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Although mRNA SARS-CoV-2 vaccines are generally safe and effective, in certain immunocompromised individuals they can elicit poor immunogenic responses. Among these individuals, people living with HIV (PLWH) have poor immunogenicity to several oral and parenteral vaccines. As the gut microbiome is known to affect vaccine immunogenicity, we investigated whether baseline gut microbiota predicts immune responses to the BNT162b2 mRNA SARS-CoV-2 vaccine in healthy controls and PLWH after two doses of BNT162b2. Individuals with high spike IgG titers and high spike-specific CD4 + T-cell responses against SARS-CoV-2 showed low α-diversity in the gut. Here, we investigated and presented initial evidence that the gut microbial composition influences the response to BNT162b2 in PLWH. From our predictive models, Bifidobacterium and Faecalibacterium appeared to be microbial markers of individuals with higher spike IgG titers, while Cloacibacillus was associated with low spike IgG titers. We therefore propose that microbiome modulation could optimize immunogenicity of SARS-CoV-2 mRNA vaccines., (© 2023. The Author(s).)

Chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from people living with HIV, here we expose the T cells to planar lipid bilayers containing ligands for T-cell receptor and a T-cell integrins and analyze the cellular morphology, dynamics of synaptic interface formation and patterns of the cellular degranulation. We find a large fraction of phenotypically naive T cells from chronically infected people are capable to form mature synapse with focused degranulation, a signature of a differentiated T cells. Further, differentiation of aberrant naive T cells may lead to the development of anomalous effector T cells undermining their capacity to control HIV and other pathogens that could be contained otherwise., (© 2022. The Author(s).)

Background: Mucosa-associated invariant T (MAIT) cells are innate-like T cells with specialized antimicrobial functions. Circulating MAIT cells are depleted in chronic human immunodeficiency virus (HIV) infection, but studies examining this effect in peripheral tissues, such as the female genital tract, are lacking., Methods: Flow cytometry was used to investigate circulating MAIT cells in a cohort of HIV-seropositive (HIV+) and HIV-seronegative (HIV-) female sex workers (FSWs), and HIV- lower-risk women (LRW). In situ staining and quantitative polymerase chain reaction were performed to explore the phenotype of MAIT cells residing in paired cervicovaginal tissue. The cervicovaginal microbiome was assessed by means of 16S ribosomal RNA gene sequencing., Results: MAIT cells in the HIV+ FSW group were low in frequency in the circulation but preserved in the ectocervix. MAIT cell T-cell receptor gene segment usage differed between the HIV+ and HIV- FSW groups. The TRAV1-2-TRAJ20 transcript was the most highly expressed MAIT TRAJ gene detected in the ectocervix in the HIV+ FSW group. MAIT TRAVJ usage was not associated with specific genera in the vaginal microbiome., Conclusions: MAIT cells residing in the ectocervix are numerically preserved irrespective of HIV infection status and displayed dominant expression of TRAV1-2-TRAJ20. These findings have implications for understanding the role of cervical MAIT cells in health and disease., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Time to AIDS in HIV-2 infection is approximately twice as long compared to in HIV-1 infection. Despite reduced viremia, HIV-2-infected individuals display signs of chronic immune activation. In HIV-1-infected individuals, B-cell hyperactivation is driven by continuous antigen exposure. However, the contribution of viremia to B-cell perturbations in HIV-2-infected individuals remains largely unexplored. Here, we used polychromatic flow cytometry, consensus hierarchical clustering and pseudotime trajectory inference to characterize B-cells in HIV-1- or HIV-2-infected and in HIV seronegative individuals. We observed increased frequencies of clusters containing hyperactivated T-bet high CD95 high CD27 int and proliferating T-bet + CD95 high CD27 + CD71 + memory B-cells in viremic HIV-1 ( p < 0.001 and p < 0.001, respectively), viremic HIV-2 ( p < 0.001 and p = 0.014, respectively) and in treatment-naïve aviremic HIV-2 ( p = 0.004 and p = 0.020, respectively)-infected individuals, compared to seronegative individuals. In contrast, these expansions were not observed in successfully treated HIV-1-infected individuals. Finally, pseudotime trajectory inference showed that T-bet-expressing hyperactivated and proliferating memory B-cell populations were located at the terminal end of two trajectories, in both HIV-1 and HIV-2 infections. As the treatment-naïve aviremic HIV-2-infected individuals, but not the successfully ART-treated HIV-1-infected individuals, showed B-cell perturbations, our data suggest that aviremic HIV-2-infected individuals would also benefit from antiretroviral treatment.

HIV-2 is less pathogenic compared to HIV-1. Still, disease progression may develop in aviremic HIV-2 infection, but the driving forces and mechanisms behind such development are unclear. Here, we aimed to reveal the immunophenotypic pattern associated with CD8 T-cell pathology in HIV-2 infection, in relation to viremia and markers of disease progression. The relationships between pathological differences of the CD8 T-cell memory population and viremia were analyzed in blood samples obtained from an occupational cohort in Guinea-Bissau, including HIV-2 viremic and aviremic individuals. For comparison, samples from HIV-1- or dually HIV-1/2-infected and seronegative individuals were obtained from the same cohort. CD8 T-cell exhaustion was evaluated by the combined expression patterns of activation, stimulatory and inhibitory immune checkpoint markers analyzed using multicolor flow cytometry and advanced bioinformatics. Unsupervised multidimensional clustering analysis identified a cluster of late differentiated CD8 T-cells expressing activation (CD38+, HLA-DR int/high ), co-stimulatory (CD226+/-), and immune inhibitory (2B4+, PD-1 high , TIGIT high ) markers that distinguished aviremic from viremic HIV-2, and treated from untreated HIV-1-infected individuals. This CD8 T-cell population displayed close correlations to CD4%, viremia, and plasma levels of IP-10, sCD14 and beta-2 microglobulin in HIV-2 infection. Detailed analysis revealed that aviremic HIV-2-infected individuals had higher frequencies of exhausted TIGIT+ CD8 T-cell populations lacking CD226, while reduced percentage of stimulation-receptive TIGIT-CD226+ CD8 T-cells, compared to seronegative individuals. Our results suggest that HIV-2 infection, independent of viremia, skews CD8 T-cells towards exhaustion and reduced co-stimulation readiness. Further knowledge on CD8 T-cell phenotypes might provide help in therapy monitoring and identification of immunotherapy targets., 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 Scharf, Pedersen, Johansson, Lindman, Olsen, Buggert, Wilhelmson, Månsson, Esbjörnsson, Biague, Medstrand, Norrgren, Karlsson, Jansson and the SWEGUB CORE Group.)

While the relationship of protective human leukocyte antigen (HLA) class I alleles and HIV progression is well defined, the interaction of HLA-mediated protection and CD8 T-cell exhaustion is less well characterized. To gain insight into the influence of HLA-B*57:01 on the deterioration of CD8 T-cell responses during HIV infection in the absence of antiretroviral treatment, we compared HLA-B*57:01-restricted HIV-specific CD8 T-cell responses to responses restricted by other HLA class I alleles longitudinally after control of peak viremia. Detailed characterization of polyfunctionality, differentiation phenotypes, transcription factor, and inhibitory receptor expression revealed progression of CD8 T-cell exhaustion over the course of the infection in both patient groups. However, early effects on the phenotype of the total CD8 T-cell population were apparent only in HLA-B*57-negative patients. The HLA-B*57:01-restricted, HIV epitope-specific CD8 T-cell responses showed beneficial functional patterns and significantly lower frequencies of inhibitory receptor expression, i.e., PD-1 and coexpression of PD-1 and TIGIT, within the first year of infection. Coexpression of PD-1 and TIGIT was correlated with clinical markers of disease progression and declining percentages of the T-bet hi Eomes dim CD8 T-cell population. In accordance with clinical and immunological deterioration in the HLA-B*57:01 group, the difference in PD-1 and TIGIT receptor expression did not persist to later stages of the disease. IMPORTANCE Given the synergistic nature of TIGIT and PD-1, the coexpression of those inhibitory receptors should be considered when evaluating T-cell pathogenesis, developing immunomodulatory therapies or vaccines for HIV, and when using immunotherapy or vaccination for other causes in HIV-infected patients. HIV-mediated T-cell exhaustion influences the patient´s disease progression, immune system and subsequently non-AIDS complications, and efficacy of vaccinations against other pathogens. Consequently, the possibilities of interfering with exhaustion are numerous. Expanding the use of immunomodulatory therapies to include HIV treatment depends on information about possible targets and their role in the deterioration of the immune system. Furthermore, the rise of immunotherapies against cancer and elevated cancer incidence in HIV-infected patients together increase the need for detailed knowledge of T-cell exhaustion and possible interactions. A broader approach to counteract immune exhaustion to alleviate complications and improve efficacy of other vaccines also promises to increase patients' health and quality of life., (Copyright © 2020 Scharf et al.)

The functional properties of circulating CD8 + T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8 + T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8 + T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8 + T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4 + T cell-associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8 + T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Disease progression of human immunodeficiency virus type 1 (HIV-1) is delayed by HIV type 2 (HIV-2) in individuals with dual HIV-1/HIV-2 infection. The protective mechanisms, however, are still to be revealed. In the current study we examined type-specific and cross-reactive antibody-dependent cellular cytotoxicity (ADCC) in HIV-1 and HIV-2 monoinfection or dual infection. Of note, intertype cross-reactive antibodies that mediated HIV-1 envelope glycoprotein (Env)-targeted ADCC were frequently identified in HIV-2-infected individuals. Furthermore, the magnitude of HIV-1 cross-reactive ADCC activity during HIV-2 infections depended on the HIV-1 Env origin and was associated with the duration of infection. These results suggest that preexisting antibodies against HIV-2, which mediate intertype ADCC, might contribute to control of HIV-1 during dual infection., (The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Purpose of Review: To introduce emerging concepts in tissue resident CD8 T cell immunosurveillance and their relevance to control HIV infection., Recent Findings: It is well appreciated that HIV preferentially infects and persists in CD4 T cells located in gut and in lymphoid tissue, yet the majority of known immunological correlates of HIV control are derived from peripheral blood. Instead, tissue-based immunological surveillance likely dictates the course of infection. Recent studies have established that nonrecirculating resident memory CD4 and CD8 T cells can be found in virtually every human tissue. These cells bear a transcriptional profile of tissue retention and immediate effector function, suggesting a pivotal role in protective immunity. Resident memory CD8 T cells specific for HIV have been found in higher numbers in sites of HIV persistence (gut and lymph nodes), and are inversely associated with HIV viral titers. These findings, along with previous studies on tissue-derived cells now known to include resident memory cells, shed new light on the compartmentalization of the immune response against HIV and its correlates of protection., Summary: Resident memory CD8 T cells represent a critical unexplored component of immune surveillance in the setting of HIV infection. Understanding the induction, dynamics, and functional properties of HIV-specific resident memory T cells in relevant tissues will better inform efforts in the treatment, control, and potential cure of HIV infection.

Background: Genital mucosa is the main portal of entry for various incoming pathogens, including human immunodeficiency virus (HIV), hence it is an important site for host immune defenses. Tissue-resident memory T (TRM) cells defend tissue barriers against infections and are characterized by expression of CD103 and CD69. In this study, we describe the composition of CD8+ TRM cells in the ectocervix of healthy and HIV-infected women., Methods: Study samples were collected from healthy Swedish and Kenyan HIV-infected and uninfected women. Customized computerized image-based in situ analysis was developed to assess the ectocervical biopsies. Genital mucosa and blood samples were assessed by flow cytometry., Results: Although the ectocervical epithelium of healthy women was populated with bona fide CD8+ TRM cells (CD103+CD69+), women infected with HIV displayed a high frequency of CD103-CD8+ cells residing close to their epithelial basal membrane. Accumulation of CD103-CD8+ cells was associated with chemokine expression in the ectocervix and HIV viral load. CD103+CD8+ and CD103-CD8+ T cells expressed cytotoxic effector molecules in the ectocervical epithelium of healthy and HIV-infected women. In addition, women infected with HIV had decreased frequencies of circulating CD103+CD8+ T cells., Conclusions: Our data provide insight into the distribution of CD8+ TRM cells in human genital mucosa, a critically important location for immune defense against pathogens, including HIV.

Current paradigms of CD8 + T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8 + T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (T RMs ). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8 + T cells in LTs also resemble T RMs Moreover, high frequencies of HIV-specific CD8 + T RMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific T RMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-T RMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8 + T cell responses resident within LTs., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

CD4+ T cells subsets have a wide range of important helper and regulatory functions in the immune system. Several studies have specifically suggested that circulating effector CD4+ T cells may play a direct role in control of HIV replication through cytolytic activity or autocrine β-chemokine production. However, it remains unclear whether effector CD4+ T cells expressing cytolytic molecules and β-chemokines are present within lymph nodes (LNs), a major site of HIV replication. Here, we report that expression of β-chemokines and cytolytic molecules are enriched within a CD4+ T cell population with high levels of the T-box transcription factors T-bet and eomesodermin (Eomes). This effector population is predominately found in peripheral blood and is limited in LNs regardless of HIV infection or treatment status. As a result, CD4+ T cells generally lack effector functions in LNs, including cytolytic capacity and IFNγ and β-chemokine expression, even in HIV elite controllers and during acute/early HIV infection. While we do find the presence of degranulating CD4+ T cells in LNs, these cells do not bear functional or transcriptional effector T cell properties and are inherently poor to form stable immunological synapses compared to their peripheral blood counterparts. We demonstrate that CD4+ T cell cytolytic function, phenotype, and programming in the peripheral blood is dissociated from those characteristics found in lymphoid tissues. Together, these data challenge our current models based on blood and suggest spatially and temporally dissociated mechanisms of viral control in lymphoid tissues.

Elimination of lymphoid tissue reservoirs is a key component of HIV eradication strategies. CD8 + T cells play a critical role in control of HIV, but their functional attributes in lymph nodes (LNs) remain unclear. Here, we show that memory, follicular CXCR5 + , and HIV-specific CD8 + T cells from LNs do not manifest the properties of cytolytic CD8 + T cells. While the frequency of follicular CXCR5 + CD8 + T cells was strongly inversely associated with peripheral viremia, this association was not dependent on cytolytic CXCR5 + CD8 + T cells. Moreover, the poor cytolytic activity of LN CD8 + T cells was linked to a compartmentalized dissociation between effector programming and the transcription factor T-bet. In line with this, activation of LN CD8 + T cells only partially induced the acquisition of cytolytic functions relative to peripheral blood CD8 + T cells. These results suggest that a state of immune privilege against CD8 + T cell-mediated cytolysis exists in lymphoid tissue, potentially facilitating the persistence of HIV., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

HIV-specific CD8 + T cells demonstrate an exhausted phenotype associated with increased expression of inhibitory receptors, decreased functional capacity, and a skewed transcriptional profile, which are only partially restored by antiretroviral treatment (ART). Expression levels of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), the co-stimulatory receptor CD226 and their ligand PVR are altered in viral infections and cancer. However, the extent to which the TIGIT/CD226/PVR-axis is affected by HIV-infection has not been characterized. Here, we report that TIGIT expression increased over time despite early initiation of ART. HIV-specific CD8 + T cells were almost exclusively TIGIT + , had an inverse expression of the transcription factors T-bet and Eomes and co-expressed PD-1, CD160 and 2B4. HIV-specific TIGIT hi cells were negatively correlated with polyfunctionality and displayed a diminished expression of CD226. Furthermore, expression of PVR was increased on CD4 + T cells, especially T follicular helper (Tfh) cells, in HIV-infected lymph nodes. These results depict a skewing of the TIGIT/CD226 axis from CD226 co-stimulation towards TIGIT-mediated inhibition of CD8 + T cells, despite early ART. These findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that could hinder future "cure" strategies requiring potent HIV-specific CD8 + T cells.

Objective: HIV type 2 (HIV-2) represents an attenuated form of HIV, in which many infected individuals remain 'aviremic' without antiretroviral therapy. However, aviremic HIV-2 disease progression exists, and in the current study, we therefore aimed to examine if specific pathological characteristics of CD4 T cells are linked to such outcome., Design: HIV-seronegative (n = 25), HIV type 1 (HIV-1) (n = 33), HIV-2 (n = 39, of whom 26 were aviremic), and HIV-1/2 dually (HIV-D) (n = 13)-infected study participants were enrolled from an occupational cohort in Guinea-Bissau., Methods: CD4 T-cell differentiation, activation, exhaustion, senescence, and transcription factors were assessed by polychromatic flow cytometry. Multidimensional clustering bioinformatic tools were used to identify CD4 T-cell subpopulations linked to infection type and disease stage., Results: HIV-2-infected individuals had early and late-differentiated CD4 T-cell clusters with lower activation (CD38HLA-DR) and exhaustion programmed death-1 (PD-1) than HIV-1 and HIV-D-infected individuals. We also noted that aviremic HIV-2-infected individuals possessed fewer individuals. CD4 T cells with pathological signs compared to other HIV-infected groups. Still, compared to HIV-seronegative individuals, aviremic HIV-2-infected individuals had T-bet CD4 T cells that showed elevated immune activation/exhaustion, and particularly the frequencies of PD-1 cells were associated with a suboptimal percentage of CD4 T cells., Conclusion: Increased frequencies of CD4 T cells with an activated/exhausted phenotype correlate with exacerbated immunodeficiency in aviremic HIV-2-infected individuals. Thus, these findings encourage studies on the introduction of antiretroviral therapy also to individuals with aviremic HIV-2 infection.

The loss of HIV-specific CD8+ T cell cytolytic function is a primary factor underlying progressive HIV infection, but whether HIV-specific CD8+ T cells initially possess cytolytic effector capacity, and when and why this may be lost during infection, is unclear. Here, we assessed CD8+ T cell functional evolution from primary to chronic HIV infection. We observed a profound expansion of perforin+ CD8+ T cells immediately following HIV infection that quickly waned after acute viremia resolution. Selective expression of the effector-associated transcription factors T-bet and eomesodermin in cytokine-producing HIV-specific CD8+ T cells differentiated HIV-specific from bulk memory CD8+ T cell effector expansion. As infection progressed expression of perforin was maintained in HIV-specific CD8+ T cells with high levels of T-bet, but not necessarily in the population of T-betLo HIV-specific CD8+ T cells that expand as infection progresses. Together, these data demonstrate that while HIV-specific CD8+ T cells in acute HIV infection initially possess cytolytic potential, progressive transcriptional dysregulation leads to the reduced CD8+ T cell perforin expression characteristic of chronic HIV infection.

Objective: In this study, we aimed to investigate the frequency and activation of invariant natural killer T (iNKT) cells and natural killer (NK) cells among HIV-1, HIV-2, or dually HIV-1/HIV-2 (HIV-D)-infected individuals, in relation to markers of disease progression., Design: Whole blood samples were collected from treatment-naive HIV-1 (n = 23), HIV-2 (n = 34), and HIV-D (n = 11) infected individuals, as well as HIV-seronegative controls (n = 25), belonging to an occupational cohort in Guinea-Bissau., Methods: Frequencies and activation levels of iNKT and NK cell subsets were analysed using multicolour flow cytometry, and results were related to HIV-status, CD4 T-cell levels, viral load, and T-cell activation., Results: HIV-1, HIV-D, and viremic HIV-2 individuals had lower numbers of CD4 iNKT cells in circulation compared with seronegative controls. Numbers of CD56 NK cells were also reduced in HIV-infected individuals as compared with control study participants. Notably, iNKT cell and NK cell activation levels, assessed by CD38 expression, were increased in HIV-1 and HIV-2 single, as well as dual, infections. HIV-2 viremia was associated with elevated activation levels in CD4 iNKT cells, CD56, and CD56 NK cells, as compared with aviremic HIV-2 infection. Additionally, disease markers such as CD4 T-cell percentages, viral load, and CD4 T-cell activation were associated with CD38 expression levels of both iNKT and NK cells, which activation levels also correlated with each other., Conclusion: Our data indicate that elevated levels of iNKT-cell and NK-cell activation are associated with viremia and disease progression markers in both HIV-1 and HIV-2 infections.

Monocyte activation has been identified as a predictor of mortality and morbidity in HIV-1 infection. This study investigated translocated bacterial flagellin as a potential contributor to systemic monocyte activation via Toll-like receptor 5 (TLR5) stimulation.We demonstrated that circulating flagellin correlated to anti-flagellin, which was associated with soluble markers of microbial translocation (LPS, LBP) and monocyte activation (sCD14, sCD163). Flagellin exposure in vitro reduced monocyte TLR5 expression and the magnitude of reduction was correlated to anti-flagellin levels, indicative of previous flagellin exposure. Circulating anti-flagellin and basal TLR5 expression were both associated with basal and flagellin-stimulated monocyte cytokine production, where HIV + and HIV − differed in their cytokine patterns (IL-1β, IL-6, IL-8).Our results suggest that translocated flagellin contributes to systemic immune activation in HIV-1 infection and reduces monocyte surface TLR5 expression resulting in a hyperactivated state with elevated basal cytokine production and reduced ability to respond to further TLR5 stimulation.

Understanding the mechanisms involved in cellular immune responses against control of human immunodeficiency virus (HIV) infection is key to development of effective immunotherapeutic strategies against viral proliferation. Clear insights into the regulation of cytotoxic CD8+ T cells is crucial to development of effective immunotherapeutic strategies due to their unique ability to eliminate virus-infected cells during the course of infection. Here, we reviewed the roles of transcription factors, co-inhibitory molecules and regulatory cytokines following HIV infection and their potential significance in regulating the cytotoxic potentials of CD8+ T cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

HIV infection provokes a myriad of pathological effects on the immune system where many markers of CD4+ T cell dysfunction have been identified. However, most studies to date have focused on single/double measurements of immune dysfunction, while the identification of pathological CD4+ T cell clusters that is highly associated to a specific biomarker for HIV disease remain less studied. Here, multi-parametric flow cytometry was used to investigate immune activation, exhaustion, and senescence of diverse maturation phenotypes of CD4+ T cells. The traditional method of manual data analysis was compared to a multidimensional clustering tool, FLOw Clustering with K (FLOCK) in two cohorts of 47 untreated HIV-infected individuals and 21 age and sex matched healthy controls. In order to reduce the subjectivity of FLOCK, we developed an "artificial reference", using 2% of all CD4+ gated T cells from each of the HIV-infected individuals. Principle component analyses demonstrated that using an artificial reference lead to a better separation of the HIV-infected individuals from the healthy controls as compared to using a single HIV-infected subject as a reference or analyzing data manually. Multiple correlation analyses between laboratory parameters and pathological CD4+ clusters revealed that the CD4/CD8 ratio was the preeminent surrogate marker of CD4+ T cells dysfunction using all three methods. Increased frequencies of an early-differentiated CD4+ T cell cluster with high CD38, HLA-DR and PD-1 expression were best correlated (Rho = -0.80, P value = 1.96×10-11) with HIV disease progression as measured by the CD4/CD8 ratio. The novel approach described here can be used to identify cell clusters that distinguish healthy from HIV infected subjects and is biologically relevant for HIV disease progression. These results further emphasize that a simple measurement of the CD4/CD8 ratio is a useful biomarker for assessment of combined CD4+ T cell dysfunction in chronic HIV disease.

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.

CD8(+) T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8(+) T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8(+) T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8(+) T cells was elevated in chronically infected individuals and highly associated with a T-bet(dim)Eomes(hi) expressional profile. Interestingly, both resting and activated HIV-specific CD8(+) T cells in chronic infection were almost exclusively T-bet(dim)Eomes(hi) cells, while CMV-specific CD8(+) T cells displayed a balanced expression pattern of T-bet and Eomes. The T-bet(dim)Eomes(hi) virus-specific CD8(+) T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8(+) T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8(+) T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8(+) T cells to control the viral replication post-ART cessation.

Viral escape from HIV-1-specific CD8(+) T cells has been demonstrated in numerous studies previously. However, the qualitative features driving the emergence of mutations within epitopes are still unclear. In this study, we aimed to distinguish whether specific functional characteristics of HLA-B*5701-restricted CD8(+) T cells influence the emergence of mutations in high-risk progressors (HRPs) versus low-risk progressors (LRPs). Single-genome sequencing was performed to detect viral mutations (variants) within seven HLA-B*5701-restricted epitopes in Gag (n = 4) and Nef (n = 3) in six untreated HLA-B*5701 subjects followed from early infection up to 7 y. Several well-characterized effector markers (IFN-γ, IL-2, MIP-1β, TNF, CD107a, and perforin) were identified by flow cytometry following autologous (initial and emerging variant/s) epitope stimulations. This study demonstrates that specific functional attributes may facilitate the outgrowth of mutations within HLA-B*5701-restricted epitopes. A significantly lower fraction of IL-2-producing cells and a decrease in functional avidity and polyfunctional sensitivity were evident in emerging epitope variants compared with the initial autologous epitopes. Interestingly, the HRPs mainly drove these differences, whereas the LRPs maintained a directed and maintained functional response against emerging epitope variants. In addition, LRPs induced improved cell-cycle progression and perforin upregulation after autologous and emerging epitope variant stimulations in contrast to HRPs. The maintained quantitative and qualitative features of the CD8(+) T cell responses in LRPs toward emerging epitope variants provide insights into why HLA-B*5701 subjects have different risks of HIV-1 disease progression.

HIV disease progression is characterized by numerous pathological changes of the cellular immune system. Still, the CD4 cell count and viral load represent the laboratory parameters that are most commonly used in the clinic to determine the disease progression. In this study, we conducted an interdisciplinary investigation to determine which laboratory parameters (viral load, CD4 count, CD8 count, CD4 %, CD8 %, CD4/CD8) are most strongly associated with pathological changes of the immune system. Multiparametric flow cytometry was used to assess markers of CD4(+) and CD8(+) T cell activation (CD38, HLA-DR), exhaustion (PD-1, Tim-3), senescence (CD28, CD57), and memory differentiation (CD45RO, CD27) in a cohort of 47 untreated HIV-infected individuals. Using bioinformatical methods, we identified 139 unique populations, representing the "combined T cell pathogenesis," which significantly differed between the HIV-infected individuals and healthy control subjects. CD38, HLA-DR, and PD-1 were particularly expressed within these unique T cell populations. The CD4/CD8 ratio was correlated with more pathological T cell populations (n = 10) and had a significantly higher average correlation coefficient than any other laboratory parameters. We also reduced the dimensionalities of the 139-unique populations by Z-transformations and principal component analysis, which still identified the CD4/CD8 ratio as the preeminent surrogate of combined T cell pathogenesis. Importantly, the CD4/CD8 ratio at baseline was shown to be significantly associated with CD4 recovery 2 y after therapy initiation. These results indicate that the CD4/CD8 ratio would be a suitable laboratory predictor in future clinical and therapeutic settings to monitor pathological T cell events in HIV infection.

We aimed to investigate whether the character of the immunodominant HIV-Gag peptide (variable or conserved) targeted by CD8(+) T cells in early HIV infection would influence the quality and quantity of T cell responses, and whether this would affect the rate of disease progression. Treatment-naive HIV-infected study subjects within the OPTIONS cohort at the University of California, San Francisco, were monitored from an estimated 44 days postinfection for up to 6 years. CD8(+) T cells responses targeting HLA-matched HIV-Gag-epitopes were identified and characterized by multicolor flow cytometry. The autologous HIV gag sequences were obtained. We demonstrate that patients targeting a conserved HIV-Gag-epitope in early infection maintained their epitope-specific CD8(+) T cell response throughout the study period. Patients targeting a variable epitope showed decreased immune responses over time, although there was no limitation of the functional profile, and they were likely to target additional variable epitopes. Maintained immune responses to conserved epitopes were associated with no or limited sequence evolution within the targeted epitope. Patients with immune responses targeting conserved epitopes had a significantly lower median viral load over time compared to patients with responses targeting a variable epitope (0.63 log(10) difference). Furthermore, the rate of CD4(+) T cell decline was slower for subjects targeting a conserved epitope (0.85% per month) compared to subjects targeting a variable epitope (1.85% per month). Previous studies have shown that targeting of antigens based on specific HLA types is associated with a better disease course. In this study we show that categorizing epitopes based on their variability is associated with clinical outcome.

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8(+) T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4(+) T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8(+) T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8(+) T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.

CD4+ T cells orchestrate immunity against viral infections, but their importance in HIV infection remains controversial. Nevertheless, comprehensive studies have associated increase in breadth and functional characteristics of HIV-specific CD4+ T cells with decreased viral load. A major challenge for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73% of the predicted peptides were found to induce HIV-specific CD4+ T cell responses. The Gag and Nef peptides induced most responses. The vast majority of the peptides (93%) had predicted restriction to the patient's HLA alleles. Interestingly, the viral load in viremic patients was inversely correlated to the number of targeted Gag peptides. In addition, the predicted Gag peptides were found to induce broader polyfunctional CD4+ T cell responses compared to the commonly used Gag-p55 peptide pool. These results demonstrate the power of the PopCover method for the identification of broadly recognized HLA class II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines.

The functional properties of circulating CD8+ T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8+ T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8+ T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8+ T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4+ T cell-associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8+ T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV.

Current paradigms of CD8+ T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs.

HIV-specific CD8+ T cells demonstrate an exhausted phenotype associated with increased expression of inhibitory receptors, decreased functional capacity, and a skewed transcriptional profile, which are only partially restored by antiretroviral treatment (ART). Expression levels of the inhibitory receptor, T cell immunoglobulin and ITIM domain (TIGIT), the co-stimulatory receptor CD226 and their ligand PVR are altered in viral infections and cancer. However, the extent to which the TIGIT/CD226/PVR-axis is affected by HIV-infection has not been characterized. Here, we report that TIGIT expression increased over time despite early initiation of ART. HIV-specific CD8+ T cells were almost exclusively TIGIT+, had an inverse expression of the transcription factors T-bet and Eomes and co-expressed PD-1, CD160 and 2B4. HIV-specific TIGIThi cells were negatively correlated with polyfunctionality and displayed a diminished expression of CD226. Furthermore, expression of PVR was increased on CD4+ T cells, especially T follicular helper (Tfh) cells, in HIV-infected lymph nodes. These results depict a skewing of the TIGIT/CD226 axis from CD226 co-stimulation towards TIGIT-mediated inhibition of CD8+ T cells, despite early ART. These findings highlight the importance of the TIGIT/CD226/PVR axis as an immune checkpoint barrier that could hinder future "cure" strategies requiring potent HIV-specific CD8+ T cells.

Current paradigms of CD8+ T cell-mediated protection in HIV infection center almost exclusively on studies ofx peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs. Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs. [ABSTRACT FROM AUTHOR]

The loss of HIV-specific CD8+ T cell cytolytic function is a primary factor underlying progressive HIV infection, but whether HIV-specific CD8+ T cells initially possess cytolytic effector capacity, and when and why this may be lost during infection, is unclear. Here, we assessed CD8+ T cell functional evolution from primary to chronic HIV infection. We observed a profound expansion of perforin+ CD8+ T cells immediately following HIV infection that quickly waned after acute viremia resolution. Selective expression of the effector-associated transcription factors T-bet and eomesodermin in cytokine-producing HIV-specific CD8+ T cells differentiated HIV-specific from bulk memory CD8+ T cell effector expansion. As infection progressed expression of perforin was maintained in HIV-specific CD8+ T cells with high levels of T-bet, but not necessarily in the population of T-betLo HIV-specific CD8+ T cells that expand as infection progresses. Together, these data demonstrate that while HIV-specific CD8+ T cells in acute HIV infection initially possess cytolytic potential, progressive transcriptional dysregulation leads to the reduced CD8+ T cell perforin expression characteristic of chronic HIV infection. [ABSTRACT FROM AUTHOR]