This study included 52 HIV-infected individuals in four different subject groups: 16 elite controllers, 14 viremic controllers, 14 noncontrollers, and 8 individuals on HAART with undetectable plasma viral loads. The median viral loads for viremic controllers and noncontrollers were 286 copies/ml and 27,590 copies/ml, respectively (Table ). The median blood CD4 counts were 746, 448, 411, and 488 for elite controllers, viremic controllers, noncontrollers, and HAART-suppressed groups, respectively (Table ). Elite controllers had significantly higher CD4 counts than the viremic controllers and the noncontrollers (P < 0.05). A summary of major histocompatibility complex (MHC) class I genotypes is presented in Table .
Immunodominant responses are shared between peripheral blood and rectal mucosa.
In order to provide sufficient cells to map CD8+
T-cell responses by IFN-γ ELISPOT, CD8+
T cells were polyclonally expanded from peripheral blood mononuclear cells (PBMC) and rectal mononuclear cells (RMC). Previous studies have shown this method to expand cells nonspecifically without introducing bias toward particular peptides or T-cell receptor clonotypes (2
) and also revealed similar targeting and magnitudes of HIV-specific T-cell responses in fresh and expanded CD8+
T cells (29
We sought to determine the extent to which responses to individual epitopes were shared between peripheral blood and rectal mucosa. Epitopes recognized by both PBMC and RMC were termed “concordant,” while those recognized by only PBMC or RMC were termed “discordant.” When the magnitudes of concordant and discordant responses were compared, concordant responses tended to be immunodominant (i.e., of higher magnitude) while discordant responses tended to be of lower magnitude (Fig. ). This trend was most obvious in responses to HIV Gag, with significant differences between concordant versus discordant responses in controllers (peripheral blood, P < 0.01) and noncontrollers (rectal mucosa, P < 0.05) (Fig. ). Similar but weaker trends were observed for Env-specific responses (rectal mucosa in controllers, P < 0.05) (Fig. ). The magnitudes of concordant and discordant Nef-specific responses were similar in controllers, whereas in noncontrollers concordant responses appeared to be immunodominant (Fig. ).
FIG. 1. Concordant and discordant CD8+ T-cell responses in peripheral blood and rectal mucosa of controllers and noncontrollers. Concordant (shared between peripheral blood [PB] and rectal mucosa [RMC]) and discordant (unique to PB or RMC) responses for (more ...)
A previous study by Ibarrondo et al. attributed discordant responses to culture artifacts, such as differential expansion or starting frequencies of epitope-specific cells, resulting in low or discordant responses near the limit of detection. While some discordant responses in our study were near the limit of detection for our assay (50 SFC/million), many discordant responses, particularly those in rectal mucosa, were well above this limit, with 84% of Gag responses and 85% of Env and Nef responses being 100 SFC/million or greater (Fig. ). Another technical issue that may have contributed to discordant responses is the limitation imposed by biopsy specimen sampling due to variable localization of inductive and effector sites within the GI tract. Given the limited number of biopsies performed, it may be difficult to capture all antigen-specific T-cell populations with comparable efficiency, although this remains the best available method for acquiring tissue at this time.
HIV controllers frequently target HIV Gag p24 (capsid) and p7 (nucleocapsid).
T cells in HIV controllers may target highly conserved regions of HIV structural proteins, leading to a potentially high fitness cost associated with cytotoxic T-lymphocyte (CTL) escape mutations (8
). In order to better visualize regions targeted by the mucosal CD8+
T-cell response in controllers and noncontrollers, we generated peptide cluster maps for Gag, Env, and Nef (Fig. to D). We determined the percentage of subjects in each group that responded to individual peptides, irrespective of MHC restriction.
FIG. 2. CD8+ T-cell targeting of HIV Gag and Nef. Peptide cluster maps were constructed to visualize the frequency of subjects targeting specific areas of Gag and Nef. (A) Gag p17, (B) Gag p24, (C) Gag p2, p7, p1, and p6, and (D) Nef. Each peptide numbered (more ...)
Both controllers and noncontrollers appeared to target HIV Gag p17 (matrix) equally well and in similar locations (Fig. ). A higher percentage of controllers responded to p24 (capsid) peptides than noncontrollers (Fig. ). These responses were generally concordant and, since a majority of controllers were HLA-B57+, frequently targeted immunodominant, HLA-B57-restricted epitopes (described in detail below). Controllers also more frequently targeted the zinc finger domains of p7 (nucleocapsid) than noncontrollers (Fig. ). In contrast, noncontrollers preferentially targeted Nef, particularly regions in the folded core domain (Fig. ). HIV Env was sporadically targeted in our assays (data not shown); however, the targeting of Env peptides in these individuals may have been underestimated due to the use of consensus clade B peptides in our assay as opposed to autologous viral sequences.
Immunodominant responses to HLA-B27- and HLA-B57-restricted epitopes in rectal mucosa of HIV controllers.
HLA-B27 and HLA-B57 are enriched in HIV controller populations (10
). Immunodominant Gag epitopes restricted by these two alleles have been well defined: KRWIILGLNK (B27-KK10, p24 amino acids [aa] 131 to 140) (1
), KAFSPEVIPMF (B57-KF11, p24 aa 30 to 40) (22
), ISPRTLNAW (B57-IW9, p24 aa 15 to 23) (22
), QASQEVKNW (B57-QW9, p24 aa 176 to 184) (22
), and TSTLQEQIGW (B57-TW10, p24 aa 108 to 117) (8
). Our cohort included two controllers positive for HLA-B27, one positive for both HLA-B27 and HLA-B57, and 11 positive for HLA-B57.
B57-KF11 and B57-IW9 were the most frequently targeted HLA-B57-restricted epitopes among controllers in our study (Fig. ). B57-KF11 was also the most consistently immunodominant (Fig. ; Table ), eliciting the highest median responses in blood and rectal mucosa of any HLA-B57-restricted epitope. Furthermore, both HLA-B27- and HLA-B57-restricted CD8+
T cells elicited significantly higher-magnitude responses in controllers, compared to the median responses to non-HLA-B27- or HLA-B57-restricted epitopes (P
< 0.05; Fig. ). For comparison, the well-defined HLA-A2-restricted epitope SLYNTVATL (A2-SL9, p17 aa 77 to 85) (30
), which is not associated with controller status, elicited responses among controllers that were similar in magnitude to those for other non-HLA-B27- and HLA-B57-restricted epitopes (Fig. ; Table ).
FIG. 3. Strength of the IFN-γ response to HLA-B27- and HLA-B57-restricted Gag epitopes in HIV controllers. (A) IFN-γ response to five immunodominant HLA-B27- and HLA-B57-restricted Gag epitopes compared to that for an immunodominant HLA-A2-restricted (more ...)
Median IFN-γ response magnitudes for HLA-B27 and HLA-B57 epitopes
Median mucosal T-cell responses to Gag peptides are stronger in controllers than in noncontrollers, while Env and Nef responses show the opposite trend.
In general, and consistent with our earlier studies (20
), controllers (elite and viremic controllers combined) had significantly higher median responses to Gag p55 in rectal mucosa than HAART-suppressed individuals (P
< 0.05; Fig. A). Controllers also showed a trend toward higher mucosal Gag-specific responses than noncontrollers (Fig. ). In PBMC, controllers had significantly higher median IFN-γ responses to HIV Gag than noncontrollers (P
< 0.001, Fig. ).
FIG. 4. Strength of the IFN-γ response to HIV Gag, Env, and Nef in rectal mucosa and peripheral blood. Median IFN-γ response to Gag, Env, and Nef in rectal mucosa (A) and peripheral blood (B), as measured by ELISPOT assay. All data are presented (more ...)
The peripheral blood IFN-γ responses to Env and Nef were slightly greater in controllers than in noncontrollers or HAART-suppressed subjects (Fig. ). However, median Env and Nef responses in rectal mucosa were stronger in noncontrollers than either controllers or individuals on HAART (P > 0.05, Fig. ). Thus, mucosal responses to HIV Gag peptides tended to be stronger in controllers than in noncontrollers (in terms of IFN-γ SFC/million), while mucosal responses to Env and Nef revealed the opposite trend.
There was also a trend toward higher-magnitude Gag-, Env-, and Nef-specific responses in rectal mucosa than in PBMC in both controllers and noncontrollers (Fig. ). This trend reached significance only in Env-specific responses among noncontrollers (P
< 0.05, Fig. ). This observation recalls previous findings and is a likely consequence of the higher frequency of antigen-experienced, memory T cells in gastrointestinal lamina propria than in peripheral blood (12
Viremic controllers show a greater breadth of response to HIV Gag than elite controllers, noncontrollers, and individuals on HAART.
The total ELISPOT response breadth for each subject was calculated by summing the number of epitopes recognized within Gag, Env, and Nef by PBMC or RMC. For these calculations, responses to overlapping peptides spanning a single known epitope were counted as a single response. Total response breadth was similar in controllers and noncontrollers (Fig. A and B). However, controllers had broader Gag-specific responses in rectal mucosa than subjects on HAART (median, 4 versus 2 epitopes) (Fig. ). Controllers also had broader Gag-specific responses in PBMC than noncontrollers (median, 3 versus 1.5 epitopes) (Fig. ). Subdividing the controller group into elite (i.e., those with VL <75 copies/ml) and viremic controllers (i.e., those with VL <2,000 copies/ml), we found that viremic controllers had significantly broader Gag-specific responses in rectal mucosa than elite controllers, noncontrollers, and individuals on HAART (median, 5 versus 3, 3, and 2 epitopes, respectively; P < 0.05) (Fig. ), suggesting that the presence of ongoing low-level viral replication in these individuals may support continued targeting of HIV Gag.
FIG. 5. Breadth of the CD8+ T-cell response. The number of epitopes (breadth) recognized within Gag, Env, and Nef by CD8+ T cells in rectal mucosa (A) and peripheral blood (B), as measured by IFN-γ ELISPOT assay. Horizontal bars represent (more ...) Reduced breadth and magnitude of mucosal CD8+ T-cell responses in individuals on HAART.
Subjects on HAART with undetectable viral loads generally showed lower-magnitude CD8+ T-cell responses to Gag, Env, and Nef than HIV controllers, with many subjects on HAART showing no detectable responses at all (Fig. ). Likewise, response breadth was also low in these individuals (Fig. ). As a percentage of the total response in HAART-suppressed individuals (Gag, Env, and Nef combined), Gag epitopes were most frequently targeted in both rectal mucosa and blood (60 and 75% of targeted epitopes, respectively; data not shown). Env epitopes elicited fewer responses in subjects on HAART (40% and 25% of targeted epitopes in mucosa and blood, respectively) (data not shown). Only one sample from a HAART-suppressed individual in our study targeted Nef (Fig. ).
Gag-specific responses dominate in rectal mucosa of HIV controllers.
The pie charts in Fig. A show median ELISPOT response magnitudes partitioned into Gag, Env, and Nef. In HIV controllers, responses in rectal mucosa and blood showed a slight bias toward Gag (39 to 42% of the total response), with the remaining 60% divided approximately evenly between Env and Nef. In contrast, in HIV noncontrollers, less than 25% of the response in either tissue was directed toward Gag, while 40% of the mucosal response and 49% of the PBMC response were directed toward Nef.
FIG. 6. Proportion of the CD8+ T-cell response attributed to Gag, Env, or Nef. Each pie shows the proportion of the measured response magnitude (A) or breadth (B) attributed to Gag, Env, and Nef in either peripheral blood or rectal mucosa of controllers (more ...)
The pie charts in Fig. show the median response breadth (number of epitopes targeted) for each tissue and subject group. In controllers, at least 50% of the epitopes targeted by CD8+ T cells in both rectal mucosa and PBMC were located within HIV Gag, compared to fewer than 20% for HIV Env (Fig. ). In noncontrollers, Gag, Env, and Nef were targeted at similar frequencies in blood.
Taken together, the data in Fig. reveal that Gag responses were the largest contributor to mucosal and blood CD8+ T-cell responses in HIV controllers, while Nef and Env-specific responses dominated in HIV noncontrollers.