Fluctuations of functionally distinct CD8+ T-cell clonotypes demonstrate flexibility of the HIV-specific TCR repertoire

Abstract

T-cell receptor (TCR) diversity of virus-specific CD8+ T cells likely helps prevent escape mutations in chronic viral infections. To understand the dynamics of the virus-specific T cells in more detail, we followed the evolution of the TCR repertoire specific for a dominant HLA-B*08-restricted epitope in Nef (FLKEKGGL) in a cohort of subjects infected with HIV. Epitope-specific CD8+ T cells used structurally diverse TCR repertoires, with different TCRbeta variable regions and with high amino acid diversity within antigen recognition sites. In a longitudinal study, distinct Vbeta populations within the HIV-specific TCR repertoire expanded simultaneously with changes in plasma viremia, whereas other Vbeta populations remained stable or even decreased. Despite antigenic variation in some subjects, all subjects had the consensus sequence present during the study period. Functional analysis of distinct Vbeta populations revealed differences in HIV-specific IFN-gamma secretion ex vivo as well as differences in tetramer binding, indicating functional heterogeneity among these populations. This contrasts with findings in a subject on antiretroviral therapy with suppression of viremia to less than 50 copies/mL, where we observed long-term persistence of a single clonotype. Our findings illustrate the flexibility of a heterogeneous HIV-1-specific CD8+ TCR repertoire in subjects with partial control of viremia.

Figure 1.

HIV-specific CD8⁺ T-cell repertoire in HIV-infected HLA-B*08⁺ individuals. (A) HIV-specific CD8⁺ T-cell repertoire tested by IFN-γ ELISpot in 8 HLA-B*08⁺ HIV-infected individuals as described in “Materials and methods.” CTL magnitudes are expressed as spot-forming cells per million PBMCs (SFC/million PBMCs). (B) Pie charts display the hierarchy of IFN-γ⁺ CD8⁺ T-cell responses in the respective individual.

Figure 2.

Vβ repertoire of B8-FL8 tetramer–positive CD8⁺ T cells. Vβ usage of unselected peripheral CD8⁺ T cells (□) and B8-FL8 tetramer–positive CD8⁺ T cells (▪) in 8 HIV-infected individuals. Percentages indicate the frequency of Vβ⁺ cells within the respective T-cell population.

Figure 3.

Longitudinal analysis of viral load, B8-FL8 tetramer expression, and B8-FL8–specific Vβ repertoire. (A) Longitudinal analysis of HIV-1 RNA copies per milliliter of plasma, absolute numbers of CD4⁺ and CD8⁺ T cells, percentage of B8-FL8 tetramer–positive cells per peripheral CD8⁺ T cells, as well as the relative and absolute number of B8-FL8–specific Vβ populations in 3 untreated HIV-infected individuals (AC-14, AC-15, and AC-47). Days indicate time after diagnosis of acute HIV infection. (B) Longitudinal analysis of HIV-1 RNA copies per milliliter of plasma, percentage of B8-FL8 tetramer–positive cells per peripheral CD8⁺ T cells, and absolute number of B8-FL8–specific Vβ populations for an HIV-infected patient (AC-42) under antiretroviral treatment. (C) Longitudinal analysis as in panel A for an HIV-infected patient (AC-46) undergoing STI after a phase of optimal viral suppression under antiretroviral treatment.

Figure 4.

Tetramer expression patterns of distinct Vβ populations. Frequency and staining patterns of B8-FL8 tetramer–positive CD8⁺ T cells in subjects AC-46 (A), AC-15 (B), and AC-14 (C). Percentages indicate the frequency of tetramer-positive cells per CD8⁺ T cells (left) or the frequency of Vβ⁺ cells within the B8-FL8 tetramer–positive population (middle and top).

Figure 5.

Functional analysis of HIV tetramer–positive cells and their Vβ populations ex vivo. (A) IFN-γ secretion of Vβ2 and Vβ14⁺ CD8⁺ T cells in AC-46 ex vivo. Percentages indicate the frequency of IFN-γ⁺ cells per CD8⁺ T cells (left) or the frequency of Vβ⁺ cells within the IFN-γ⁺ population (right). (B) Kinetics of B8-FL8 tetramer–positive cells and IFN-γ⁺ cells after stimulation with B8-FL8 peptide in AC-46 during STI. Bars indicate relative frequency (%) of IFN-γ⁺ cells within the B8-FL8 tetramer⁺ population. (C) Costaining of B8-FL8 tetramer and intracellular IFN-γ secretion in Vβ2 and Vβ14⁺ CD8⁺ T-cell populations in AC-46 ex vivo after stimulation with B8-FL8 peptide or PMA/ionomycin. (D) Summary of the IFN-γ secretion of B8-FL8 tetramer⁺ Vβ2⁺ and Vβ14⁺ CD8⁺ T cells after stimulation with B8-FL8 peptide in 3 independent experiments. (E) Costaining of B8-FL8 tetramer and intracellular IFN-γ secretion in 4 Vβ⁺ populations in AC-15 ex vivo after stimulation with B8-FL8 peptide. (F) Bar graph indicates the frequency of IFN-γ⁺ cells per Vβ population.

Figure 6.

Sequencing of autologous virus. Bulk sequencing of autologous viral sequences derived from AC-14, AC-15, AC-46, and AC-47 covering the B8-FL8 epitope and its flanking regions derived from different time points over the course of the study period.