Immune correlates of protection for HIV-1 infection are poorly defined, presenting a significant challenge to the development of an effective vaccine.1 The difficulty in defining these correlates relates to the fact that, without antiretroviral therapy (ART), more than 99% of HIV-1-infected individuals develop AIDS, as they lack an antiviral response that affords long-term protection from disease progression.2,3 Failure of the HIV-1-specific immune response is attributed to on-going depletion of CD4 T-cell populations,4,5 an established HIV-1 reservoir in latently infected cells,6,7 and virus escape from the host response.8-10 However, evidence of durable control exists among individuals identified as long-term nonprogressors or elite controllers (EC), who maintain low plasma viral loads (pVL) for many years without the use of ART.11-13 Hence, understanding the mechanisms that underlie delayed disease progression amongst controllers will aid in the identification of correlates of protection and design of a therapeutic HIV-1 vaccine.
The critical role CD8 T cells play in controlling virus replication has been documented in several studies of HIV and simian immunodeficiency virus (SIV) infection.14-19 Furthermore, the strong association between certain major histocompatibility complex class I molecules and delayed disease progression suggests CD8 T cells contribute to viral control.20 CD8 T cells that are polyfunctional21 and target multiple Gag epitopes22-25 correlate with improved disease outcome, suggesting that the quality of response is an important determinant of their ability to restrict HIV-1 replication.
The Step Study, the first trial to evaluate a T-cell-based HIV-1 vaccine, was closed when interim analysis revealed a possible enhanced risk of infection in vaccine recipients.26 Although the trivalent MRKAd5 construct used in this trial was highly immunogenic as analyzed by IFN-γ ELISPOT assays, this effector response did not equate with protection from infection or decreased viral load (VL) upon seroconversion.27,28 These results may be explained by a number of factors including poor induction of CD8 T cells and a limited quality of response. The outcome of the Step Study underscores the need for improved methods to evaluate vaccine-induced T-cell responses.29
Several groups have developed quantitative assays to measure inhibition of HIV-1 and SIV replication.30-36 Some studies have shown an association between enhanced antiviral efficacy and Gag specificity.32,37 Recently, analysis of HLA-B*2705-restricted responses indicated that inhibition of HIV-1 replication was related to the kinetics of infected target cell recognition by epitope-specific CD8 T cells.38 Despite these reports, the CD8 T-cell phenotype associated with enhanced control of virus replication remains unclear. Based on previous reports,21,39 we hypothesized that IL-2 production would identify a population of HIV-1-specific CD8 T cells with enhanced suppressive capacity and this population would be increased amongst controllers. To address this question, we sought to quantitatively assess CD8 T-cell-mediated suppression of HIV-1 replication using a modified in vitro suppression assay (iVSA).
Samples were collected from HIV-1-infected controllers (n = 4) and progressors (n = 5) enrolled at the University of Alabama at Birmingham 1917 Clinic. Progressors had a pVL greater than 2000 copies per milliliter, and controllers were defined as having a pVL less than 2000 copies per milliliter (see Table, Supplemental Digital Content 1,http://links.lww.com/QAI/A187). CD4 T-cell counts were determined as previously described.24 Plasma HIV-1 RNA levels were measured using the Amplicor Ultra Sensitive HIV-1 Monitor assay (Version 1.5; Roche Diagnostic Systems, Indianapolis, IN). All patients were off ART for at least 6 months at the evaluated time points. Genomic DNA extracted from peripheral blood mononuclear cells (PBMCs) was used for PCR-based genotyping of HLA class I alleles.40 Informed consent was obtained from all participants and the University of Alabama at Birmingham Institutional Review Board approved the study.
IFN-γ ELISPOT Assay
Participants were screened for HIV-1-specific responses based on IFN-γ production. The ELISPOT assay was performed as previously described.41 Peptides were selected based on optimized CD8 epitopes predicted by each subject's HLA class I genotype and described in the LANL HIV Molecular Immunology Database (www.hiv.lanl.gov/content/immunology/index.html). A positive response was defined as values twice background (unstimulated cells) and greater than 55 SFC/106 PBMCs.
Expansion of CD8 T-Cell Lines
To generate antigen-specific CD8 T-cell lines, immunodominant responses detected by ELISPOT were expanded in vitro. PBMCs were resuspended in serum-free media and plated at 1.2 × 106 cells per well. Nonadherent cells were removed after 2-hour incubation at 37°C, 5% CO2. Adherent cells, previously shown to be monocytes,42,43 were irradiated (33 gray), pulsed with HIV-1 peptide (10 μg/mL) for 2 hours and washed to remove excess peptide. Autologous CD8 T cells were negatively isolated using the MACS CD8+ T cell isolation kit (Miltenyi Biotec, Gladbach, Germany), resuspended in complete media supplemented with IL-7 (25 ng/mL), and plated onto the peptide-pulsed monocytes at 0.5 × 106 cells per well. On day 7, CD8 T cells were restimulated with peptide-pulsed monocytes. Expanded CD8 T-cell lines were tested for HIV-1-specific function on day 14 or 15.
Intracellular Cytokine Staining Assay
Intracellular cytokine staining (ICS) was performed as previously described.44 Approximately 0.5-1 × 106 cells were incubated with appropriate HIV-1 peptide (10 μg/mL) for 6 hours in the presence of anti-CD107a. Cells were labeled with LIVE/DEAD fluorescent reactive dye (Invitrogen, Carlsbad, CA) and stained with antibodies against CD3, CD4, and CD8. Cells were then fixed, permeabilized (FIX & PERM, Invitrogen, Carlsbad, CA), and stained with antibodies for intracellular markers IFN-γ, TNF-α, IL-2, and perforin. A minimum of 100,000 events were acquired on an LSRII (BD Immunocytometry Systems, San Jose, CA) and data analyzed using FlowJo software (v7.6.1, TreeStar, Ashland, OR). Responses greater than 0.02% and twice the background response (negative control) were considered positive. Boolean gating was used to generate polyfunctional subsets. Analysis and presentation of distributions was performed using SPICE version 5.1, downloaded from <http://exon.niaid.nih.gov/spice>.45
CD3-Pacific blue, CD8-PerCP-Cy5.5, CD107a-FITC, IFN-γ-Alexa 700, IL-2-APC, TNF-α-PE-Cy7 or PerCP-Cy5.5 (all from BD Biosciences, San Diego, CA), CD4-Qdot 605, CD8-Qdot 655 (both from Invitrogen, Carlsbad, CA), anti-perforin-PE (Cell Sciences, Canton, MA).
In Vitro Suppression Assay
HIV-1-specific CD8 T-cell lines were used as effector (E) cells in the iVSA. To generate target cells (T), PBMCs were depleted of CD8+ cells using CD8 Dynabeads (Invitrogen, Carlsbad, CA). Enriched CD4+ cells were activated with IL-2 (50 U/mL) and phytohemagglutinin (2 μg/mL) for 72 hours. Cell infection with HIV-1NL4-3 was optimized at a multiplicity of infection of 0.001, minimizing virus-induced cytotoxicity. Effector cells were co-cultured with autologous and non-autologous targets at multiple E:T ratios (range 0:1 to 5:1) in a 96-well plate for 7 days. Supernatant was collected on days 0, 1, 3, 5, 7 and stored at −80°C until analysis. The TZM-bl reporter cell line was used for analysis of HIV-1 replication, in which luciferase expression is highly sensitive and linearly related to the quantity of infectious HIV-1.46 Luciferase was quantified in relative luciferase units using an automated luminometer (Microplate Luminometer, Applied Biosystems, Foster City, CA). All cell lines tested in the iVSA were run in duplicate. CD8 T-cell-mediated suppression was quantified as percent suppression on the day of maximum HIV-1 replication.
Percent suppression = [1 - (relative luciferase units of sample with E)/(relative luciferase units of sample without E)] × 100.
Clinical markers and T-cell functional responses between progressors and controllers were compared using the nonparametric Mann-Whitney test. Spearman rank correlation coefficient was calculated to analyze the relationship between suppression (E:T, 1:1) and functional/clinical parameters. Statistical analyses were performed with Prism software (GraphPad Software, La Jolla, CA). Comparison of distributions generated with SPICE was performed using a partial permutation test as described.45
Identification and Expansion of Immunodominant HIV-1-Specific Responses
To determine if there is a signature phenotype for CD8 T cells capable of virus suppression, we analyzed effector function in controllers with superior HIV-1 control, off ART, compared with individuals with progressive disease. Immunodominant CD8 T-cell responses are major determinants of CTL escape and such responses could be important mediators of virus suppression.47,48 We, therefore, identified immunodominant responses in each subject using the IFN-γ ELISPOT assay. IFN-γ production by virus-specific CD8 T cells is often the last function detected before cells are fully exhausted, making it a reliable marker to identify HIV-1-specific responses during chronic infection.49,50 A positive HIV-1 response to at least one 9-11mer was detected in all individuals (range, 1-13 positive responses), with the magnitude ranging from 55 to 1438 SFC/106 PBMC in progressors and 55 to 4083 SFC/106 PBMCs in controllers (see Figure, Supplemental Digital Content 2,http://links.lww.com/QAI/A188). Despite a significant difference in burden of disease as evidenced by pVL (P = 0.02), there was no difference in the magnitude of response between progressors and controllers. Interestingly, the lowest ELISPOT responses were detected in an EC, C1655, who has maintained undetectable pVL for more than 20 years, highlighting the limited utility of ELISPOT measurements as a correlate of an efficacious HIV-1-specific response.
Due to low frequencies of HIV-1-specific CD8 T cells in controllers such as C1655,51 the potentially important information gleaned from ex vivo functional analysis of these cells is infrequently attempted. We, therefore, used a 14-day stimulation protocol to expand epitope-specific CD8 T cells from cryopreserved PBMCs. Immunodominant responses detected by ELISPOT (see open circles, Figure, Supplemental Digital Content 2, http://links.lww.com/QAI/A188) were selected for expansion. Of 15 CD8 T-cell lines, 9 targeted epitopes restricted by HLA class I alleles associated with delayed disease progression (Table 1). Consistent with the dominance of Gag targeting amongst controllers,24,52 all lines obtained from this group were specific to epitopes within p24.
Phenotypic Analysis of HIV-1-Specific CD8 T Cells
Although expansion of epitope-specific CD8 T cells is frequently performed, few studies have analyzed functional changes that occur after in vitro expansion. To establish the utility in analyzing expanded HIV-1-specific CD8 T cells, we used ICS to measure multiple effector functions and compared responses both ex vivo (Fig. 1A) and in vitro (Fig. 1B). We did not detect a significant difference between the median ex vivo or in vitro responses in progressors compared with that of controllers for any of the functions analyzed. As expected, in vitro expansion increased the frequency of antigen-specific CD8 T cells (CD107a, IFN-γ, TNF-α, perforin, Fig. 1C). In fact, in some ECs, this was the only method of detecting HIV-1-specific effector function. Interestingly, enhanced perforin mobilization was detected after expansion in progressors and controllers despite the fact that the former had few perforin-producing cells when analyzed ex vivo. Although the frequency of epitope-specific responses increased after in vitro expansion, there was no preferential expansion of CD8 T cells within either group.
Polyfunctional responses are a hallmark of nonprogressive HIV-1 disease;21,53 we, therefore, analyzed these responses ex vivo and noted that controllers had an increased frequency of epitope-specific CD8 T cells with 3 or more functions compared with progressors (Fig. 2A). After in vitro expansion, however, these differences disappeared as cell lines derived from progressors were also polyfunctional (Fig. 2B). This improved function was particularly prominent with perforin-producing cells (orange arcs in Figs. 2A, 2B), especially those that also expressed IFN-γ and CD107a (Fig. 2D). There was no significant difference in the median frequency of IL-2+ cells between the 2 groups (Fig. 1C). IL-2+ CD8 T cells tended to be monofunctional in progressors (black arcs border gray slices, Figs. 2A, 2B), although this finding was not significant (P = 0.145). Amongst controllers, black arcs border the yellow, blue, and red pie slices, indicating IL-2 is being produced by cells capable of 3 or more functions. Figures 2C and 2D depict 31 unique combinations of CD8 T-cell responses detected ex vivo and after in vitro expansion. Analysis of these subsets revealed that polyfunctional CD8 T cells which produce IL-2 tended to be enriched in controllers after expansion (P = 0.066).
Epitope-Specific CD8 T-Cell Lines Derived From Controllers Demonstrate Increased HIV-1 Suppression
Despite similar cytokine profiles among the expanded CD8 T-cell lines, we investigated whether these cells varied in their ability to suppress HIV-1 replication. CD8 T-cell line P7, specific for B57-IW9, was derived from progressor P2824 and tested in the iVSA. Autologous, HIV-1-infected CD4 T cells cultured without effector cells reached maximal HIV-1 replication on day 3, a 2-log increase above day 0 (Fig. 3A). As CD8 T cells were added at increasing concentrations, we observed a slight diminution in HIV-1 replication as quantified by luciferase expression. At an E:T ratio of 1:1, cell line P7 demonstrated 54.5% suppression of virus replication. Analysis of CD8 T cells derived from controllers showed a significantly different pattern of suppression. As the cell line C6 (B57-TF11-specific; derived from controller C1655) was added at increasing concentrations, we observed a dose-dependent decrease in HIV-1 replication (Fig. 3C), with substantial suppression at the lowest concentration of effector cells (97.2%, 0.2:1) and complete virus suppression (99%) at a ratio of 1:1. When cell lines C6 and P7 were cultured with nonautologous, infected targets (Figs. 3B, 3D), there was no inhibition of virus replication, indicating an major histocompatibility complex class I-dependent mechanism of suppression.
Total analysis of HIV-1-specific CD8 T-cell lines revealed that controllers effectively suppress virus replication compared with progressors (Fig. 3E). The median percent suppression in progressors was 73.7% at an E:T ratio of 0.2:1, 76.0% at 0.5:1, compared with 92.9% and 98.8%, respectively, in controllers. When the total analysis was adjusted by removing outlier data points (P6, P7, P9), we were still able to detect significant differences in suppressive capacity, with controllers exhibiting enhanced virus suppression (0.2:1, P = 0.017; 0.5:1, P = 0.004, P = 0.0087). At the highest effector cell concentration (E:T, 5:1), the difference in suppression of autologous and nonautologous targets was no longer distinct, suggesting nonspecific targeting of infected cells (Figs. 3B, 3D).
Polyfunctional CD8 T Cells That Maintain IL-2 Production are Associated With HIV-1-Suppressive Capacity
We sought to identify a more readily measured correlate of antiviral function. No single marker, when analyzed ex vivo or after in vitro expansion, was associated with enhanced virus suppression (Table 2). This was true even for perforin, regardless of its production with other effector molecules. Polyfunctionality, without an IL-2 response, did not correlate with CD8 T-cell-mediated suppression of HIV-1 replication. Rather, CD8 T-cell lines that were polyfunctional and positive for IL-2 production, as measured after expansion, were associated with enhanced in vitro virus suppression (r = 0.56; P = 0.03, Table 2). The same function measured ex vivo tended to correlate with suppression, although it did not reach significance (r = 0.51; P = 0.09).
The qualitative features of virus-specific CD8 T cells that contribute to protection from HIV-1 disease have not been clearly defined. In this study, we quantified the ability of epitope-specific CD8 T cells to suppress HIV-1 replication, which may represent a more direct marker of antiviral effector function.29,54 When tested in the iVSA, CD8 T-cell lines derived from HIV-1-infected controllers showed significantly increased suppressive capacity compared with those from progressors. CD8 T cells were tested against autologous CD4 T cells, revealing a dose-dependent decrease in virus replication as effectors were added at increasing concentrations. As others have shown, our data provides further evidence that controllers maintain CD8 T-cell populations with potent antiviral function. At high E:T ratios, virus suppression between the 2 groups was indistinguishable. As previously observed, this decreased sensitivity at high E:T ratios is likely due to nonspecific targeting when CD8 T cells are in excess.32,36,38 Furthermore, increased suppressive capacity may reflect the highly avid CD8 T-cell responses often detected in those with superior viral control.55 Thus, progressors may require more CD8 T cells to attain similar protection due to low avidity populations. Enhanced suppressive capacity is an identifiable marker of an effective HIV-1-specific CD8 T-cell response and is one plausible mechanism by which controllers delay disease progression.
For this study, expanded CD8 T-cell lines underwent 2 rounds of in vitro stimulation with peptide-pulsed autologous monocytes. This protocol provides a method of expanding populations of low-frequency antigen-specific cells, thereby permitting evaluation of CD8 T cells that would otherwise not be analyzed ex vivo, but may play an important role in the host immune response. This is particularly important in view of the fact that many HIV-1-infected individuals with excellent viral control off ART have low to undetectable frequencies of HIV-specific CD8 T cells.51 A potential caveat of this protocol is that we may have enriched for epitope-specific CD8 T cells with increased survival capacity.
Although several groups have demonstrated that HIV-1-specific CD8 T cells obtained from controllers are more efficient at viral suppression, a phenotypic profile of the CD8 T-cell capable of enhanced HIV-1 suppression has yet to be well defined.30-32,38 Results from a recent study suggested that antiviral function is dependent on antigen specificity, having observed increased HIV-1 inhibition by Gag-specific CD8 T cells compared with Env-specific cells.32 Our study did not address Env-specific CD8 T cells; however, Gag specificity was not a universal predictor of HIV-1 suppressive capacity. In fact, Gag-specific CD8 T cells derived from progressors lacked suppressive function. Although suppressive capacity may depend upon which epitopes are targeted, a larger sample size will be needed to address this question.
Production of soluble effector molecules may be an important predictor of antiviral efficacy as polyfunctional HIV-1-specific CD8 T cells are more frequently observed among controllers.21 Comparison of ex vivo CD8 T-cell responses between progressors and controllers did not reveal a significant difference in production or mobilization of any individual function (CD107a, IFN-γ, TNF-α, IL-2, perforin). This was also true for responses detected after in vitro expansion in both groups. As previously reported,38 we observed that expanded HIV-1-specific CD8 T cells had an enhanced functional phenotype when compared with ex vivo responses, with increased production of CD107a, IFN-γ, TNF-α, and perforin. Polyfunctional, IL-2-producing CD8 T cells were present in controllers after expansion, in contrast to progressors in whom IL-2 producing CD8 T cells were predominantly monofunctional. CD8 T-cell lines able to produce IL-2 in combination with at least 2 other effector molecules exhibited increased virus suppression (r = 0.56; P = 0.03). The same function, measured ex vivo, trended toward an association with virus suppression, which may be related to the relatively small sample size and limited detection of IL-2-producing CD8 T cells.
Earlier studies have associated IL-2 production by HIV-1-specific CD8 T cells with viral control.39,56 More recently, polyfunctional CD8 T-cell responses were associated with increased suppressive capacity37; however, it remains unclear if IL-2 production, polyfunctionality, or both are involved in viral control or if these phenotypes are the result of controlled virus replication. Although our association of virus suppression with polyfunctional IL-2 production likely represents a means to identify CD8 T cells that have matured in the context of viral control, it may indicate that IL-2 is involved in this control. One potential mechanism is that IL-2-producing CD8 T cells have better proliferative capacity,57,58 and these may transition to an effector phenotype thus providing a stable population of cells able to maintain control of HIV-1 replication in vivo.
Other groups have been able to correlate degranulation and mobilization of perforin and granzymes with enhanced suppression in vitro.34 In contrast, we found that expanded CD8 T cells obtained from progressors have no difficulty increasing perforin production after cell expansion. These observed differences might be due, in part, to the fact that previous studies used a monoclonal antibody recognizing pre-formed perforin (clone δG9), but not newly synthesized perforin. We utilized a perforin antibody (clone D48), able to detect de novo synthesis of the protein.59 Incomplete staining with clone δG9 antibody would make it difficult to completely quantify upregulation of perforin after stimulation. Furthermore, our method of expansion, using autologous monocytes compared with bulk PBMCs for antigen presentation is significantly different from that of other groups, and it has been demonstrated that the type of antigen presenting cell used for stimulation can impact functional development of effector CD8 T cells.60,61
Studies in SIV-infected rhesus macaques have yielded varying results, unable to clearly define the CD8 T-cell phenotype capable of restricting virus replication. Suppression of SIV replication by CD8 T cells from infected macaques in earlier studies showed associations with host level of viral control33 and CD8 T-cell epitope specificity,62 although a more recent study could not identify a correlate of SIV suppression despite measuring a number of immune parameters.63 However, vaccine-induced responses in rhesus macaques immunized with a DNA prime/Ad5 boost vaccine demonstrated that CD8 T cells were able to suppress SIV replication in vitro, which trended toward both a lower peak VL during acute infection and a lower VL setpoint.64 These inconsistencies may relate to differences in which effector cells were derived. Yet, we report, as others have consistently observed, that HIV-1-specific CD8 T cells from controllers have an enhanced ability to inhibit virus replication when compared with CD8 T cells from patients who lack control, despite varying methods used to isolate effector cells.30-32,65
CD8 T-cell recognition and elimination of infected cells is a critical component of the host immune response to viral infection. However, this effector function is not routinely analyzed in preclinical studies of candidate HIV-1 vaccines. We demonstrate that increased in vitro suppression of HIV-1 replication is a hallmark of nonprogressive disease. This enhanced suppression was associated with a polyfunctional IL-2+ CD8 T-cell response, which may have implications for analysis of vaccine-induced responses. Although these studies were completed in the context of chronic HIV-1 infection, and may not translate to protection mediated by vaccine-induced responses, our data suggest that quantification of additional effector functions, especially virus suppression, should be incorporated to evaluate HIV-1 vaccines in a comprehensive manner.
We would like to thank staff and patients of the University of Alabama at Birmingham 1917 and Alabama Vaccine Research Clinics. We thank Dr June Kan-Mitchell for helpful discussions regarding CD8 T-cell expansion. We are grateful to Marion Spell for assistance with flow cytometric acquisition and Juliette Easlick for technical assistance with the iVSA.
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