T-cell responses to HIV-1 infection have been associated with resistance to infection, control of viremia, and the clinical course of disease progression [1–4]. However, the magnitude (e.g. the number of clonal expansions) and diversity (e.g., the number of epitopes recognized) of HIV-specific clones in the T-cell repertoire and their associations with clinical outcomes remain uncertain. Investigation of the immune response to HIV-1 infection has been hindered by the lack of methodologies to measure the HIV-1-specific T-cell response. For example, the use of MHC-peptide tetrameric complexes to characterize HIV-specific T cells has provided a very incomplete description of the T-cell response, whereas other methods that provide a broader assessment of the T-cell repertoire, such as T-cell receptor spectratyping, are not HIV-1 specific [5,6]. Comprehensive assessment of the HIV-specific T-cell response may provide prognostic value and contribute to the development of more effective therapeutic and vaccine strategies by identifying immune assets and defects.
Acquired immunity, a fundamental component of the cell-mediated immune response, relies on the clonal expansion of antigen-specific T cells . As a result, many mammalian viral infections induce robust CD8+ and more limited CD4+ T-cell proliferative responses [8–13]. Antigen-specific T-cell responses are mediated through T-cell receptor (TCR) recognition of peptides presented by human leukocyte antigens (HLA) [14,15]. The T-cell receptor expressed on the vast majority of T cells is a heterodimer of an alpha (α) and a beta (β) polypeptide chain . The TCRβ chain consists of variable (V), joining (J), diversity (D), and constant (C) regions encoded by gene segments spanning 685 kilobases of human chromosome seven . Diversity and antigen specificity of T-cell antigen recognition is primarily attributed to the hypervariable, or third complementarity-determining region (CDR3), of the TCRβ chain, and is achieved by non-templated nucleotide additions or deletions during TCR gene rearrangements [15,18,19]. Antigen driven T-cell responses are observable as perturbations in the Gaussian-like distributions of CDR3 gene fragment lengths of TCR families, because responding antigen-specific cells are retained in the memory lymphocyte pool, permanently altering the distributions of cells within the T-cell repertoire [18,20–23]. Therefore, analysis of CDR3 lengths provides an assessment of the frequency and diversity of the T-cell response to antigenic exposure.
To assess the degree of T-cell clonality attributable to HIV infection, we investigated the TCRβ CDR3 length profiles, commonly referred to as spectratypes, of low-risk (LR) and at-risk seronegative (AR), and HIV-1 seropositive men (SP).
Materials and methods
Background clinical data on the subjects in this study are provided in Table 1. Two groups of men were selected from participants in the Los Angeles center of the Multicenter AIDS Cohort Study (MACS) . HIV-1 seropositive men (SP; n = 13) were selected from HIV-1-infected individuals in the chronic stage of infection (mean CD4 : CD8 ratio was 0.55) who were not receiving highly active antiretroviral therapy (HAART). At-risk HIV-1 seronegative (AR; n = 20) men were selected based on their self-reported anal-receptive sexual exposures adjusted by frequency of condom usage upon enrollment in the MACS . Within the AR group, thirteen men in 90th percentile (AR-90) and seven men in the 10th percentile (AR-10) of sexual risk behavior were selected. A third group of men at low-risk of HIV-1 infection (LR; n = 12) was identified through a local blood bank. The LR men were screened by a questionnaire to ascertain prior history of disease and exposure. Based on their questionnaire results and on their blood bank eligibility status, individuals in the LR group had HIV risk scores of 0. Individuals in the LR and AR groups were matched to the SP group on age, race, and residence (Los Angeles, California, USA). No statistically significant age differences were present across groups.
Cryopreserved peripheral blood mononuclear cells (PBMC) and frozen serum specimens were obtained from the UCLA MACS repository for subjects in the AR and SP groups . Whole blood was collected from subjects in the LR group and processed and stored in accordance with MACS protocols. The cryopreserved PBMC were used for the TCRVβ flow cytometry and spectratyping assays. The frozen serum was used for serological assays. T-lymphocyte percentages and absolute counts were obtained from fresh whole blood measurements. Complete blood count (CBC) and automated differential were performed with a Coulter (Hialeah, Florida, USA) STKS hematology analyzer.
Serologic testing for hepatitis B, hepatitis C, cytomegalovirus, and herpes simplex virus
Routine clinical serological testing was performed by the University of California, Los Angeles (UCLA) clinical laboratories. Chronic hepatitis B (HBV) infection status was determined by anti-core antibody. IgG antibody levels were assessed for hepatitis C virus (HCV) and herpes simplex viruses 1 and 2 (HSV-1, HSV-2). Both IgM and IgG measurements were collected for cytomegalovirus (CMV).
T-lymphocyte phenotyping by flow cytometry
Two-color and four-color flow cytometric analyses were performed on FACScan and FACSCalibur (Becton Dickinson, San Jose, California, USA) cytometers respectively. Anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies (BDIS, San Jose, California, USA) were purchased as conjugates of fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Two-color (FITC/PE) combinations were used to analyze CD3/CD4 and CD3/CD8. Four-color flow cytometric assessments of the TCRVt distributions within CD4+ and CD8+ T-cell subsets were conducted using a panel of monoclonal antibodies specific for 22 different TCRVβ families as previously described .
CDR3 length analyses using reverse transcriptase-polymerase chain reaction
Positive selection for CD8+ and CD4+ T-lymphocytes was performed by incubating PBMC with anti-CD8 or anti-CD4 immunomagnetic beads (Dynal, Great Neck, New York, USA) in accordance with conditions recommended by the manufacturer. The average purity of the separated CD4+ and CD8+ cells was > 99%. TCRβ CDR3 spectratyping assays were performed as previously described [28,29]. Briefly, RNA was extracted from CD4+ and CD8+ T cells suspended in TRI Reagent (Molecular Research Center Inc., Cincinnati, Ohio, USA), reverse-transcribed using a Cβ-specific oligonucleotide, and subsequently used for the polymerase chain reaction (PCR) amplification of the CDR3 regions of 24 TCRVβ families. The nucleotide lengths and intensities of the fluorescently labeled CDR3 region amplicons were measured using a 310 Genetic Analyzer (Applied Biosystems, Foster City, California, USA). PCR reactions and CDR3 measurements were performed in duplicate.
Statistical analyses of T-cell subset differences were performed using analysis of variance (ANOVA) in S-PLUS 6.1 (Insightful, Seattle, Washington, USA) and Bonferroni corrections were applied to account for the multiple comparisons performed on five variables across two groups.
Clonal T-cell expansions were identified using a previously described statistical approach to analyze CDR3 distributions . Briefly, within TCRVβ family, the relative proportion of each CDR3 length was calculated as a fraction of the total fluorescence intensity (area). The means and standard deviations of all CDR3 length-relative proportions were calculated from the LR values. Next, for each subject and TCRVβ family, the median ratio of the respective subject to LR relative CDR3 length proportion was calculated. Using the median ratio, CDR3 distributions for each subject and TCRVβ family were standardized to the mean CD4 CDR3 length proportions derived from the LR group. Expansions were identified as CDR3 lengths whose adjusted proportions exceeded the mean CD4 value by at least three standard deviations. For each subject and T-cell subset, a ratio of the number of expansions per number of CDR3 lengths measured, called the expansion ratio (ER), was computed. Statistical comparisons of expansion ratios between groups were assessed using ANOVA in S-PLUS.
To estimate the extent of clonality attributable to HIV-1 infection in at-risk men, we calculated the 95% confidence interval (95% CI) of the mean CD8+ expansion ratio difference between the AR and SP groups. The estimated standard error of the difference between means of the two groups, sMd, was calculated as follows:
where M is the mean, MSE is the mean square error and SSE is the sum of squared errors. The harmonic mean (nh) of the sample sizes for the SP (n1) and AR (n2) groups was computed to account for unequal sample sizes. The critical t value (two-tailed), specific for the confidence level and degrees of freedom (d.f.), was identified using a t table.
Serological testing for common viral infections
Serologic testing was performed on all available specimens to determine each individual's serostatus for several common chronic viral infections. The clinical serology results are presented in Table 1. All individuals tested were hepatitis C negative. The majority of individuals tested in the AR (16 of 20 = 80%) and SP (12 of 13 = 92%) groups were HBV seropositive, as evidenced by positive HBV core antigen results. Only one individual in the LR group (one of 11 = 9%) was HBV seropositive. Taken together, almost all of the subjects had evidence of CMV infection (CMV IgG positive, 34 of 36 = 94%). The majority of individuals tested were positive for herpes simplex viruses HSV-1 (28 of 36 = 78%) and HSV-2 (22 of 36 = 61%). Interestingly, significant differences were observed for HSV-2 seropositivity between the AR-10 (one of seven) and AR-90 (nine of 13) subgroups (P = < 0.05). These results suggest that riskful sexual activity is associated with exposure to HBV and HSV-2 infection among the men in this study.
We evaluated T-lymphocyte subsets to assess whether these exposures resulted in significant differences between the groups of men. T-cell phenotype distributions for each group are shown in Table 2. CD4+ and CD8+ T-cell percentage and absolute count and CD4 : CD8 ratio were each statistically significant (P < 0.05) in AR versus SP comparisons. No statistically significant differences were observed between the AR-10 and AR-90 subgroups for these parameters. The AR group revealed significantly higher CD8+ T-cell percentages and absolute counts and a corresponding significantly lower CD4 : CD8 ratio than the LR group. The CD4+ T-cell percentage and absolute count were not significantly different between the LR and AR groups. These data demonstrate that the groups had significant differences in CD8+ T-cell levels, suggesting that CD8+ T-cell counts are associated with exposure risk histories.
TCR repertoire comparisons by flow cytometry
Flow cytometric assessments of the T-cell repertoire have been used to identify T-cell responses to many viral infections including HIV [30,31], CMV , Epstein-Barr virus (EBV) , HBV and HSV . Therefore, we compared CD4+ and CD8+ TCRVβ family percentages between risk groups. The mean T-cell percentages we observed for each Vβ family were not significantly different across groups and were similar to those previously reported . However, in light of the role played by human leukocyte antigen (HLA) genetics in governing antigen presentation and ensuing T-cell responses, identifying commonly recruited TCRVβ families within groups of HLA unmatched individuals is unlikely. Therefore, we evaluated TCRVβ expansions in each individual by using the CD4+ and CD8+ mean percentage and standard deviation of each TCRVβ family derived from the LR group for comparison. Based on these reference values, we identified expansions as percentages that exceeded the LR mean values by more than three standard deviations. This evaluation revealed that individuals in the AR and SP groups exhibited a significantly greater number of expansions in both the CD4+ (P < 0.05) and CD8+ (P < 0.05) T-cell compartments than individuals in the LR group. No significant differences were found between the AR and SP groups with respect to the number of TCRVβ expansions determined by flow cytometry. Reference ranges for each TCRVβ antibody as well as the results of this analysis are shown in Table 3. These findings demonstrate a possible association between risk of HIV infection and increased percentages of select TCRVβ families in peripheral blood.
TCRβ CDR3 length distributions in CD4+ and CD8+ T cells
We compared the TCRβ CDR3 distributions of CD4 and CD8 subsets across the three groups of men. Representative CDR3 profiles are shown in Figure 1. Within each of the three groups studied, the CD4+ subset was found to exhibit significantly fewer expansions than the CD8+ subset. Figure 2a and b shows box-plot comparisons of the expansion ratios for each group. The 95% CI of the expansion ratios for the LR, AR, and SP groups were 0.03 ± 0.02, 0.08 ± 0.02, and 0.10 ± 0.03 for CD4+ T cells and 0.18 ± 0.02, 0.22 ± 0.02, and 0.28 ± 0.02 for CD8+ T cells, respectively. Both the CD4+ and CD8+ profiles exhibit significantly more clonal dominance in the AR and SP groups than in the LR group. The CD8+ T-cell expansion ratios of all three groups were statistically significantly different from each other (P < 0.05) with the AR group intermediate between the LR and SP groups. Although the CD4+ expansion ratios of the AR and SP groups were not found to be statistically significantly different from each other, the mean and all quartiles of the expansion ratio were higher in the SP group. Comparisons of the number of CD4+ and CD8+ clonal expansions between the AR-10 and AR-90 subgroups revealed no statistically significant positive associations. No common expansion trends of specific TCRVβ subfamilies were observed among subjects. For example, expansions in Vβ3 were not found to be preferred over expansions in other TCRVβ families. Given the likely heterogeneous genetic backgrounds (e.g. HLA types) of the subjects, these findings are not surprising.
Estimation of T-cell clonality attributable to chronic HIV-1 infection in at-risk men
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.
Longitudinal assessments of the TCR repertoires of HIV-seronegative homosexual men
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:
Baltimore: The Johns Hopkins University School of Hygiene and Public Health: J. B. Margolick (principal investigator), H. Armenian, B. Crain, A. Dobs, H. Farzadegan, N. Kass, S. Lai, J. McArthur, S. Strathdee, E. Taylor. Chicago: Howard Brown Health Center and Northwestern University Medical School: J. P. Phair (principal investigator), J. S. Chmiel, B. Cohen, M. O'Gorman, D. Variakojis, S. M. Wolinsky. Los Angeles: University of California, UCLA Schools of Public Health and Medicine: Principal investigators – R. Detels and B. Jamieson, B. R. Visscher (co-principal investigator), A. Butch, J. Fahey, O. Martínez-Maza, E. N. Miller, J. Oishi, P. Satz, E. Singer, H. Vinters, O. Yang, S. Young. Pittsburgh: University of Pittsburgh, Graduate School of Public Health: C. R. Rinaldo (principal investigator), L. Kingsley (co-principal investigator), J. T. Becker, P. Gupta, J. Mellors, S. Riddler, A. Silvestre. Data Coordinating Center: The Johns Hopkins University School of Hygiene and Public Health: A. Mu oz (principal investigator), L. P. Jacobson (co-principal investigator), L. Ahdieh, S. Cole, S. Gange, C. Kleeberger, S. Piantadosi, E. Smit, S. Su, P. Tarwater. NIH: National Institute of Allergy and Infectious Diseases: C. Williams, P. Miotti. National Cancer Institute: S. Melnick.
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