Marcus Buggert

T-bet and Eomes Are Differentially Linked to the Exhausted Phenotype of CD8+ T Cells in HIV Infection.

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-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi 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.

Arming of MAIT Cell Cytolytic Antimicrobial Activity Is Induced by IL-7 and Defective in HIV-1 Infection

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.

Multiparametric Bioinformatics Distinguish the CD4/CD8 Ratio as a Suitable Laboratory Predictor of Combined T Cell Pathogenesis in HIV Infection

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.

Combined immunodeficiency and Epstein-Barr virus?induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity.

Interdisciplinary Analysis of HIV-Specific CD8+ T Cell Responses against Variant Epitopes Reveals Restricted TCR Promiscuity

HIV-1–specific CTL responses play a key role in limiting viral replication. CTL responses are sensitive to viral escape mutations, which influence recognition of the virus. Although CTLs have been shown to recognize epitope variants, the extent of this cross-reactivity has not been quantitatively investigated in a genetically diverse cohort of HIV-1–infected patients. Using a novel bioinformatic binding prediction method, we aimed to explain the pattern of epitope-specific CTL responses based on the patients’ HLA genotype and autologous virus sequence quantitatively. Sequences covering predicted and tested HLA class I-restricted epitopes (peptides) within the HIV-Gag, Pol, and Nef regions were obtained from 26 study subjects resulting in 1492 patient-specific peptide pairs. Epitopes that were recognized in ELISPOT assays were found to be significantly more similar to the autologous virus than those that did not elicit a response. A single substitution in the presented epitope decreased the chance of a CTL response by 40%. The impact of sequence similarity on cross-recognition was confirmed by testing immune responses against multiple variants of six selected epitopes. Substitutions at central positions in the epitope were particularly likely to result in abrogation of recognition. In summary, the presented data demonstrate a highly restricted promiscuity of HIV-1–specific CTL in the recognition of variant epitopes. In addition, our results illustrate that bioinformatic prediction methods are useful to study the complex pattern of CTL responses exhibited by an HIV-1–infected patient cohort and for identification of optimal targets for novel therapeutic or vaccine approaches.

Characterization of HIV-Specific CD4+ T Cell Responses against Peptides Selected with Broad Population and Pathogen Coverage

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.

Combination of Immune and Viral Factors Distinguishes Low-Risk versus High-Risk HIV-1 Disease Progression in HLA-B*5701 Subjects

ABSTRACT 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.

T-bet+ B cells are induced by human viral infections and dominate the HIV gp140 response

Humoral immunity is critical for viral control, but the identity and mechanisms regulating human antiviral B cells are unclear. Here, we characterized human B cells expressing T-bet and analyzed their dynamics during viral infections. T-bet+ B cells demonstrated an activated phenotype, a distinct transcriptional profile, and were enriched for expression of the antiviral immunoglobulin isotypes IgG1 and IgG3. T-bet+ B cells expanded following yellow fever virus and vaccinia virus vaccinations and also during early acute HIV infection. Viremic HIV-infected individuals maintained a large T-bet+ B cell population during chronic infection that was associated with increased serum and cell-associated IgG1 and IgG3 expression. The HIV gp140-specific B cell response was dominated by T-bet-expressing memory B cells, and we observed a concomitant biasing of gp140-specific serum immunoglobulin to the IgG1 isotype. These findings suggest that T-bet induction promotes antiviral immunoglobulin isotype switching and development of a distinct T-bet+ B cell subset that is maintained by viremia and coordinates the HIV Env-specific humoral response.

Perturbed CD8(+) T cell TIGIT/CD226/PVR axis despite early initiation of antiretroviral treatment in HIV infected individuals.

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.

Temporal Dynamics of CD8+ T Cell Effector Responses during Primary HIV 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.