechanism to clear HIV. How HIV-specific ADP responses mature during HIV infection or in response to vaccinations administered, including the partially successful RV144 HIV vaccine, is not known. We established a modified ADP assay to measure internalisation of HIV antibody (Ab)-opsonised targets using a specific hybridisation internalisation probe. Labelled beads were coated with both biotinylated HIV gp140 envelope protein and a fluorescent internalisation probe, opsonised with Abs and incubated with a monocytic cell line. The fluorescence derived from the fluorescent internalisation probe on surface-bound beads, but not from internalised beads, was quenched by the addition of a complementary quencher probe. HIV Env-specific ADP was measured in 31 subjects during primary infection and early chronic HIV infection. Although ADP responses were present early during HIV infection, a significant increase in ADP responses in all 31 subjects studied was detected (P<0.001). However, when we tested 30 HIV-negative human subjects immunised with the Canarypox/gp120 vaccine regimen (subjects from the RV144 trial) we did not detect HIV-specific ADP activity. In conclusion, a modified assay was developed to measure HIV-specific ADP. Enhanced ADP responses early in the course of HIV infection were observed but no ADP activity was detected following the vaccinations administered in the RV144 trial. Improved vaccine regimens may be needed to capitalise on ADP-mediated immunity against HIV.

Natural killer T (NKT) cells bridge across innate and adaptive immune responses and have an important role in chronic viral infections such as human immunodeficiency virus (HIV). NKT cells are depleted during chronic HIV infection, but the timing, drivers and implications of this NKT cell depletion are poorly understood. We studied human peripheral blood NKT cell levels, phenotype and function in 31 HIV-infected subjects not on antiretroviral treatment from a mean of 4 months to 2 years after HIV infection. We found that peripheral CD4(+) NKT cells were substantially depleted and dysfunctional by 4 months after HIV infection. The depletion of CD4(+) NKT cells was more marked than the depletion of total CD4(+) T cells. Further, the early depletion of NKT cells correlated with CD4(+) T-cell decline, but not HIV viral levels. Levels of activated CD4(+) T cells correlated with the loss of NKT cells. Our studies suggest that the early loss of NKT cells is associated with subsequent immune destruction during HIV infection.

The OX40/CD25 assay is a novel technique that assesses antigen-specific CD4(+) T-cell responses. To unequivocally demonstrate that responding cells are memory cells that become activated after secondary stimulation, naïve CD45RA(+) and memory CD45RO(+) populations were stimulated with cytomegalovirus (CMV) lysate and the combined expression of CD25 and OX40 measured. As expected, the naïve population showed very little response, whereas there was a higher response from the memory counterpart. To further elucidate CD4(+) memory T-cell subsets involved in recall responses, CD4(+) T cells were separated into central memory (Tcm) and effector memory (Tem) subsets and stimulated with antigen-pulsed antigen-presenting cells (APCs). CMV responses in healthy donors showed a Tem-dominant response with a Tem/Tcm ratio of 1.2, whereas the tetanus toxoid responses were dominated by a Tcm response with a Tem/Tcm ratio of 0.35. To determine memory response in the chronic of HIV infection, patient samples were used. A similar pattern to healthy donors was observed in seven chronic HIV+ patients at week 4 after anti-retroviral therapy who responded to CMV with a larger response coming from Tem. The pattern was similar after 48 weeks of therapy but the responses were lower in magnitude. In chronic HIV+ patients who respond to Gag peptides, following institution of therapy there was an inversion of the ratio of the responding memory subsets compared with week 4, with a greater response from Tcm at week 48. This result was concordant with reduction in antigen load. As immune activation decreased there was also a decrease in the percentage of responding effector memory cells and maintenance of long-term central memory.

Anal cancer is one of the most common non-AIDS-defining malignancies in the era of combination antiretroviral therapy. Its precursor lesion, anal intraepithelial neoplasia (AIN), is highly prevalent in HIV-infected populations. More than 90% of anal squamous cell cancers are attributable to human papillomavirus (HPV). While the biology of HPV-related intraepithelial neoplasia is consistent across lower anogenital sites, the natural history of AIN is not well established and cannot be assumed to be identical to that of cervical intraepithelial neoplasia. Screening strategies to prevent anal cancer should be developed based on robust natural history data in HIV-infected and uninfected populations. Likewise, treatments need to be tested in randomized clinical trials, and reserved for those at significant risk of progression to cancer. This review covers the epidemiology, pathogenesis and immunology of HPV infection, AIN and anal cancer, and summarizes the current diagnosis, screening and treatment strategies in HIV-infected adults., (© 2013 British HIV Association.)

Background: Simple and effective delivery methods for cellular immunotherapies are needed. We recently published on the effectiveness of using ex vivo pulsing of overlapping SIV Gag 15mer peptides onto fresh peripheral blood cells in 32 SIV(mac251)-infected pigtail macaques., Methods: We now report on the safety of this approach, analysis of a novel assay for immunogenicity, the effect of an MHC allele, Mane-A*10, on CD8 T cell escape occurring and disease outcome., Results: The vaccine strategy was safe, with no perturbations in weight or hematological profiles in comparison to controls. The high levels of SIV-specific T cell immunogenicity of this approach was confirmed using a novel assay measuring upregulation of surface CD134 of CD4 T cells. A substantial effect of the Mane-A*10 allele in reducing SIV viral load of pigtail macaques was observed in both vaccinees and controls; the virologic efficacy of the immunotherapy in comparison to controls was greatest in Mane-A*10- animals. Escape mutations at several new CD8 T cell epitopes throughout the SIV proteome were observed, primarily in animals with poorer virologic control., Conclusions: In summary, we provide further information that peptide-pulsed PBMC are a safe, immunogenic and effective immunotherapy. The observed influence of MHC alleles and immune escape allows us to design more insightful future immunotherapy studies.

Human immunodeficiency virus (HIV) infection causes chronic progressive immunodeficiency and immune dysregulaton. Although simple depletion of the major target of HIV infection, the CD4+ T cell, can explain much of the immunosuppression seen, there are multiple other factors contributing to the immune dysregulation. CD4+ T-cell depletion induces a range of homeostatic mechanisms that contribute to immune activation and cell turnover, providing a milieu conducive to further viral replication and cell destruction, resulting in functional defects in various lymphoid organs. These changes are progressive and in turn compromise the homeostatic processes. Further, the infection, like any other viral infection, provokes an active immune response consisting of both CD4+ and CD8+ T-cell responses. Both appear compromised, displaying aberrant memory cell production. While some of these defects result from viral variation and the chronicity of antigen presentation, other defects of memory cell production appear very early during the primary immune response limiting the viral specific T-cell responses from the outset. This, combined with the ability of the virus to escape any successful immune responses, results in an attenuated immune response that eventually becomes exhausted, characterized by progressive deficits in T-cell repertoire. Furthermore, negative regulatory mechanisms that normally control the immune response may be aberrantly invoked, perhaps directly by the virus, further compromising the efficacy of the immune response. Rational design of effective immunotherapies depends on a clear understanding of the processes compromising the immune response to HIV.

Analysing T-cell receptor excision circle numbers in healthy individuals we find a marked change in the source of naive T cells before and after 20 years of age. The bulk of the naive T cell pool is sustained primarily from thymic output for individuals younger than 20 years of age whereas proliferation within the naive phenotype is dominant for older individuals. Over 90% of phenotypically naive T cells in middle age are not of direct thymic origin. Moreover, this change in source of naive T cells is accompanied either by an increased death rate of T cells from the thymus or reduced thymic export. Modelling of these processes shows that new naive T cells of a thymic origin have a half-life of approximately 50 days before this change occurs, and that either the life-span of recent thymic emigrants (but not necessarily of all naive cells) decreases approximately threefold in middle age, or thymic production drops by this same amount. The decay rate of T-cell receptor excision circle levels for individuals over 20 years of age is consistent with the decay rate of the productive thymus. Our modelling suggests that at age 25, thymic export is responsible for 20% of naive T-cell production and that this percentage decreases with the 15.7 year half-life of the productive thymus so that by age 55 only 5% of naive production arises from thymic export.