Immunology

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Analysis of viral inhibitory activity of cytotoxic T. Lymphocytes targeting identical epitopes restricted by different class 1 HLA alleles from the same HLA supertype.
(2015) Ogunshola, Funsho Japhet.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
Human leukocyte antigen (HLA) polymorphism and the genetic diversity of human immunodeficiency virus (HIV) are the major obstacles for designing an effective HIV Cytotoxic T Lymphocytes (CTLs) based vaccine. Interestingly, recent studies have demonstrated that multiple class I alleles can recognize common epitopes “supertopes” due to the homology of amino acids within the major binding pockets of the peptide binding cleft. The implications of this for vaccine design is that a vaccine containing a small number of highly promiscuous supertopes can confer protection against a wide range of HIV variants. This notion makes supertopes immunogen design an attractive option. However, it is not clear whether supertopes presented in the context of different class I HLA alleles would induce functional equivalent CTL responses. In this study, we investigated the inhibitory activity of CTLs targeting identical epitopes presented by class I HLA alleles from the same superfamily. The viral inhibitory activity was measured using a newly developed CEM-GFP reporter T-cell line (GXR-cell) as target cell. We first compared the inhibitory activity of CTLs from 8 subjects targeting TPQDLNTML (Gag p24 residue 180-188-TL9) epitope presented by HLA-B*81:01 or B*42:01 alleles. We then assessed the inhibitory activity of the 8 subjects’ CTLs when presented with in-vivo occurring mutant (Q182S)-TL9 epitope by HLA-B*81:01 or B*42:01 alleles. Furthermore, we compared the inhibitory activity of CTLs from 4 subjects targeting ISPRTLNAW (Gag p24 residue 147-155-IW9) epitope presented by HLA-B*57:03 or B*58:01 alleles. Comparative analysis of the inhibitory activity of the 8 subjects’ CTLs showed no statistical significant difference when TL9 epitope was presented by HLA-B*81:01 or B*42:01 alleles (1:1; p-value = 0.8785, paired t test), even at low target to effector ratio (1:8; p-value = 0.4418). No statistical significant difference was observed in the inhibitory activity of the 8 subjects’ CTLs when mutant (Q182S)-TL9 epitope was presented by HLA-B*81:01 or B*42:01 alleles (1:1; p-value = 0.8042), same result was observed at low target to effector ratio (1:8; p-value = 0.9396). Comparative analysis of the inhibitory activity of the 4 subjects’ CTLs targeting identical IW9 epitopes presented by HLA-B*57:03 or B*58:01 alleles showed a trend towards significance at target to effector ratio 1:1 (1:1; p-value = 0.0924), but at low target to effector ratio, no significance difference was observed (1:8; p-value = 0.1496). In conclusion, we have demonstrated that there is no observable significant difference in the inhibitory activity of CTLs targeting wildtype TL9 or mutant (Q182S)-TL9 epitopes presented in the context of HLA-B*81:01 or B*42:01 alleles. Thus, TL9 epitope could be immunogenic for individuals expressing HLA-B*81:01 or B*42:01 alleles. We have also shown that the inhibitory activity of CTLs targeting identical IW9 epitopes presented by HLAB* 57:03 or B*58:01 alleles is comparable. Indicating that IW9 epitope could be included in immunogen design for individuals expressing HLA-B*57:03 or B*58:01 alleles. These findings are relevant for HIV vaccine approach that seeks to identify immunogenic supertopes that can be cross-presented in a broadly cross-reactive T cell based vaccine design.
Characterizing the role of CD4+ T cell immunoregulatory networks in peripheral blood and lymphoid tissue during HIV-1 clade C infection.
(2018) Laher, Faatima.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
HIV eradication efforts have been unsuccessful due to virus persistence in cellular and tissue reservoirs. Recent evidence suggests that germinal centers (GCs) within lymph nodes (LN) contain a novel subset of regulatory T cells (TREGs), termed follicular regulatory T (TFR) cells. These cells control the magnitude and specificity of the GC response and like TREGs are essential for the maintenance of self-tolerance and immune homeostasis. However, the exact role of TFR cells in HIV infection and their contribution to viral control is not completely understood, possibly due to their low frequency, heterogeneity and more so, the difficulty in accessing human lymphoid tissue samples to fully study them. Thus, we set out to comprehensively investigate TFR cells in LN and peripheral blood (PB) samples, using a multifaceted approach including flow cytometry, MHC class II tetramers, immunofluorescence microscopy (IF), ELISA, digital droplet PCR and singlecell RNA sequencing (SeqWell), in HIV-1 clade C infection. Furthermore, we aimed to determine the effect of very early treatment on the frequency and function of this cell subset. Overall, our studies contributed various notable findings to the field. Firstly, we were able to develop MHC class II tetramers, specific in our HIV-1 clade C setting, as a more sensitive method of identifying very low cell frequency antigen-specific CD4+ T cells without relying on function. Tetramers eliminate the bias associated with in vitro stimulation required for functional assays and the limitation associated with only detecting subsets of cells capable of secreting a cytokine. Notably, we used class II tetramers to demonstrate that HIV-specific CD4+ T cell responses restricted to DRB1*11-Gag41 are associated with immune control of HIV-1 infection. We next focused on understanding the role of CD4+ regulatory cells during HIV-1 infection. Firstly, we showed that TFR cell frequencies were significantly higher in LN compared to PB samples. Secondly, TFR are a phenotypically and transcriptionally distinct subset compared to regulatory T cells (TREGs) and T Follicular Helper cells (TFH). Thirdly, we were able to detect HIV-specific TFR using our newly synthesized MHC class II tetramers, and showed higher frequencies observed in LNs during untreated HIV infection. Fourthly, as measured by both flow cytometry and IF, most of TFR localized outside of the GC, with very early ART initiators displaying larger proportions of TFR within the GC. Lastly, TFR cells exhibited a potential suppressive functional capacity as they produced IL-10, which is a canonical suppressive cytokine and they were also positively associated with gp41 IgG antibodies titers. Overall, the data presented in this thesis highlights the advantage of MHC class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. More so, the results give important insights into regulatory cells within lymph nodes; their biology, function and their role in the setting of very ART initiation.
Development and optimization of real-time PCR assays to detect anti-microbial immune factors and their response to type I and II interferons.
(2016) Kieswetter, Nathan Scott.; Ndung'u, Peter Thumbi.; Wong, Emily.
Abstract available in PDF file.
Mapping immunodominant patterns and HLA class II restriction characteristics of HIV-specific CD4+ T cell responses in acute and chronic HIV-1 subtype C infection.
(2014) Laher, Faatima.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
Increasing evidence suggests that virus-specific CD4+ T cells contribute to immune-mediated control of HIV-1 infection. However, precise details of CD4+ T cell contribution to immune protection against HIV have not been adequately defined and most of the existing data was predominantly generated in clade B HIV-1 infection. Understanding the contribution of CD4+ T cell responses in clade C infection is important for developing vaccines that would be efficacious in sub-Saharan Africa which carries the highest burden of the HIV epidemic in the world. Therefore this study focused on the role of CD4+ T helper cells in the immune response to clade C HIV-1 infection. We tested the hypothesis that HIV-1-specific CD4+ T cell responses and protective class II HLA alleles are important determinants of effective immunological control of HIV-1 infection. Firstly, CD8 depleted PBMCs were used in an IFN-γ ELISPOT assay to conduct a comprehensive analysis of virus-specific CD4+ T cell responses in acute and chronic HIV-1 clade C infection. Thereafter the host genetic effects of class II HLA-DRB1 alleles on HIV viremia were assessed using the HLA-DRB1 restriction assay, where HLA class II-restriction characteristics of detectable responses were defined. Lastly, functional differences of HIV-specific CD4+ T cells were further characterized using flow cytometric analysis. In our study, Gag and Pol regions of the HIV proteome were found to be the most frequently targeted in acute HIV-1 infection (69% of total responses), with CD4+ T cell targeting across the proteome remaining relatively stable over time. In chronic HIV-1 clade C infection, dominant HIV-1-specific CD4+ T cell responses were detectable against a limited number of epitopes. Epitopes in the Gag region were the most targeted by CD4+ T cells (30/40 peptides), with OLP 41 in the Gag p24 region being the most dominant epitope targeted (15% of responses). There were no significant differences observed between total or Gag-specific CD4+ T cell responses and contemporaneous viral load. Interestingly, responses rarely targeted the envelope region in clade C infection, in contrast to multiple epitopes targeted in this protein in previous clade B studies. Functional analysis demonstrated that IFN-γ, IL-2 and TNFα were the most secreted cytokines by HIV-specific CD4+ T cells in 18/25 individuals, with IFN-γ being the most dominant response in individual subjects. The HLA class II DRB1 restriction in clade C HIV infected individuals showed epitope promiscuity, consistent with previous studies in clade B infection. The HLA-DRB1*13:01 allele variant was associated with the highest frequency of responders (22%) in our cohort and restricted the highest number of HIV-specific peptides (9/15). Together, our data identify immunodominant regions of HIV-specific CD4+ T cell responses and their association with viral control during clade C infection. Furthermore, our findings will inform studies aimed at elucidating the underlying mechanism by which CD4+ T cells modulate effective CD8+ T cell and B cell responses. Additionally, these data suggest that epitope promiscuity among class II HLA molecules should be taken into account for vaccines designed to induce CD4+ T cell responses. This information will be critical to vaccine efforts designed to induce these responses, as well as potential therapeutic manipulation of immunity in persons with acute and chronic HIV-1 infection.
Molecular mechanisms of CD8+ T cell mediated control of HIV-1 infection in peripheral blood and lymphoid tissues.
(2019) Ogunshola, Funsho Japhet.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
Naturally induced CD8+ T cells do not clear human immunodeficiency virus (HIV) infection, partly because the virus rapidly escapes CD8+ T cell responses and the effector cells are excluded from HIV reservoirs sites. However, optimizing CD8+ T cell responses could potentially be leveraged in HIV vaccine or cure efforts if epitope escape and barriers to effector CD8+ T cells infiltrating the sites of HIV reservoirs are overcome. In our first study, we described a potential mechanism of HIV-1 control by CD8+ T cells targeting different variants in individuals infected with HIV-1. Our second study focused on describing the molecular regulation of CXCR5 expression in human CD8+ T cells. Study 1 HLA-B*81 is associated with control of HIV-1 subtype C infection, while the closely related allele B*42 is not. Interestingly, both alleles present the immunodominant Gag TL9 epitope, and the magnitude of this response correlates negatively with viral load. To examine the role of T cell receptor (TCR) in this process, we characterized the sequence and function of TL9-specific CD8+ TCR in B*81 and B*42 individuals. TL9-specific CD8+ T cells were identified and isolated using B*81 and/or B*42 TL9 tetramers. TCR beta genes were amplified from single sorted cells and sequenced. Paired alpha genes were identified for selected clones. TCR function was tested using a reporter cell assay where TCR+ Jurkat cells were co-cultured with peptide-pulsed or HIV-1 infected B*81 or B*42 target cells, and signalling quantified by luminescence. TCR recognition was assessed against all single amino acid TL9 variants and results were compared to HIV-1 subtype C sequences. A population of dual-reactive T cells was detected by both B*81- and B*42-TL9 tetramers in 7/9 (78%) B*81 and 4/11 (36%) B*42 individuals; and this population was associated with lower viremia. Mono- and dual-reactive TCR beta sequences were collected from six individuals. In B*81 individuals, all TCRs were highly restricted to TRBV12-3. In B*42 individuals, mono-reactive TCRs encoded a variety of V beta genes, while dual-reactive TCRs were restricted to TRBV12-3 and enriched for public clones. Functional analyses indicated that B*81 TCRs (1 mono, 2 dual) and a dualreactive public B*42 TCR displayed similar TL9 cross-reactivity profiles and enhanced capacity to recognize HIV-1 escape mutations compared to mono-reactive B*42 TCRs. This work highlights the impact of TCR promiscuity on T cell-mediated control of HIV-1. Study 2 HIV-1 infection is difficult to cure even with effective antiretroviral therapy (ART) because of persistent viral replication in immune privileged sites such as the B cell follicles of secondary lymphoid tissues. CD8+ T cells are generally excluded from B cell follicles, partially due to a lack of expression of the follicular homing receptor CXCR5. Recent murine studies have identified CXCR5+ CD8+ T cells, referred to as follicular CD8+ T cells (fCD8s), that localize in B cell follicles. However, the mechanisms governing expression of CXCR5 on human CD8+ T cells are not known. We investigated the epigenetic and transcriptional mechanisms involved in the regulation of CXCR5 expression in human CD8+ T cells. We FACS-sorted CXCR5+CD8+ (fCD8s), CXCR5-CD8+ (non-fCD8s), naïve CD8+ T cells and germinal center T follicular helper cells (GCTfh) from the lymph node of HIV- 1 infected individuals and performed RNA-sequencing (RNA-Seq), DNA methylation assays and the assay for transposase-accessible chromatin using sequencing (ATACSeq). RNA-Seq was used to quantify the expressed genes in FACS-sorted subsets and to determine transcriptional modules governing CXCR5 expression in CD8+ T cells. ATAC-Seq was used to quantify accessible genes, identify the transcriptional factors footprinting and determine epigenetic modules governing CXCR5 expression. DNA methylation, a major epigenetic gene silencing mechanism, was used to profile methylation pattern of the CXCR5 gene region in the sorted subsets. We observed hypermethylation of DNA around the transcriptional start site (TSS) of the CXCR5 gene in non-fCD8s but not in fCD8s. ATAC-Seq analysis revealed a closed chromatin conformation at the TSS in non-fCD8s, but not in fCD8s. Our gene expression data revealed significant differences in the CXCR5 associated factors between GCTfh and fCD8s. Computational analysis further revealed the presence ofa nucleosome at the TSS of fCD8s, which could be a plausible explanation for lower expression of CXCR5 in fCD8s as compared to GCTfh. Together, we identified epigenetic regulations involved in CXCR5 expression in human CD8+ T cells and propose that DNA methylation, chromatin structure and nucleosome positioning cooperatively regulate the expression of CXCR5 in CD8+ T cells. Our data open up the possibility of using epigenetic manipulation as a novel strategy for redirecting CD8+ T cells to B cell follicles where they are needed to eradicate HIV-1 infected cells.
Patterns and features of HIV-1 specific CD8+ T-cell responses during acute HIV-1 infection and their association with viral control.
(2015) Radebe, Mopo.; Ndung'u, Peter Thumbi.
Evidence suggests that CD8+ T-‐cells play a major role in the control of HIV-‐1 viremia and apply significant immune pressure on HIV-‐1 replication. However, the presence of virus-‐specific CD8+ T-‐cells in individuals with varying levels of viral control suggests that CD8+ T-‐cells may differ in their antiviral function or efficacy. The mechanisms underlying differences in the control of viremia, particularly the reasons why particular individuals experience more effective acute viremia resolution, which is a good correlate of the subsequent rate of disease progression, are still not well understood. In order to uncover some of the features of CD8+ T-‐cell subsets responsible for the control of HIV replication, particularly during the critical early infection phase, we investigated the patterns and features of HIV-‐1-‐specific CD8+ T-‐ cell responses during acute and primary HIV-‐1 infection and their association with viral control. We also sought to determine the impact of acute phase immune activation on the acute HIV-‐1-‐specific CD8+ T-‐cell response and on disease progression. We hypothesized that protein-‐specific and epitope-‐specific immunodominance patterns during the first 12 weeks of HIV-‐1 infection are associated with subsequent disease progression. Our data show the presence of HIV-‐1 specific CD8+ T-‐cells with limited breadth during acute HIV-‐1 infection and also demonstrate that the magnitude and breadth of interferon gamma (IFN-‐γ) ELISPOT assay responses measured within 12 weeks post-‐infection are unrelated to the course of disease in the first year of infection. During the first weeks of infection Nef protein was most frequently recognized by T-‐ xv cells and was the target for the earliest response. Although initially subdominant, there was a broadening of the Gag-‐specific T-‐cell immune response such that these responses became immunodominant by one year post infection. The broadening and preservation of early Gag–specific T-‐cell responses during the follow up period was associated with better control of viremia and lower viral load set point. Although many of the acute/early HIV-‐1-‐specific IFN-‐γ enzyme linked immunospot assay (ELISPOT) CD8+ T-‐cell responses targeting Gag and Pol persisted, the majority of acute and early T-‐cell responses targeting Env, Nef and other regulatory proteins waxed and waned over time and could not be detected at the last time point evaluated. Some of the early T-‐cell responses which where no longer detectable when using overnight ELISPOT assay were detectable when PBMCs were stimulated with corresponding peptides and cultured for 10 days before measuring IFN-‐γ secretion via the ELISPOT assay. The presence of these cultured ELISPOT central memory type T-‐cell responses targeting epitopes in Pol, Env, Nef, Regulatory and Accessory proteins were not significantly associated with viral set point. However, cultured ELISPOT Gag-‐specific responses correlated with low plasma viremia, thus further providing evidence for the favourable role of Gag-‐specific T-‐cell responses in the control of viral replication. We also show that three cytokines IL-‐10, IP-‐10 and IL-‐ 12 were associated with changes in viral load set point and/or CD4+ T-‐cell dynamics during the first year of HIV-‐1 infection. Interestingly, the activation of the PD-‐1 inhibitory pathway in acute HIV-‐1 infection was associated with a slower disease progression.
Protective HLA class I alleles : investigation of viral control and lack of control in chronic HIV-1 subtype C infection.
(2015) Koofhethile, Catherine Kegakilwe.; Ndung'u, Peter Thumbi.; Thobakgale, Christina Fanesa.
Abstract available in PDF file.
Sex differences in the Kinetics of immune reconstitution under antiretroviral therapy.
(2017) Mazibuko, Noluthando Y.; Thobakgale, Christina Fanesa.; Ndung'u, Peter Thumbi.
The human immunodeficiency virus type 1 (HIV-1) remains a global health threat and is increasingly becoming a female epidemic due to gender inequalities. The introduction of antiretroviral therapy (ART) has greatly increased the life span while reducing AIDS-related deaths in HIV-1-infected people. However, some patients experience adverse effects during ART due to an acute inflammatory response termed immune reconstitution inflammatory syndrome (IRIS), which is a paradoxical clinical worsening upon initiation of ART that is thought to be due to a hyperactive or uncontrolled immune restoration. Studies have shown that females infected with HIV-1 elicit a stronger immune response and faster disease progression compared to men with the same viral load. Given the above mentioned higher baseline levels of immune activation in HIV-1 infected females, we hypothesized that immune restoration during ART will have significant sex-based differential outcomes. The aim of this project was to understand the impact of immune reconstitution in HIV-1-infected men and women during ART by investigating antigen presenting cells (monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic (pDCs) cells) phenotype and function. In total, we investigated cryopreserved samples from eleven HIV-1-infected males and thirteen HIV-1-infected females from which peripheral blood mononuclear cells (PBMCs) and plasma samples were longitudinally collected at defined time points, including before the initiation of ART (TP01), after 1-4 months (TP02) and after 5-8 months of treatment initiation. Changes in toll-like receptor (TLR) responsiveness were analyzed following stimulation with the following toll-like receptor (TLR) ligands: TLR4 ligand Lipopolysaccharide, TLR7/8 ligand CL097, and the TLR9 ligand ODN2216/ CpG. Multiparameter flow cytometry was used to analyze the cytokine production upon TLR stimulation as well as the level of immune activation by analyzing phenotypic characteristics of antigen-presenting cells including monocytes subsets. In addition, multiplex analysis was used to determine levels of plasma pro-inflammatory cytokines (IFN-ɣ, IFN-α, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, TNF-α, IL-12p70, GM-CSF, MIP-1β and IP-10), in males and females at all time-points. Our results show that the expression of immune activation markers (measured by HLA-DR+ and CD38+ on T cells) did not significantly differ between males and females, although females showed elevated levels of both activated CD8+ and CD4+ T cells at baseline, the activation levels decreased upon ART initiation. Furthermore, our results demonstrated an increase in the proportion of classical monocytes (CD14++CD16-) in females compared to males at baseline (p=0.03). We did not observe any significant differences in the percentage of intermediate (CD14++CD16+) and non-classical monocytes (CD14+CD16++) between males and females at any time point analyzed. Similarly, no sex differences were evident after TLR ligand stimulation on monocytes in terms of cytokines measured (IFN-α, TNF-α, MIP-1β and IL-12). Interestingly, upon TLR9 stimulation, a significantly higher percentage of pDCs from females produced IFN-α (p=0.001, TP03), MIP-1β (p=0.001, at TP02) and TNF-α (p˂0.01, p˂0.001, TP02 and TP03 respectively) during ART compared to males. In addition, females had increased IFN-α (p=0.01) and TNF-α (p=0.004) production on pDCs during ART compared to baseline following TLR9 stimulation. Taken together, our data suggest sex-specific differences in the level of immune reconstitution during ART, as females show signs of elevated immune response and inflammation compared to males Therefore, these findings may provide a basis for future studies in larger cohorts aimed at adapting ART therapy based on sex differences in disease progression rates in men and women.
T follicular helper cell dynamics during acute HIV-1 subtype C infection and relevance for T-helper cell-dependent responses.
(2018) Baiyegunhi, Omolara Olujimi.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
Abstract available in pdf.

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