JAIDS Journal of Acquired Immune Deficiency Syndromes:
Phenotypic and Functional Characterization of HIV-1-Specific CD4+CD8+ Double-Positive T Cells in Early and Chronic HIV-1 Infection
Howe, Rawleigh MD, PhD*; Dillon, Stephanie PhD*; Rogers, Lisa BS*; Palmer, Brent PhD*; MaWhinney, Samantha ScD*; Blyveis, Naomi MS*; Schlichtemeier, Rick BS*; D'Souza, Michelle MS*; Ingoldby, Laura BS*; Harwood, Jeri E F PhD*; Rietmeijer, Cornelis MD, PhD, MSPH†; Ray, Graham RN, MSN*; Connick, Elizabeth MD*; Wilson, Cara C MD*
From the *University of Colorado Denver, Aurora, CO; and †Denver Department of Public Health, Denver, Colorado.
Received for publication August 25, 2008; accepted December 23, 2008.
Supported by funding from National Institutes of Health grant P01 (AI55356), with additional support from the University of Colorado Center for AIDS Research (AI 51550) and the University of Colorado General Clinical Research Center (CA 46934) grants.
Presented in part at the Conference for Retroviruses and Opportunistic Infections, February 25-28, 2007, Los Angeles, CA.
Correspondence to: Cara C. Wilson, MD, Division of Infectious Diseases, Department of Medicine, University of Colorado Denver, Mail Stop #B168, Building P15, 12700 East 19th Avenue, Aurora, CO 80045 (e-mail: cara.wilson@UCHSC.edu).
Objective: CD4+CD8+ double-positive (DP) T cells represent a poorly characterized population of effector T cells found at low frequencies in the peripheral blood. Virus-specific DP T cells have been identified in HIV-1-infected patients but their origin, relationship to conventional CD4+ and CD8+ single-positive (SP) T cells, and role in disease pathogenesis are unclear.
Methods: In this study, peripheral blood T cells were analyzed for cytokine production, maturation, and cytolytic marker expression by polychromatic flow cytometry in subjects with both early (n = 27) and chronic (n = 21) HIV-1 infection.
Results and Conclusions: HIV-1-specific interferon gamma (IFN-γ)-producing DP T cells were identified at a median frequency of 0.48% compared with 1.08% and 0.02% for CD8 and CD4 SP cells, respectively, in response to pooled HIV-1 peptides. HIV-1-specific DP T cells exhibited polyfunctionality with characteristics of both CD4 and CD8 SP T cells, including coproduction of IFN-γ and IL-2 and expression of cytolytic-associated lysosomal-associated membrane protein. No differences in frequencies of unstimulated DP T cells were observed in early compared with chronic infection. However, chronic infection was associated with higher frequencies of HIV-specific, IFN-γ-producing DP T cells and higher fractions of effector memory and lysosomal-associated membrane protein expression among these cells, suggesting an effect of cumulative viral antigen burden on DP T-cell function.
Although most T cells express CD4 and CD8 molecules in a mutually exclusive fashion, a small percentage of CD4+CD8+ [double positive (DP)] T cells have been identified in the periphery of both humans and animals.1-3 Unlike immature thymic DP T cells, peripheral T cells exhibit functional properties of mature T cells including antigen-dependent cytokine production and cytolytic activity and express markers associated with a memory phenotype.4-7 Several observations suggest that such cells may represent the progeny of antigen-stimulated conventional single-positive (SP) CD4+ and CD8+ T cells. Human DP T cells can be readily generated in vitro from SP precursors,8-11 and adoptive transfer experiments in animals suggest that at least some DP T cells can arise from CD4+ SP precursors in vivo.12,13 Elevated percentages of DP T cells are seen in the setting of autoimmune diseases, and in some acute and chronic viral infections.6,13-23
The expression levels of CD4 and CD8 molecules on DP T cells are variable, and 2 subpopulations have commonly been identified: one population expressing CD8 at high levels but reduced (dull) amounts of CD46,8-11 and the other expressing high CD4 but reduced CD8.6,7,24-28 The former population, hereafter DP 4d, has been primarily characterized in vitro.8-11 DP 4d T cells may arise from as many as 30%-50% of naive (NV) CD8 SP cells upon T-cell receptor ligation but are less efficiently generated from memory CD8 SP cells.10 This finding predicts that early after infection, DP 4d T cells should be more prominent than later in chronic infections, but this possibility has not been addressed in vivo. The other major subset, expressing dull levels of CD8 with normal CD4 (hereafter DP 8d), is generally better characterized and is found at elevated frequencies both in acute and chronic infections in humans and animal models.8 Cells with a similar phenotype are present in high frequencies in intestinal tissue, particularly the lamina propria of jejunum, and are notable for the expression of alpha/alpha CD8 homodimers rather than the conventional alpha/beta heterodimers of CD8 SP cells.6,7,24-30 In addition to these 2 subsets, some studies have resolved additional subpopulations of DP T cells, implying an even greater complexity.2,31
The expression of CD4-bearing DP T cells is relevant in HIV-1 disease because such cells would be predicted to be infectible with HIV-1. Indeed, results from in vitro studies suggest that DP T cells can be infected by both X4-tropic and R5-tropic virus, and ex vivo isolation of DP 4d T cells from HIV-1-infected patients directly demonstrated HIV-1 provirus, suggesting infectibility in vivo.10,32-36 Studies in both simian immunodeficiency virus (SIV) and HIV-1 infections have demonstrated that intestinal DP T cells are at least as (if not more) susceptible to SIV/HIV-1-mediated depletion as conventional CD4 T cells.37-39 Moreover, peripheral blood DP T cells in SIV-infected macaques are also reduced relative to uninfected animals. At odds with these findings, levels of DP 8d T cells in peripheral blood of HIV-1-infected patients have not been observed to be significantly depressed, and some authors have reported expansions of DP T cells in some patients.8,40 Few studies have addressed functional properties of DP 8d T cells in HIV-1-infected patients. A study of a limited number of HIV-1 subjects receiving a therapeutic vaccine revealed the important finding that DP 8d T cells exhibit polyfunctionality in that both substantial cytokine production and Cytotoxic T Lymphocyte (CTL) activity were observed,5 a feature previously described in other antigen systems.4 Importantly, polyfunctionality is a feature of CD8 T cells in HIV-1-infected patients which correlated with lower viral loads and nonprogressive disease.41,42 However, no studies have addressed the antigen-specific functional features of DP T cells in larger numbers of unvaccinated HIV-1-infected individuals.
Most studies of HIV-1 pathogenesis in man have focused on studying responses in chronically infected patients mainly because the majority of patients are not identified early after infection. Recent studies in both experimental macaque models and in naturally infected humans have highlighted the dramatic depletion of CD4 T cells in the gut mucosa early during the course of SIV and HIV-1 infection.37-39,43 In addition, it has long been appreciated that the HIV-1 viral set point which emerges relatively early after infection predicts later disease progression,44 hence it follows that study of immune mechanisms in early or recently infected patients, including the aforementioned polyfunctionality, may reveal important features correlated with viral set point and hence disease progression. Because of an incomplete understanding of DP T cells in general and in the setting of HIV-1 infection, in the present study, we sought (1) to compare the phenotypic and functional properties of DP T cells with those of SP CD4 and CD8 T cells, emphasizing HIV-1-specific responses, and (2) to compare these properties of DP T cells among patients with early vs. chronic HIV-1 infection.
MATERIALS AND METHODS
Twenty-seven adults of 18 years of age or older with acute or recent HIV-1 infection were recruited from the University of Colorado Denver, the Denver Health Medical Center, and Denver Public Health, Denver, CO. Acute HIV-1 infection was defined as detectable plasma HIV-1 RNA ≥2000 copies per milliliter within 14 days of study entry and 1 of the following: (a) a negative enzyme-linked immunosorbent assay (ELISA) for HIV-1 antibodies, (b) a positive ELISA but negative or indeterminate Western blot (≤2 bands) within 14 days of study entry, or (c) a positive ELISA and Western blot but a negative ELISA or plasma HIV-1 RNA (<2000 copies/mL) within 30 days before study entry. Recent HIV-1 infection was defined as either (a) a positive ELISA and Western blot (>2 bands) within 14 days of study entry but a negative ELISA or plasma HIV-1 RNA (<2000 copies/mL) within days 31-90 before study entry or (b) a positive ELISA and Western blot within 14 days of study entry but a nonreactive 3A11-LS ELISA (Abbott Laboratories, Abbott Park, IL)45 documented within 14 days of study entry. Among these 27 individuals, only 2 met the criteria for acute infection, therefore, we combined all acute or recently infected individuals into a single cohort, hereafter termed “early HIV-1-infected” individuals. Of these, 26 were male, 1 was female, median age was 38 years (range 24-52 years), the median CD4 count was 598 cells per microliter (range 37-1091 cells/μl), and median viral load was 4.6 log10 HIV-1 RNA copies per milliliter plasma (range 3.2-6.2 copies/mL). Twenty-one subjects with chronic untreated HIV-1 infection, 6 female and 14 male, were recruited from the Adult Infectious Diseases Group Practice clinic at University of Colorado Health Sciences Center. Among these subjects, the median age was 36 years (range 24-65 years), median viral load 5.0 log10 HIV-1 RNA copies per milliliter plasma (range 3.3 to >5.8 copies/mL), and median CD4 count 391 cells per microliter (range 10-836 cells/μl). Twelve University of Colorado Health Sciences Center employees self-identifying as HIV-1 seronegative, 6 female and 6 male, median age 35 years (range 24-55 years) were recruited as healthy controls. All study subjects participated voluntarily and gave written informed consent to participate. The study was approved by the Colorado Multiple Institutional Review Board at University of Colorado Denver.
Assays for Recent HIV-1 Infection
The 3A11-LS ELISA (Abbott Laboratories) was performed at the Blood Centers of the Pacific, San Francisco, CA, in accordance with the manufacturer's instructions.
Assays for Plasma HIV-1 RNA
Plasma HIV-1 RNA levels were determined using the Amplicor HIV-1 Monitor test (Roche Diagnostics Corporation, Indianapolis, IN). When HIV-1 RNA levels were below 400 copies per milliliter, the ultrasensitive version of this assay was used.
HIV-1 Gag peptides (123 peptides) consisted of 15mers overlapping by 11 amino acids and corresponding to sequences of clade B HXB2 strain HIV-1 [National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, Germantown, MD, catalog # 5107]. HIV-1 consensus clade B Nef peptides (49 peptides) consisted of 15mers overlapping by 11 amino acids (NIH AIDS Research and Reference Reagent Program, catalog # 5189). HIV-1 consensus clade B Pol peptides (249 peptides) consisted of 15mers overlapping by 11 amino acids (NIH AIDS Research and Reference Reagent Program, catalog # 6208). Lyophilized peptides were reconstituted in dimethyl sulfoxide (Sigma, Saint Louis, MO).
Peripheral Blood Mononuclear Cell Preparation, Cell Culture, and Cell Staining
Approximately 40 mL of blood was obtained by venipuncture, and peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Hypaque centrifugation. Five million cells in 1 mL of RPMI supplemented with 1% glutamine, 1% penicillin, 1% streptomycin, and 10% heat-inactivated normal human serum were cultured in slanted polypropylene tubes. After overnight incubation, tubes were stimulated with 1 of 5 stimuli: Gag, Nef, or Pol peptide pools, staphylococcal enterotoxin B (SEB, 1 μg/mL; Toxin Technologies, Sarasota, FL), or media plus 0.25% dimethyl sulfoxide. Each peptide preparation represented pooled peptides corresponding to nucleic acid sequences spanning gag, nef, or pol HIV-1 genes with each peptide present at 1 μg/mL final concentration. Anti-CD28 (3 μg/mL; BD Biosciences, San Jose, CA), anti-CD49d (3μg/mL; BD Biosciences), and fluorescein-Isothiocyanate-conjugated anti-CD107a and anti-CD107b [lysosomal-associated membrane protein (LAMP); BD Biosciences] were also added at this time to all conditions. After 2 hours, brefeldin A (GolgiPlug; BD Biosciences) and monensin (GolgiStop; BD Biosciences) were added at 0.1% final concentration, and the cultures continued for an additional 4 hours. Cells were washed with 1% bovine serum albumin in phosphate-buffered saline before staining. Cells were initially stained with unconjugated anti-CCR7 antibodies (BD Biosciences) followed by biotinylated anti-mouse IgM (BD Biosciences) and then streptavidin-PE (Rockland, Gilbertsville, PA). Cells were then stained with anti-CD4-APC-Cy7 (BD Biosciences), anti-CD8-Alexa Fluor-405 (Caltag, Burlingame, CA), anti-CD3-PE-Texas Red (Beckman Coulter, Fullerton, CA), and anti-CD45RA-PECy5 (BD Biosciences). After an additional wash with bovine serum albumin in phosphate-buffered saline, cells were fixed with formaldehyde (medium A, Caltag) and left overnight at 4°C. The following morning cells were washed, permeabilized (medium B, Caltag), and stained with anti-IL-2-APC (Caltag) and anti-IFN-γ PE-Cy7 (BD Biosciences). Antibodies were used at doses recommended by the manufacturer. After a final wash, cells were fixed with 2% formaldehyde. Cells (median 772,000, range 117,000 to 2 million) were acquired on a FACSAria flow cytometer (BD Biosciences), and subsequent analysis was performed with FloJo software (Treestar, Ashland, OR). Two subjects with chronic infection were excluded from cytokine analysis for technical reasons, and LAMP expression was assayed for 17 early patients and 14 chronically infected patients.
A lymphocyte gate was selected by forward and perpendicular light scatter characteristics. A narrow gate was used to select small lymphocytes to minimize artifactual CD4+CD8+ staining due to doublets of CD4+CD8− and CD4+CD8− SP cells and nonspecific staining of dead cells. This gating strategy was based on extensive preliminary experiments with vital dyes to identify dead cells and the doublet discrimination capacity of FACSAria based on height-to-area or height-to-width perpendicular light scatter. Thereafter, CD3+ subsets of CD8+CD4−, CD8-CD4+, and CD4+CD8+ or DP 4d or DP 8d subsets of CD4+CD8+ cells were further defined. Frequencies of cytokine-producing cells within the IFN-γ+IL-2−, IFN-γ+IL-2+, or IFN-γ−IL-2+ quadrants were determined on gated T-cell subsets. Total IFN-γ responses were determined by summing the aforementioned 2 frequencies, and total cytokine responses were calculated by summing all 3 frequencies. Frequencies of naive (NV), central memory (CM), effector memory (EM), and terminally differentiated effector memory (TDEM) cells were defined as the fraction of gated cells falling within the CCR7+CD45RA+, CCR7+CD45RA−, CCR7−CD45RA−, and CCR7−CD45RA+ quadrants, respectively. LAMP-positive cells were defined as percentage of gated cells positive for the CD107a+CD107b+ markers and were defined only among gated total IFN-γ+ cells. Because TDEM cells were generally of very low frequency and we were principally interested in memory rather than NV cells, an index of the fraction of CM cells relative to that of EM cells [relative central memory (RCM)] was calculated as the frequency of CM cells divided by the sum of the frequencies of CM and EM cells.
SEB-reactive or HIV-reactive cytokine-producing frequencies among a given gated T-cell subset were calculated by subtracting the percentage of cytokine-producing cells in samples cultured in the presence of media alone from those cultured in the presence of each stimulus. The net HIV-1-specific response was calculated by summing the individual net Gag, Nef, and Pol frequencies. In some analyses, positive HIV-1-specific cytokine responses were arbitrarily defined as those with frequencies ≥0.1%, and the percentage of subjects with such positive responses was determined among all subjects.
The fraction of IFN-γ-producing cells among total cytokine-producing cells responding to SEB was calculated as the SEB reactive total IFN-γ response divided by the SEB reactive total cytokine response. The RCM fraction among SEB-specific or HIV-specific IFN-γ-producing cells was calculated by first determining the frequency of stimulus-specific IFN-γ-producing CM cells (after subtracting media control values) and then dividing by the sum of the stimulus-specific IFN-γ-producing EM plus CM cells. The fraction of SEB-specific and HIV-1-specific cells expressing LAMP was defined by calculating the frequency of stimulus-specific IFN-γ+ cells expressing LAMP divided by the frequency of total stimulus-specific IFN-γ-producing cells. Samples with no IFN-γ-positive events in response to HIV-1 peptides were excluded from analysis. This resulted in exclusion of HIV-1-specific memory or LAMP marker data from 6 early infected subjects for the DP 4d subset and from no more than 1 early or chronic subjects for all other T-cell subsets. For purposes of graphing results, values less than 0 or greater than 1 were set equal to 0 and 1, respectively, though these adjustments had minimal effect on the nonparametric statistical methods employed. Tests of statistical significance using alpha = 0.05 were determined by JMP 6 software (SAS, Cary, NC). Statistical correlations were assessed by the nonparametric Spearman test. Statistical significance for comparison of responder frequencies was determined by χ2 test. All other analyses described herein utilized the nonparametric Wilcoxon rank sum test for comparisons of 2 samples and Kruskal-Wallis test for comparisons of more than 2 samples. Because of the exploratory nature of this research, no adjustments were made for multiple comparisons.
Distribution of T-Cell Subsets in HIV-1-Infected and HIV-1-Uninfected Subjects
Our initial objective was to enumerate the frequencies of CD4+CD8− (CD4), CD4−CD8+ (CD8), CD4+CD8+ (DP) T cells, and the CD4 dull (DP 4d) and CD8 dull (DP 8d) subsets of DP T cells among HIV-1-infected patients with early (n = 27) and chronic (n = 21) infection and in healthy uninfected donors (n = 12). The flow cytometric gating strategy is illustrated in Figure 1A, and the results are summarized in Figure 2. As shown in Figures 2A and C, although patients with early HIV-1 infection exhibited median CD8 and CD4 T-cell percentages similar to levels of the control group, chronically infected individuals had significantly higher fractions of CD8 (P < 0.0001) and lower percentages of CD4 T cells (P = 0.0001) than early infected patients. In contrast, the fraction of DP T cells among the groups was virtually identical (Fig. 2B; median 0.94%, 0.89%, and 0.84% for early, chronic, and control groups, respectively). However, a much greater range of DP T cells was seen in subjects with early infection, consistent with the possibility that this subset might be expanded in some persons with early HIV-1 infection. When DP T cells were further delineated into DP 4d and DP 8d T-cell subsets, as depicted in Figures 2D and E, it was clear that DP 8d T cells were more frequent (median = 0.64% among all subject groups) than DP 4d T cells (median = 0.15% among all subject groups). Although no differences in the median percentages of either DP T-cell subset were observed between patients with early and chronic HIV-1 infection, the range of DP 8d T-cell frequencies was greater among subjects with early infection (Fig. 2E). Of note, a median of 12.8% of all gated DP did not reside within either the DP 4d or DP 8d subsets, consistent with previous observations that additional heterogeneity exists within DP cells.2,31 There was no statistically significant difference between these non-DP 4d and non-DP 8d cells among early (median 10.6%) or chronically infected (median 12.9%) patients (P = 0.67). Finally, the absolute count of each of these single and DP T-cell subsets was determined per cubic millimeter blood for early and chronically infected patients. Although the absolute count of CD4 T cells decreased and that of CD8 T cells increased in chronic relative to early infected patients (P = 0.0008 and 0.0033, respectively), consistent with the percentage differences, no statistically significant differences were observed among absolute counts of total DP T cells or the 2 DP T-cell subsets (data not shown).
Distribution of Memory Cell Populations Among Subsets of T Cells
Figure 3 depicts the distribution of cell surface markers defining NV, (Fig. 3A), CM, (Fig. 3B), EM (Fig. 3C), and TDEM (Fig. 3D) phenotypes among unstimulated CD4, CD8, and DP 4d and DP 8d T cells from pooled early and chronically HIV-infected patients. Although the fraction of NV cells did not differ substantially (P = 0.07) across T-cell subsets (Fig. 3A), CM cells were much more prominently represented among CD4 than CD8 T cells, with DP 4d and DP 8d T cells exhibiting intermediate percentages (Fig. 3B). Conversely, CD8 T cells exhibited higher fractions of EM and TDEM cells than CD4, with DP 4d and DP 8d T cells again expressing intermediate values (Figs. 3C, D). The memory phenotypic differences between these T-cell subsets were highly significant (Figs. 3B-D). To simplify the comparison of these memory subsets and because frequencies of TDEM were low, we calculated an index of RCM (see Materials and Methods) as % CM/(%CM + %EM). The RCM of CD4 and CD8 SP T cells was compared with the DP 4d and DP 8d T-cell subsets (Fig. 3E). The results indicate that among DP T cells, the 4d and 8d subsets were clearly distinct from CD8 and CD4 T cells, with DP 4d T cells (median RCM 0.55) and DP 8d T cells (median RCM 0.62) exhibiting values intermediate to that of CD8 T cells (RCM 0.26) and CD4 T cells (RCM 0.83) (P < 0.0001, across all groups).
We next compared memory phenotypes of these unstimulated T-cell subsets between the 2 HIV-1-infected patient cohorts. Memory phenotype data based on CCR7 and CD45RA expression for each T-cell subset are shown for subjects with early (open circles) and chronic (closed circles) infection in Figure 3. For CD8 T cells, a higher percentage of EM cells were identified among patients with chronic (median 56.2%) vs. early HIV-1 infection (median 39.8%, P = 0.007). No differences in memory subsets of CD4 T cells were observed between the 2 patient cohorts (data not shown). Although small differences among DP 4d T cells were noted between early and chronically infected patients within NV cells (medians 22.9% and 16.1%, respectively, P = 0.1), CM cells (medians 31.5% and 42.0%, respectively, P = 0.06), the EM subset (medians 22.3% and 30.4%, respectively, P = 0.40), and TDEM cells (medians 9.7% and 5.9%, respectively, P = 0.27), these differences did not reach statistical significance at the 0.05 level. Similarly, there were no statistically significant differences comparing early and chronic infection within the DP 8d subset among NV cells (medians 17.5% and 14.5%, respectively, P = 0.96), among CM cells (medians 41.4% and 48.1%, P = 0.17) and among EM cells (medians 30.8% and 23.0%, P = 0.76). A small difference in the TDEM subset among DP 8d cells was observed comparing early (median 3.6%) and chronic (median 1.4%) subjects (P = 0.02). Moreover, the RCM did not differ appreciably between early and chronically infected patients for DP 4d cells (medians 0.52 and 0.59, respectively, P = 0.52) or for DP 8d cells (medians 0.60 and 0.69, respectively, P = 0.44) (Fig. 3E). Collectively, these results indicate that although dramatic differences between the relative distribution of memory subpopulations were observed between unstimulated single and DP T-cell subsets, these differences were largely independent of clinical status.
Frequency of HIV-1-Specific Cytokine-Producing Cells in T-Cell Subsets
To assess HIV-1-specific responses, PBMC were stimulated with Gag, Nef, and Pol peptide pools, intracytoplasmic IFN-γ-synthesizing and/or IL-2-synthesizing cells were identified, and summed HIV-1-specific responses were determined as described in Materials and Methods according to gating strategies depicted in Figure 1C. Responses from all HIV-1-infected patients are illustrated in Figure 4. Readily detectable frequencies of HIV-1-specific IFN-γ+IL-2− cells were observed among total DP T cells (median 0.48%, range 0%-13.3%; not shown). Such cytokine-producing cells were apparent within both DP 4d (median 1.17%, range 0%-23.4%) and DP 8d T-cell subsets (median 0.23%, range 0%-8.2%) and among CD8 T cells (median 1.08%, range 0%-10.1%) (Fig. 4A). HIV-1-specific CD4 T-cell frequencies were generally much lower (median 0.02%, range 0%-0.24%). The difference between HIV-specific frequencies of CD4 and DP 8d cells and that of DP8d and DP 4d T cells were highly significant (P = 0.007 and P = 0.006, respectively). HIV-1-specific IFN-γ-cosynthesizing and IL-2-cosynthesizing cells were observed at very low frequencies among CD4 (median 0.005%) and CD8 (median 0.007%) T cells (Fig. 4B). In contrast, the total DP T-cell subset exhibited significantly higher levels of HIV-specific IFN-γ+IL-2+ cells than either SP subset (median 0.068%, P = 0.016 and 0.05 vs. that of CD4 and CD8 T cells, respectively). When total DP cells were split into DP 4d and 8d subsets as shown in Figure 4B, IFN-γ+IL-2+-coproducing cells were clearly apparent in both subsets. However, the responses of many subjects were negligible so that the median responses of these 2 subsets did not differ significantly from the essentially negative CD4 and CD8 T-cell responses. Therefore, we arbitrarily stratified subjects into responders or nonresponders based on the presence or absence of at least 0.1% HIV-specific IFN-γ+IL-2−-producing or IFN-γ+IL-2+-producing cells. As shown in Figure 4C, positive IFN-γ+IL-2+-coproducing responses were significantly more prominent within either DP 4d or DP 8d subsets than within SP CD4 or CD8 T cells although a low response frequency was revealed within the latter cell population. In contrast, positive IFN-+IL-2-γ responses were most prominent among CD8 T cells with DP 4d, DP 8d, and CD4 T cells exhibiting progressively fewer responses, mirroring the hierarchy apparent in Figure 4A. HIV-1-specific IFN-γ−IL-2+ frequencies were low in all T-cell subsets, and significant differences among these T-cell subsets were not apparent (data not shown).
We then compared frequencies of HIV-1-specific T-cell subsets in subjects with early vs. chronic infection. As depicted in Figure 4D, within the CD8 T-cell subset, frequencies of IFN-γ+IL-2− cells were significantly higher in subjects with chronic infection (median 2.45%) than with early infection (median 0.52%, P = 0.0005). Similarly, DP 4d and DP 8d T cells exhibited higher HIV-1-specific IFN-γ+IL-2− frequencies in chronically infected subjects (medians 1.65% and 0.59%, respectively) compared with early infection (0.76% and 0.16%, respectively), and these increases in frequency in chronic infection were of borderline statistical significance for both DP subsets (P = 0.04 and 0.08, respectively). No significant differences between early and chronic infection were observed for HIV-specific IFN-γ+IL-2−-synthesizing CD4 T cells (P = 0.41). Positive HIV-1-specific IFN-γ+IL-2+ cell frequencies (those exceeding 0.1%) among DP 4d T cells were much more prominent in chronically infected subjects (63%) than in those with early infection (18%, P = 0.002). In contrast, there was only a modest difference in the percentage of coproducing responders between early (30%) and chronic (17%) infection subjects among the DP 8d subset (P = 0.30). The low CD8 responder frequency was principally seen among chronically infected individuals (chronic 26%, early 0%, P = 0.002).
HIV-1-Specific DP T-Cell Frequencies in Patients With Higher or Lower Percentages of Total DP T Cells
As noted above, a subset of patients with early HIV-1 infection exhibited elevated frequencies of total DP T cells and high levels of the DP 8d T-cell subset (Figs. 2B, E). We arbitrarily stratified early infected patients into 2 groups, those with higher DP T cells (total DP T cells >2.7% of total T cells, n = 6, hereafter “DP high”) or lower DP T cells (<2.7% of total T cells, n = 21, hereafter “DP low”). None of the subjects from the chronically HIV-1-infected or control group had DP T cells at levels corresponding to the DP high group. Among the DP high group, the majority of DP, 90.3%, were DP 8d cells, whereas only 3.8% were DP 4d. In contrast, among the DP low group, DP 8d comprised only 66.0% of total DP cells and the DP 4d subset 15.4%. Frequencies of HIV-1-specific IFN-γ-producing cells within total DP (median 0.18%), DP 8d (median 0.16%), and DP 4d (median 0.95%) T-cell subsets from the DP high group were clearly not higher than frequencies within total DP (median 0.54%), DP 8d (median 0.25%), and DP 4d (median 1.01%) T-cell subsets among patients in the DP low group. Furthermore, we found no evidence of enhanced frequencies of HIV-1-specific IL-2-producing cells among the DP high group (data not shown). These results clearly indicate that the high fractions of DP T cells seen in some subjects with early HIV-1 infection (the DP high group) are not due to selective expansions of HIV-1-specific IFN-γ-producing or IL-2-producing cells, at least as defined by reactivity to the pooled HIV peptides used in this study.
Expression of Memory Markers and LAMP on HIV-1-Specific Cytokine-Producing Cells
CM, EM, and TDEM phenotypes were assessed on HIV-1-specific IFN-γ-producing T-cell subsets. Virtually all of the memory cells resided within either CM or EM populations. The highest percentage of TDEM cells was exhibited by the CD8 T-cell subset (median 4.5%). The other T-cell subsets had median TDEM percentages of less than 1% (data not shown). The RCM index for total HIV-specific IFN-γ+ cells among CD4, CD8, DP 4d, and DP 8d T-cell subsets are illustrated for all HIV-infected subjects in Figure 5A. Although DP 8d T cells had the highest RCM index (median 0.85), CD8 T cells had the lowest (0.08), and CD4 and DP 4d T cell subsets exhibited intermediate values (medians 0.35 and 0.36, respectively). These differences were highly significant statistically (P < 0.0001, across all groups).
The RCM indices of HIV-1-reactive CD4, DP 4d, and CD8 T cells were all reduced among subjects with chronic infection when compared with those with early infection (Fig. 5C). In contrast, DP 8d T cells retained a high relative fraction of CM cells in both early and chronically HIV-1-infected patients.
The expression of LAMP is associated with cytolytic effector activity and thus is considered a convenient flow cytometric surrogate for such activity.46 Accordingly, as shown in Figure 5B, we assessed the fraction of HIV-specific IFN-γ-producing cells that expressed LAMP among the various T-cell subsets. Although a small portion of CD4 cells expressed LAMP (median 22%), a very high fraction of CD8 T cells were LAMP+ (median 89%), and DP 4d and DP 8d T-cell subsets exhibited intermediate proportions of LAMP+ cells (medians 69% and 34%, respectively). These differences were highly significant (P < 0.0001, across all groups). LAMP expression was also identified on IFN-γ-IL-2-coproducing cells among both DP 4d and DP 8d subsets (data not shown), but low event numbers precluded further comparative analyses.
Comparison of LAMP expression on HIV-specific IFN-γ+ cells in early vs. chronic infection indicated that T cells had higher LAMP expression in chronically infected patients (P = 0.0002, across all groups, Fig. 5D) and the 2 DP T-cell subsets show the greatest relative difference in LAMP expression based on disease stage. The increase in the fraction of LAMP-expressing cells within the DP 8d T-cell subset without a corresponding change in the fraction of CM cells in chronic patients (Fig. 5C) implies LAMP activity within the CM subset. To assess this directly, we regated all T-cell subsets and compared LAMP expression within each memory subpopulation. Differences in LAMP activity between early and chronically infected patients were apparent within each memory subset (data not shown). These observations indicate that observed increases in LAMP activity in chronic relative to early infected patients depicted in Figure 5D is not related solely to changes in the frequencies of subpopulations of CM and EM cells.
Functional Characteristics of SEB-Reactive DP T Cells
To determine whether these functional properties of T cells were unique to HIV-1-specific responses or were representative of polyclonally stimulated T cells, we performed a similar analysis with PBMCs from early and chronically infected patients which were activated with the polyclonal stimulus, SEB. Among all HIV-infected patients, the percentages of CD4, DP 8d, DP 4d, and CD8 T cells producing IFN-γ and/or IL-2 in response to SEB were 3.6%, 4.1%, 8.0%, and 5.6%, respectively. Unlike the HIV-1-specific response, which was dominated by IFN-γ-synthesizing cells, the response to SEB included both IFN-γ and significant IL-2 production. The fraction of cells producing IFN-γ among total cytokine (IFN-γ and/or IL-2)-producing cells is illustrated in Figure 6A. Among total cytokine-producing CD4 T cells, a lower fraction produced IFN-γ (median 27%), whereas among DP 8d and 4d subsets, a median of 41% and 82%, respectively, produced IFN-γ. The highest fraction of IFN-γ production was seen among CD8 T cells (median 95%).
Figure 6B examines the index of RCM among total IFN-γ-producing cells in response to SEB. As with the HIV-1-reactive cells (Fig. 5A), the median RCM of DP 8d T cells (0.42) is highest, with CD8 T cells having the lowest (0.09) and CD4 and DP 4d T cells with intermediate values (0.25 and 0.32, respectively). Finally, Figure 6C depicts the percentage of SEB-reactive IFN-γ-producing cells expressing LAMP as follows: for CD4 (median 23%), DP 8d (median 42%), DP 4d (median 78%), and CD8 (median 83%) T cells. These results indicate that SEB-reactive and HIV-reactive DP 8d and DP 4d T cells have a higher relative fraction of CM than CD4 and CD8 T cells, respectively, and generally have levels of LAMP intermediate to those of CD4 and CD8 T cells.
For a given T-cell subset, neither the IFN-γ+ fraction among SEB-reactive cytokine-producing cells nor the relative CM among SEB-stimulated IFN-γ-producing cells differed significantly between subjects with early vs. chronic HIV-1 infection (data not shown). Like that of HIV-1 peptide stimulation, SEB stimulation elicited LAMP expression, which was higher in chronically infected than in early infected patients. Although this pattern was observed for each of the 4 T-cell subsets, it reached statistical significance only for the DP 8d subset (P = 0.003, early vs. chronic).
Associations Between HIV-1-Specific DP T Cells, Plasma Viral Load, and Peripheral CD4 Count
Among all HIV-1-infected subjects, the frequency of HIV-1-specific IFN-γ-producing cells was not significantly correlated with plasma viral load for either DP 4d (Spearman r = 0.03, P = 0.82) or DP 8d T cells (r = 0.09, P = 0.54). Similarly, no significant correlation was found between these DP T-cell frequencies and patient CD4 counts for either DP 4d (r = −0.11, P = 0.48) or DP 8d (r = −0.07, P = 0.64) subsets. No significant associations between DP T-cell frequencies and clinical parameters were revealed upon independent evaluation of patients with early or chronic infection. Examination of the 6 “DP High” subjects with expanded levels of DP 8d cells revealed no difference in viral load (P = 0.38) or CD4 count (P = 0.52) when compared with the 21 “DP low” patients with early disease. Neither DP subset exhibited significant correlations between the HIV-1-specific RCM among IFN-γ-producing cells and plasma viral load (P = 0.62 and P = 0.45 for DP 4d and DP 8d, respectively) nor CD4 count (P = 0.16 and P = 0.32 for DP 4d and DP 8d, respectively). Finally, a modest association was observed between the fraction of IFN-γ+ DP 8d T cells expressing LAMP and plasma viral load (r = 0.31, P = 0.04), whereas no such association was seen for the DP 4d T-cell subset.
DP T cells are a poorly characterized cell population that represent a minor subset of peripheral blood T cells but are found at higher levels in certain tissues. Studies suggest that DP T cells may have a memory phenotype and produce cytokines in response to viral antigens.41 In addition, evidence suggests that DP T cells may be infected with HIV-1 in vitro and in vivo.10,32-36 To better understand the phenotype and function of blood DP T cells and their role in HIV-1 pathogenesis, we undertook a comprehensive analysis of DP and SP T cells in 60 subjects, including subjects with early and chronic HIV-1 infection and healthy, uninfected, control subjects. This analysis entailed evaluating functional properties of unstimulated DP T cells and those stimulated with either the polyclonal SEB stimulus or pooled HIV-1 peptides. The most notable findings of this study are that (1) HIV-1-reactive cells could be identified within 2 previously defined subpopulations of DP T cells based on varying densities of CD4 and CD8 expression, (2) these 2 subsets of DP T cells displayed functional properties of both conventional CD4 and CD8 SP T cells and hence could be considered polyfunctional, (3) no statistically significant differences were observed in the frequencies of unstimulated DP T cells across different clinical groups, and (4) HIV-1-specific IFN-γ-producing DP T cells in chronically infected patients were present at higher frequencies than in early infection patients, and a greater fraction expressed EM cell and LAMP markers.
A previous study demonstrated the presence of HIV-1-specific DP T cells in chronically HIV-infected subjects receiving a therapeutic HIV-1 vaccine, most receiving antiretroviral therapy,5 but here we detect HIV-1-specific DP 8d and DP 4d T cells in treatment NV subjects in both early and chronic stages of infection. A salient feature of the DP T-cell subsets in this study is their apparent polyfunctionality in that these cells expressed functions of each of the classically defined SP CD4 and CD8 T-cell subsets. Thus a portion of the DP T cells expressed both IFN-γ and IL-2, and cytokine coproduction was significantly more frequent among gated DP T cells than among gated CD4 and CD8 SP T cells. Moreover, analysis of LAMP expression suggested that DP T cells had substantially more LAMP activity than did CD4 SP T cells. Polyfunctionality among total CD8 T cells has previously been shown to be correlated with reduction in viral load and nonprogressive disease41 and hence has been proposed to represent a possible functional correlate of immune-mediated protection. In that most studies do not delineate CD8 SP T cells from CD8-expressing DP T cells, it is possible that DP T cells may have contributed in part to the correlations observed. The finding that DP T cells display a unique functional profile argues that independent assessment of DP T cells are thus important in future studies of HIV-1 pathogenesis.
Analysis of the 2 major subpopulations of DP T cells indicated that these 2 subsets themselves are distinct from each other, with the DP 4d T-cell subset expressing less LAMP than CD8 T cells but more than DP 8d T cells, and the latter DP T cell subpopulation expressing more SEB-induced IL-2 than either DP 4d or CD8 T cells but less than conventional CD4 T cells. The DP 8d and DP 4d T-cell subsets could also be distinguished from each other and from CD4 and CD8 T cells by their expression of memory markers, with DP 4d T cells expressing a lower fraction of CM cells than DP 8d T cells but higher than CD8 T cells and DP 8d T cells expressing higher levels of CM than CD4 T cells upon antigen stimulation. Differences in the profile of memory markers were also seen among each of the 4 subsets among unstimulated cells as well. These observations render extremely unlikely the possibility that DP T cells represent a collection of SP CD4 and CD8 T cells that were incorrectly gated. Moreover, summary analysis of all 4 subsets (Table 1) reveals a clear hierarchical pattern suggesting a functional continuum between CD8 cells at one extreme and CD4 T cells at the other, with DP 4d T cells more closely related to CD8 T cells and DP 8d T cells more closely related to CD4 T cells. A number of previous studies both in vitro and in vivo have obtained data consistent with the possibility that DP 4d T cells arise peripherally from CD8 T cells and DP 8d T cells from CD4 T cells in response to foreign antigen stimulation,8-11 and our data are consistent with this possibility. In addition, although DP cells have been most commonly delineated into 4d and 8d subsets, it is clear that other subpopulations can be resolved by varying densities of CD4, CD8, and other markers.2,31 In addition, by gating on small-sized cells to avoid analysis of artifactual doublets of SP CD4 and CD8 T cells, it is possible that we excluded some larger sized CD4+CD8+ cells with properties distinct from the cells we have described here. Hence, it will be important to define whether there exist phenotypic and ontogenic relationships between these other DP subsets and the 4 T-cell subsets defined in this study.
Given the expression of CD4 on all DP T cells and the reported susceptibility to HIV-1 infection of DP 4d and DP 8d T-cell subsets,10,32-37 we might have expected to see a substantial decrease in either or both DP T cell subsets comparable to that of CD4 T cells among chronically infected patients. However, a notable finding in this study was the lack of significant change in median frequency of unstimulated DP T cells across different clinical groups. This contrasted sharply with the decrease in the median frequencies of unstimulated CD4 T cells and increase in unstimulated CD8 T cells in chronically infected relative to early HIV-1-infected patients or to HIV-1-seronegative controls. We did note an increase in the range of frequencies of both total DP T cells and of the DP 8d T-cell subset among patients with early HIV-1 infection, consistent with an expansion of DP 8d T cells in a subset of individuals. However, the antigen specificity of the DP cells in such early infection subjects was unclear as they were not enriched for cells with reactivity to the HIV-1 peptide pools used in this study. Our findings add to a spectrum of observations about DP T cells in HIV-1 infection reported by other investigators, including infrequent increases in unstimulated DP 4d T cells,8 expansions of DP 8d T cells in a chronically infected patient,40 and modest reductions in unstimulated DP T cells in chronically infected patients with low CD4 counts relative to healthy controls.47 These variable results in the setting of HIV-1 infection may in part reflect the complexity of cellular dynamics. In that steady state levels of any cell population represent the summed effects of rates of generation, elimination and redistribution to different tissue compartments, changes in 1 or more of these parameters can lead to diverse steady state outcomes. For example, in chronically infected patients, accelerated rates of DP T-cell generation may match HIV-1-induced losses of activated DP T cells. However, because none of the aforementioned rates can be independently assessed by current approaches, such hypotheses remain speculative. Alternatively, it is possible that the apparent constancy in steady state levels of total DP in early and chronically infected patients is obscured by microheterogeneity of this subset beyond that defined in this study.2,31 Further definition of DP by other markers may reveal subsets whose levels change in opposing fashions as disease progresses.
We did observe significant differences in the frequency and functional characteristics of HIV-1 peptide-stimulated DP T cells between patients with early and chronic HIV-1 infection. A modest increase in the frequency of IFN-γ-producing HIV-1-specific DP cells was seen in chronically infected subjects relative to those with early infection, and this increase was more apparent in the DP 4d T-cell subset than in the DP 8d subset. Moreover, the DP 4d IFN-γ-producing cells shifted toward greater EM relative to CM among chronically infected compared with early infected subjects. Because no significant differences in memory phenotype were seen among unstimulated cells, or among SEB-reactive cells, this likely indicates an HIV-1-specific in vivo effect. In parallel, increased frequencies of EM cells were also observed for HIV-1-specific CD8 SP T cells in chronically infected subjects, a finding reinforcing the proposed ontogenic relationship between DP 4d and CD8 cells. Although the details of T-cell memory generation and maturation remain to be clarified, one paradigm suggests that maturation is strongly influenced by the overall strength of antigen stimuli, with increasing levels driving cells toward greater effector rather than CM.48 We and others have previously observed relative increases in EM frequencies of HIV-1-specific CD4 T cells in viremic patients vs. those on antiretroviral therapy with virologic suppression.49-53 Moreover, HIV-1-specific CD8 T cells have been shown to be enriched in EM compared with CD8 T cells specific for other viruses in HIV-1-infected patients.49-53 It is thus reasonable to propose that chronically infected patients experience a greater cumulative HIV-1 burden and hence greater antigen load than patients at earlier stages of infection and that this contributes to the emergence over time of greater EM among CD4 T cells, CD8 T cells, and DP 4d T-cell subsets.
In striking contrast to these observations, the HIV-1-specific DP 8d T-cell subset exhibited substantial skewing toward CM in both early and chronically infected patients. This enrichment in CM cells was less apparent in unstimulated or SEB-stimulated DP 8d T cells. It is tempting to speculate that this finding may reflect an HIV-1-mediated block in vivo in the normal maturational sequence involving these DP T cells, and that, rather than transitioning to EM cells, DP 8d T cells arrest or accumulate at the CM stage. Alternatively, this observation may simply reflect different thresholds of activation between DP 8d central and EM T cells such that the former are more readily activated under current in vitro assay conditions. Further studies are required to distinguish between these and other mechanisms.
We also observed increases in LAMP expression in HIV-specific IFN-γ-producing T cells among chronic compared with early infection patients. Increased LAMP expression in chronic infection was also seen among SEB-reactive cells, implying that, although related to chronic HIV-1 infection, it is not unique to HIV-1-specific cells. This pattern was apparent for all T-cell subsets but seemed to be most pronounced within the CM-predominant DP 8d T-cell subset. These observations show that mechanisms involved in generation of LAMP expression are not strictly linked to those which regulate expression of surface-homing markers (eg, CCR7) associated with EM and that such mechanisms apparently can be dissociated in chronically infected patients. Although the in vivo mechanisms of induction and regulation of cytolytic activity and homing receptors are not well defined in humans, it is known that abnormalities in lymphoid tissue occur in chronic HIV-1 infection, ultimately leading to substantial disruption of the lymphoid architecture in patients with advanced HIV-1 disease.54 Hence, it is conceivable that alternative or aberrant induction mechanisms may emerge in chronic disease.
A caveat in polychromatic flow cytometry analysis is that because so many subpopulations can be resolved, tests of statistical significance theoretically require adjustment for the number of comparisons made. This adjustment in effect lowers the P value needed to assign statistical significance to a given association. On one hand, this reduces the risk of ascribing importance to chance associations but on the other increases the likelihood of missing nonrandom associations of scientific relevance. In the present study, we elected not to make such adjustments for the latter reason and as well because nonbiased estimates of the true number of groups are difficult given the exploratory nature of this research. The finding with conventional pairwise nonparametric analysis of a large number of statistically significant differences both between DP and SP, and between DP from patients at early and chronic stages of infections, argue strongly that at least some of the observed associations are unlikely to be due to chance alone. However, it is clear that observations in this study will require confirmation in follow-up studies.
In summary, we show that HIV-1-specific polyfunctional DP 4d and DP 8d T-cell subsets can be identified in patients with early and chronic HIV-1 infection. The functional properties of DP T cells suggest a close ontogenic relationship with conventional CD8 and CD4 T cells and suggest that DP T cells arise as a consequence of in vivo antigen stimulation of SP T cells. Importantly, we find that the steady state levels of total DP T cells do not appreciably change during the disease course. We speculate that the increased generation of DP T cells as the disease progresses may generally be offset by HIV-1-mediated DP T cell losses. Detailed phenotypic and functional analysis of HIV-1-specific DP T cells, however, did reveal differences between early and chronic infection, and we suggest that these may reflect different mechanisms of induction of these cells and the effects of cumulative antigen doses at different stages of disease.
We thank the physicians, staff, and patients in the Infectious Diseases Group Practice Clinic at the University of Colorado Hospital, Denver Health Medical Center, and Denver Public Health for their assistance and participation in our study. We are grateful for the assistance of Dr Bharat Rawal of the Blood Centers of the Pacific for facilitating performance of the detuned assays. We thank the Colorado Center for AIDS Research Immunology Core for assistance with flow cytometry. Finally, we would like to acknowledge Cathi Basler, Beverly Putnam, and Dave Shugarts for their assistance with the study.
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