Masafumi Takiguchi

Escape from highly effective public CD8+ T-cell clonotypes by HIV

Mapping the precise determinants of T-cell efficacy against viruses in humans is a public health priority with crucial implications for vaccine design. To inform this effort, we performed a comprehensive analysis of the effective CD8(+) T-cell clonotypes that constitute responses specific for the HIV p24 Gag-derived KK10 epitope (KRWIILGLNK; residues 263-272) restricted by HLA-B*2705, which are known to confer superior control of viral replication in HIV-infected individuals. Particular KK10-specific CD8(+) T-cell clonotypes, characterized by TRBV4-3/TRBJ1-3 gene rearrangements, were found to be preferentially selected in vivo and shared between individuals. These public clonotypes exhibit high levels of TCR avidity and Ag sensitivity, which impart functional advantages and enable effective suppression of HIV replication. The early L(268)M mutation at position 6 of the KK10 epitope enables the virus to avoid recognition by these highly effective CD8(+) T-cell clonotypes. However, alternative clonotypes with variant reactivity provide flexibility within the overall KK10-specific response. These findings provide refined mechanistic insights into the workings of an effective CD8(+) T-cell response against HIV.

Multilayered defense in HLA-B51-associated HIV viral control

Polymorphism in the HLA region of a chromosome is the major source of host genetic variability in HIV-1 outcome, but there is limited understanding of the mechanisms underlying the beneficial effect of protective class I alleles such as HLA-B57, -B27, and -B51. Taking advantage of a unique cohort infected with clade B’ HIV-1 through contaminated blood, in which many variables such as the length of infection, the infecting viral strain, and host genetic background are controlled, we performed a comprehensive study to understand HLA-B51–associated HIV-1 control. We focused on the T cell responses against three dominant HLA-B51–restricted epitopes: Gag327-345(NI9) NANPDCKTI, Pol743-751(LI9) LPPVVAKEI, and Pol283-289(TI8) TAFTIPSI. Mutations in all three dominant epitopes were significantly associated with HLA-B51 in the cohort. A clear hierarchy in selection of epitope mutations was observed through epitope sequencing. L743I in position 1 of epitope LI9 was seen in most B51+ individuals, followed by V289X in position 8 of the TI8, and then, A328S, in position 2 of the NI9 epitope, was also seen in some B51+ individuals. Good control of viral load and higher CD4+ counts were significantly associated with at least one detectable T cell response to unmutated epitopes, whereas lower CD4+ counts and higher viral loads were observed in patients who had developed escape mutations in all three epitopes or who lacked T cell responses specific to these epitope(s). We propose that patients with HLA-B51 benefit from having multiple layers of effective defense against the development of immune escape mutations.

Effects of naturally-arising HIV Nef mutations on cytotoxic T lymphocyte recognition and Nef's functionality in primary macrophages.

BackgroundAlthough HIV can infect several cellular subsets, such as CD4+ T lymphocytes and macrophages, it remains unclear whether an HIV infection in macrophages supports cytotoxic T lymphocyte (CTL) escape. Here, we tested two naturally-arising mutations located in the well-conserved polyproline region of Nef for their effects on CTL recognition, Nefs functionality, and viral replication capacity in macrophages. These mutations were selected because they are known to cause CTL escape in the context of T lymphocytes.FindingsMonocyte-derived macrophages (MDMs) infected with the wild-type virus, but not with variant viruses, were efficiently killed by CTL clones targeting Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). The CTL-escape mutation, Arg75Thr, or Arg75Thr/Tyr85Phe double mutation, reduced the HLA class I down-regulation activity and, interestingly, increased the susceptibility of virus-infected MDMs to recognition by CTLs targeting a different epitope. The same mutations reduced the CCR5, but not CD4, down-regulation activity. Moreover, the Nef variants were impaired for Hck activation and enhancement of viral replication in MDMs.ConclusionsThese results suggest that HIV-infected MDMs are killed by CTLs targeting Nef epitopes, contributing to selection and adaptation of CTL-escape viral variants.

HLA Class I-Mediated Control of HIV-1 in the Japanese Population, in Which the Protective HLA-B*57 and HLA-B*27 Alleles Are Absent

ABSTRACT We investigated the effect of HLA class I alleles on clinical parameters for HIV-1 disease progression in the Japanese population, where two strongly protective alleles, HLA-B*57 and HLA-B*27, are virtually nonexistent. HLA-B alleles showed a dominant role, primarily through HLA-B*67:01 and the HLA-B*52:01-C*12:02 haplotype. Neither a rare-allele nor a heterozygote advantage was found, suggesting that the effect of HLA alleles in the Japanese population is either different from those observed in Africans and Caucasians or undetectable due to limited power.

Impact of Vaccination on Cytotoxic T Lymphocyte Immunodominance and Cooperation against Simian Immunodeficiency Virus Replication in Rhesus Macaques

ABSTRACT Cytotoxic T lymphocyte (CTL) responses play a central role in viral suppression in human immunodeficiency virus (HIV) infections. Prophylactic vaccination resulting in effective CTL responses after viral exposure would contribute to HIV control. It is important to know how CTL memory induction by vaccination affects postexposure CTL responses. We previously showed vaccine-based control of a simian immunodeficiency virus (SIV) challenge in a group of Burmese rhesus macaques sharing a major histocompatibility complex class I haplotype. Gag206-216 and Gag241-249 epitope-specific CTL responses were responsible for this control. In the present study, we show the impact of individual epitope-specific CTL induction by prophylactic vaccination on postexposure CTL responses. In the acute phase after SIV challenge, dominant Gag206-216-specific CTL responses with delayed, naive-derived Gag241-249-specific CTL induction were observed in Gag206-216 epitope-vaccinated animals with prophylactic induction of single Gag206-216 epitope-specific CTL memory, and vice versa in Gag241-249 epitope-vaccinated animals with single Gag241-249 epitope-specific CTL induction. Animals with Gag206-216-specific CTL induction by vaccination selected for a Gag206-216-specific CTL escape mutation by week 5 and showed significantly less decline of plasma viral loads from week 3 to week 5 than in Gag241-249 epitope-vaccinated animals without escape mutations. Our results present evidence indicating significant influence of prophylactic vaccination on postexposure CTL immunodominance and cooperation of vaccine antigen-specific and non-vaccine antigen-specific CTL responses, which affects virus control. These findings provide great insights into antigen design for CTL-inducing AIDS vaccines.

Selection of escape mutant by HLA-C-restricted HIV-1 Pol-specific cytotoxic T lymphocytes carrying strong ability to suppress HIV-1 replication

HIV‐1 mutants escaping from HLA‐A‐ or HLA‐B‐restricted CTL have been well studied, but those from HLA‐C‐restricted CTL have not. Therefore we investigated the ability of HLA‐C‐restricted CTL to select HIV‐1 escape mutants. In the present study, we identified two novel HLA‐Cw*1202‐restricted Pol‐specific CTL epitopes (Pol328‐9 and Pol463‐10). CTL specific for these epitopes were detected in 25–40% of chronically HIV‐1‐infected HLA‐Cw*1202+ individuals and had strong abilities to kill HIV‐1‐infected cells and to suppress HIV‐1 replication in vitro, suggesting that these CTL may have the ability to effectively control HIV‐1 in some HLA‐Cw*1202+ individuals. Sequence analysis of these epitopes showed that a V‐to‐A substitution at the 9th position (V9A) of Pol 463‐10 was significantly associated with the HLA‐Cw*1202 allele and that the V9A mutant was slowly selected in the HLA‐Cw*1202+ individuals. Pol 463‐10‐specific CTL failed both to kill the V9A virus‐infected cells and to suppress replication of the V9A mutant. These results indicate that the V9A mutation was selected as an escape mutant by the Pol463‐10‐specific CTL. The present study strongly suggests that some HLA‐C‐restricted CTL have a strong ability to suppress HIV‐1 replication so that they can select HIV escape mutants as in the case of HLA‐A‐restricted or HLA‐B‐restricted CTL.

Unbiased Analysis of TCRα/β Chains at the Single-Cell Level in Human CD8+ T-Cell Subsets

T-cell receptor (TCR) α/β chains are expressed on the surface of CD8+ T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5′-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8+ subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8+ subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8+ T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains.

Effective Elicitation of Human Effector CD8+ T Cells in HLA-B*51:01 Transgenic Humanized Mice after Infection with HIV-1

Humanized mice are expected to be useful as small animal models for in vivo studies on the pathogenesis of infectious diseases. However, it is well known that human CD8+ T cells cannot differentiate into effector cells in immunodeficient mice transplanted with only human CD34+ hematopoietic stem cells (HSCs), because human T cells are not educated by HLA in the mouse thymus. We here established HLA-B*51:01 transgenic humanized mice by transplanting human CD34+ HSCs into HLA-B*51:01 transgenic NOD/SCID/Jak3−/− mice (hNOK/B51Tg mice) and investigated whether human effector CD8+ T cells would be elicited in the mice or in those infected with HIV-1 NL4-3. There were no differences in the frequency of late effector memory and effector subsets (CD27lowCD28−CD45RA+/−CCR7− and CD27−CD28−CD45RA+/−CCR7−, respectively) among human CD8+ T cells and in that of human CD8+ T cells expressing CX3CR1 and/or CXCR1 between hNOK/B51Tg and hNOK mice. In contrast, the frequency of late effector memory and effector CD8+ T cell subsets and of those expressing CX3CR1 and/or CXCR1 was significantly higher in HIV-1-infected hNOK/B51Tg mice than in uninfected ones, whereas there was no difference in that of these subsets between HIV-1-infected and uninfected hNOK mice. These results suggest that hNOK/B51Tg mice had CD8+ T cells that were capable of differentiating into effector T cells after viral antigen stimulation and had a greater ability to elicit effector CD8+ T cells than hNOK ones.

Functional heterogeneity of human effector CD8 + T cells

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

Effective recognition of HIV-1-infected cells by HIV-1 integrase-specific HLA-B∗4002-restricted T cells.

HLA-B∗4002 is one of the common HLA-B alleles in the world. All 7 reported HLA-B∗4002-restricted HIV epitopes are derived from Gag, Nef, and Vpr. In the present study we sought to identify novel HLA-B∗4002-restricted HIV epitopes by using overlapping 11-mer peptides of HIV-1 Nef, Gag, and Pol, and found that 6 of these 11-mer Pol peptides included HLA-B∗4002-restricted epitopes. Analysis using truncated peptides of these 6 peptides defined 4 optimal Pol (integrase) epitopes. All epitopes previously reported had Glu at position 2 (P2), suggesting that Glu at P2 is the anchor residue for HLA-B∗4002; whereas only 2 of the integrase epitopes that we here identified had Glu at P2. CTL clones specific for the 2 epitopes effectively recognized HIV-1-infected cells whereas those for other 2 epitopes only weakly recognized them. The antigen sensitivity of the former clones for the epitope peptide was much higher than that of the latter clones, suggesting 2 possibilities: 1) the former T cells have high-affinity TCRs and/or 2) the epitope peptides recognized by the former T cells are highly presented by HLA-B∗4002 in HIV-1-infected cells. These integrase-specific T cells with high antigen sensitivity may contribute to the suppression of HIV-1 replication in HIV-1-infected HLA-B∗4002+ individuals.