To estimate the number of expansions attributable to HIV-1 infection in at-risk men, we compared the CD8+ T-cell expansion ratios of the AR and SP groups. The 95% CI of the mean CD8+ expansion ratio difference between the AR and SP groups was 0.06 ± 0.03. We interpret this finding as a crude approximation of the TCRβ CDR3 expansion ratio attributable to HIV in the CD8+ T-cell repertoire of at-risk men in the untreated chronic stage of HIV-1 infection. These numbers suggest that approximately 21% of the expanded CDR3 lengths (or 9 expansions per 150 measured CDR3 lengths) are clonal CD8+ responses to HIV-1 in the SP group.
The risks associated with unprotected sex greatly decreased during the first few years of the MACS . Therefore, we examined the CDR3 length distributions of individuals in the AR group longitudinally (mean follow-up time = 7.5 years) to assess the effect of decreased risk on the T-cell repertoire. Despite the reported decrease, CD4+ expansion ratios increased in this group (95% CIt+7.5 = 0.17 ± 0.06), whereas the group's CD8+ expansion ratios remained unchanged (95% CIt+7.5 = 0.25 ± 0.06). Sensitive T-cell repertoire stability assessments of six AR individuals were conducted by quantifying the number of CDR3 lengths that remained either unexpanded or expanded over periods of less than 1 year and greater than 7 years. Remarkable conservation of CDR3 length distributions was apparent within these individuals over time. An example of the persistency of TCR clonality is shown in Figure 1. A statistical summary of the extent to which TCR repertoires are stable over time is shown in (Figure 2c and d. All of the individuals sustained greater than 50% of their TCR expansions for more than 7 years. This analysis suggests that clonal expansions are maintained for prolonged periods.
In this study, we report five closely related observations that support our hypothesis that risks for HIV-1 infection are associated with risks for a broader spectrum of immunogenic challenges and that these challenges are reflected in the T-cell repertoires of exposed individuals: (1) altered T-cell subset distributions in at-risk men; (2) increased prevalence of viral infections in at-risk men; (3) increased percentages of select TCRVβ families in at-risk men; (4) increased clonal expansions within TCRVβ families of at-risk men; and (5) the longitudinal maintenance of clonal expansions within the T-cell repertoire.
Inspection of T-cell subset distributions revealed that the AR and SP groups had significantly elevated percentages and absolute counts of CD8+ T cells and corresponding decreased CD4 : CD8 T-cell ratios in comparison to the LR group. Our observation of gross T-cell distribution differences between LR and AR groups is consistent with previous reports  and probably indicates the increased presence of ongoing, or past, antigenic challenges, including hepatitis B infection, resulting in increased CD8+ T-cell proliferation in the AR and SP groups .
The higher prevalence of many microbial pathogens in men at increased risk for HIV infection is well documented and the self-reported sexual risk behaviors of homosexual men in the MACS indicate extensive antigenic and allogenic exposure associated with frequent sexual exposures prior to 1986 [25,37,38]. Based on the reported increased prevalences of HBV, HCV, CMV, and HSV infections among individuals with high-risk sexual exposures, we screened the individuals in this study for serostatus to these pathogens [39–46]. Evaluation for common viral infections exposed clear contrasts in the prevalence of hepatitis B infection between the LR and both the AR and SP groups. This observation clearly distinguishes the LR group from the AR and SP groups with respect to their histories of immunogenic challenges [13,47–49].
We observed that individuals in the AR and SP groups had elevated frequencies of select TCRVβ families within the CD4+ and CD8+ T-cell compartments. These flow cytometric analyses results probably reflect the increased presence of antigen-specific cellular immune responses in the AR and SP groups.
Since inequalities exist in the number of immunogenic challenges experienced by individuals with differing exposure histories, we cross-sectionally examined the T-cell repertoires of the three groups. We observed that the two groups with a history of frequent antigenic exposure (AR and SP) both exhibited greater numbers of clonal expansions in both CD4+ and CD8+ T-cell compartments than the less exposed LR group. Increased numbers of expansions in AR men are most likely due to the influence of viral pathogens and alloantigens associated with high-risk sexual exposures and perhaps antigenic exposure to HIV or even transient HIV infection . The difference in the numbers of CD8+ expansions between AR and SP groups is probably attributable to the continual replication of HIV virions and therefore ongoing T-cell responses in the SP group. This reverse transcriptase (RT)-PCR based analysis provides a sensitive examination of the clear differences between the LR, AR, and SP groups with respect to the magnitude of clonality within their T-cell repertoires.
We were unable to detect any significant differences in the numbers of clonal expansions within the AR group following stratification based on the reported frequency of anal receptive intercourse. This finding may indicate that the histories of immunogenic exposure prior to enrollment in the MACS may have been sufficiently similar among these men, as suggested by their hepatitis B serology results. Furthermore, the sample sizes may not have afforded sufficient power to detect significant differences between the AR subgroups.
Our findings suggest that only a fraction of the clonally expanded T-cell populations in HIV-1-infected homosexual men are attributable to chronic HIV-1 infection, as expansions present in the TCR repertoire may reflect prior, or even concurrent infections. Based on comparisons to at-risk seronegative men, we estimate that during chronic HIV infection, at-risk men exhibit an average of a 27% increase in the number of clonal expansions in the CD8+ subset. These expansions were broadly distributed and differed between individuals, consistent with the known variability in T-cell responses, and the variability of TCRVβ usage even for clones of the same specificity . Further studies are required to more clearly distinguish the magnitude of the HIV-1-specific T-cell response from the magnitude of T-cell responses merely associated with HIV-1 infection.
The CD4+ compartment revealed many fewer expansions than were found in the CD8+ subset. This difference is possibly due to inequities between the distribution of memory and naive cells within each compartment . We have recently observed that expansions in the CD4+ compartment are concentrated in the CD45RA−/CD62L− subset and are often obscured by the larger presence of naive cells in the CD4 subset (Killian, Hultin, Jamieson, unpublished). As the ratio of memory to naive CD4+ cells increases with HIV disease progression, expansions may become more evident. Still, in comparison to CD8+ cells, clonal proliferation of CD4+ T cells during chronic HIV infection appears to be limited, consistent with reports that HIV-1 specific CD4 T-cell responses are preferentially deleted .
With respect to the stability of clonality, we observed that clonality within the T-cell repertoire of men in the AR group remained increased for a period of greater than 7 years, despite a trend of decreased new antigenic exposure during that time. These findings suggest that antigenic exposure is positively associated with elevated clonality within the T-cell repertoire and that this clonality is maintained for extended time periods reflecting a ‘history’ of past and/or persisting antigenic stimulation. This observation is consistent with reports demonstrating that memory T cells do not require continuous exposure to their cognate antigens for long-term survival in the periphery [54–58].
In conclusion, cross-sectional evaluations of HIV-1 induced immune alterations require the use of carefully selected control groups. Our findings suggest that comparisons to low-risk individuals may result in the overestimation of TCR repertoire alterations attributable to HIV-1 infection in previously at-risk individuals, since frequent exposure to viral pathogens and immunogens appears to also be reflected in the TCR repertoires of at-risk men. Indeed, the evidence presented here suggests that the T-cell repertoire reflects a long-lived history of antigenic exposure. Thus, the immunogenic exposure histories of any individual will complicate the identification of HIV-specific responses. Importantly, these residual immuno-modulations should be considered when investigating cellular immune responses to disease, treatment, and vaccines. Although CDR3 lengths will be a useful tool for enumerating the magnitude and diversity of the T-cell response, it must be used longitudinally, or combined with other methods to characterize HIV-1 specific T-cell responses.
The authors thank Rhonda Sturgill, Marisela Killian, and Zarina Kiziloglu for their dedicated technical support.
Sponsorship: This work was supported in part by Grants UO1-AI-37613, UO1-AI-35040, and T32-AI-07481 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
This study utilized patients and specimens from the Multicenter AIDS Cohort Study (MACS), which includes the following sites and investigators:
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