Autocrine Production of β-Chemokines Protects CMV-Specific CD4+ T Cells from HIV Infection

Induction of a functional subset of HIV-specific CD4⁺ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4⁺ T cells, which are less frequently infected than HIV-specific CD4⁺ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4⁺ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4⁺ T cells rapidly up-regulated production of MIP-1α and MIP-1β mRNA, resulting in a rapid increase in production of MIP-1α and MIP-1β after cognate antigen stimulation. Production of β-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4⁺ T cells. To test whether production of β-chemokines by CD4⁺ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4⁺ T cells. We found that CMV-specific CD4⁺ T cells which produced MIP-1β contained 10 times less Gag DNA than did those which failed to produce MIP-1β. These data suggest that CD4⁺ T cells which produce MIP-1α and MIP-1β bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.     

Transient Nature of Long-Term Nonprogression and Broad Virus-Specific Proliferative T-Cell Responses with Sustained Thymic Output in HIV-1 Controllers

Background

HIV-1⁺ individuals who, without therapy, conserve cellular anti-HIV-1 responses, present with high, stable CD4⁺ T-cell numbers, and control viral replication, facilitate analysis of atypical viro-immunopathology. In the absence of universal definition, immune function in such HIV controllers remains an indication of non-progression.

Methodology/Principal Findings

CD4 T-cell responses to a number of HIV-1 proteins and peptide pools were assessed by IFN-γ ELISpot and lymphoproliferative assays in HIV controllers and chronic progressors. Thymic output was assessed by sjTRECs levels. Follow-up of 41 HIV-1⁺ individuals originally identified as “Long-term non-progressors” in 1996 according to clinical criteria, and longitudinal analysis of two HIV controllers over 22 years, was also performed. HIV controllers exhibited substantial IFN-γ producing and proliferative HIV-1-specific CD4 T-cell responses to both recombinant proteins and peptide pools of Tat, Rev, Nef, Gag and Env, demonstrating functional processing and presentation. Conversely, HIV-specific T-cell responses were limited to IFN-γ production in chronic progressors. Additionally, thymic output was approximately 19 fold higher in HIV controllers than in age-matched chronic progressors. Follow-up of 41 HIV-1⁺ patients identified as LTNP in 1996 revealed the transitory characteristics of this status. IFN-γ production and proliferative T-cell function also declines in 2 HIV controllers over 22 years.

Conclusions

Although increased thymic output and anti-HIV-1 T-cell responses are observed in HIV controllers compared to chronic progressors, the nature of nonprogressor/controller status appears to be transitory.     

Predominant Clonal Accumulation of CD8+ T Cells with Moderate Avidity in the Central Nervous Systems of Theiler's Virus-Infected C57BL/6 Mice

Induction of antigen-specific CD8⁺ T cells bearing a high-avidity T-cell receptor (TCR) is thought to be an important factor in antiviral and antitumor immune responses. However, the relationship between TCR diversity and functional avidity of epitope-specific CD8⁺ T cells accumulating in the central nervous system (CNS) during viral infection is unknown. Hence, analysis of T-cell diversity at the clonal level is important to understand the fate and function of virus-specific CD8⁺ T cells. In this study, we examined the Vβ diversity and avidity of CD8⁺ T cells specific to the predominant epitope (VP2_121-130) of Theiler''s murine encephalomyelitis virus. We found that Vβ6⁺ CD8⁺ T cells, associated with epitope specificity, predominantly expanded in the CNS during viral infection. Further investigations of antigen-specific Vβ6⁺ CD8⁺ T cells by CDR3 spectratyping and sequencing indicated that distinct T-cell clonotypes are preferentially increased in the CNS compared to the periphery. Among the epitope-specific Vβ6⁺ CD8⁺ T cells, MGX-Jβ1.1 motif-bearing cells, which could be found at a high precursor frequency in naïve mice, were expanded in the CNS and tightly associated with gamma interferon production. These T cells displayed moderate avidity for the cognate epitope rather than the high avidity normally observed in memory/effector T cells. Therefore, our findings provide new insights into the CD8⁺ T-cell repertoire during immune responses to viral infection in the CNS.Theiler''s murine encephalomyelitis virus (TMEV) is a member of the Cardiovirus genus within the Picornaviridae family (43). This virus is a common enteric pathogen among wild mice but rarely causes neurological disease (57). However, when it infects susceptible mice (e.g., the SJL/J [SJL] strain) intracerebrally, it reproducibly induces a chronic immune-mediated demyelinating disease that has been studied as an infectious model of human multiple sclerosis (MS) (10, 30). In contrast, infection of resistant mice like those of the C57BL/6 (B6) strain results in strong antiviral immune responses that clear the virus effectively and prevent disease development (24, 31). Therefore, immune responses in B6 mice have been often compared to those in susceptible SJL mice to understand the nature of protective versus pathogenic immunity in these mice.It has been shown that the major histocompatibility complex (MHC) H-2D locus is a critical genetic factor for resistance to TMEV-induced demyelinating disease (9, 49). For example, expression of the H-2D^b transgene makes susceptible FVB mice resistant by inducing strong H-2D^b-restricted VP2_121-130-specific CD8⁺ T-cell responses (36). This acquired resistance is abolished when VP2_121-130-specific T cells are tolerized by introducing the VP2 transgene (45). These results strongly suggest that CD8⁺ T cells generated in the presence of H-2D^b are critical for viral clearance from the central nervous system (CNS). Since the cardinal difference between the resistant B6 and susceptible SJL strains is the quantity, not the quality, of virus-specific CD8⁺ T cells (23, 32), strong CD8⁺ T-cell responses are probably required to prevent viral persistence and the consequent development of demyelinating disease. More than threefold more virus-specific CD8⁺ T cells were found in the CNSs of resistant B6 mice than in those of susceptible SJL mice at the acute phase of infection. Thus, the level of virus-specific CD8⁺ T cells at an early phase of the immune response may be a critical factor in resistance to the disease.Many recent investigations indicate that oligoclonal CD8⁺ T cells accumulate in the CNSs of MS patients (4, 38, 51). In addition, CD8⁺ T cells may also induce the development of experimental autoimmune encephalomyelitis (EAE) (54). Therefore, clonal expansion of certain CD8⁺ T cells may be associated with the pathogenesis of demyelinating diseases. However, B6 mice, which are resistant to TMEV-induced demyelinating disease, induce strong CD8⁺ T-cell responses to a single predominant epitope (VP2_121-130), i.e., ≥70% of CNS-infiltrating CD8⁺ T cells (41, 42). These CD8⁺ T cells result in effective viral clearance yet remain at a low level in the CNS more than 120 days postinfection (dpi) without detectable pathology (42). This inconsistency led us to investigate the shape and quality of the T-cell receptor (TCR) repertoire accumulating in the CNSs of B6 mice.The CD8⁺ T-cell responses induced after viral infection have previously been investigated with other animal viruses, including influenza virus, lymphocytic choriomeningitis virus (LCMV), mouse hepatitis virus (MHV), and Borna disease virus (11, 14, 35, 47, 58). Among these models, the detailed T-cell Vβ repertoire in the CNS was described only in the MHV model (46). CD8⁺ T-cell responses against TMEV in B6 mice are primarily against a single predominant epitope (22, 36, 41). However, virtually no study of the TCR Vβ repertoires of virus-specific CD8⁺ T cells has been reported. Furthermore, it is not yet known whether a particular TCR Vβ repertoire is associated with the avidity and/or function of CD8⁺ T cells in the CNS. Since protective versus pathogenic CD8⁺ T cells may correlate with their Vβ repertoire and T-cell function, these studies may help to elucidate the underlying mechanisms of protection versus pathogenesis of CD8⁺ T cells in the CNS.In this study, we have addressed several important questions about the CD8⁺ T-cell repertoire in the CNS. First, what is the pattern of Vβ usage in TMEV-infected B6 mice? Second, are there differences in the antigen-specific CD8⁺ T-cell clonotypes between the CNS and periphery? Third, are the T-cell clonotypes maintained in the CNS during the viral infection? Fourth, what is the functional avidity of T cells accumulating in the CNS during this virus infection? Last, what possible factors are associated with repertoire selection and expansion in the CNS? Our results show that Vβ6⁺ CD8⁺ T cells preferentially expand in the CNS during viral infection. Further analyses of the CDR3 region of antigen-specific Vβ6⁺ CD8⁺ T cells by spectratyping and sequencing indicate that distinct T-cell clonotypes are expanded in the CNS compared to those in the periphery. T cells expressing a particular Vβ6-CDR3-Jβ1.1 sequence are preferentially retained in the CNS during the course of viral infection. Interestingly, these T cells are capable of producing gamma interferon (IFN-γ) upon stimulation and display moderate avidity for the cognate epitope. We believe that our findings will provide important information regarding the CD8⁺ T-cell repertoire during viral infection and that these results may help to provide a better understanding of antiviral CD8⁺ T-cell immunity in the CNS.     

Preserved central memory and activated effector memory CD4+ T-cell subsets in human immunodeficiency virus controllers: an ANRS EP36 study

Human immunodeficiency virus (HIV) controllers are rare individuals who spontaneously control HIV type 1 replication for 10 years or more in the absence of antiretroviral treatment. In the present study, HIV controllers (n = 11) maintained potent HIV-specific CD4 responses in spite of very low antigenic loads. Their CD4⁺ central memory T (T_CM) cells were characterized by near-normal numbers and preserved interleukin-2 (IL-2) secretion in response to HIV antigens and uniformly high expression of the survival receptor IL-7 receptor α (IL-7Rα). Controllers expressed CCR7 at higher levels than uninfected controls, suggesting differences in T_CM-cell homing patterns. CD4⁺ effector memory T (T_EM)-cell responses were polyfunctional in HIV controllers, while IL-2 secretion was lost in viremic patients. Cytokine production was three times higher in controllers than in treated patients with undetectable viral loads, suggesting an intrinsically more efficient response in the former group. The total CD4⁺ T_EM-cell pool underwent immune activation in controllers, as indicated by increased HLA-DR expression, decreased IL-7Rα expression, a bias towards gamma interferon production upon polyclonal stimulation, and increased macrophage inflammatory protein 1β secretion associated with chronic CCR5 down-regulation. Thus, HIV controllers showed a preserved CD4⁺ T_CM-cell compartment and signs of potent functional activation in the CD4⁺ T_EM-cell compartment. While controllers did not show the generalized immune activation pattern associated with disease progression, they had signs of immune activation restricted to the effector compartment. These findings suggest the induction of an efficient, nondetrimental type of immune activation in patients who spontaneously control HIV.     

Gamma/Delta T-cell functional responses differ after pathogenic human immunodeficiency virus and nonpathogenic simian immunodeficiency virus infections

The objective of this study was to functionally assess gamma/delta (γδ) T cells following pathogenic human immunodeficiency virus (HIV) infection of humans and nonpathogenic simian immunodeficiency virus (SIV) infection of sooty mangabeys. γδ T cells were obtained from peripheral blood samples from patients and sooty mangabeys that exhibited either a CD4-healthy (>200 CD4⁺ T cells/μl blood) or CD4-low (<200 CD4 cells/μl blood) phenotype. Cytokine flow cytometry was utilized to assess production of Th1 cytokines tumor necrosis factor alpha and gamma interferon following ex vivo stimulation with either phorbol myristate acetate/ionomycin or the Vδ2 γδ T-cell receptor agonist isopentenyl pyrophosphate. Sooty mangabeys were observed to have higher percentages of γδ T cells in their peripheral blood than humans did. Following stimulation, γδ T cells from SIV-positive (SIV⁺) mangabeys maintained or increased their ability to express the Th1 cytokines regardless of CD4⁺ T-cell levels. In contrast, HIV-positive (HIV⁺) patients exhibited a decreased percentage of γδ T cells expressing Th1 cytokines following stimulation. This dysfunction is primarily within the Vδ2⁺ γδ T-cell subset which incurred both a decreased overall level in the blood and a reduced Th1 cytokine production. Patients treated with highly active antiretroviral therapy exhibited a partial restoration in their γδ T-cell Th1 cytokine response that was intermediate between the responses of the uninfected and HIV⁺ patients. The SIV⁺ sooty mangabey natural hosts, which do not proceed to clinical AIDS, provide evidence that γδ T-cell dysfunction occurs in HIV⁺ patients and may contribute to HIV disease progression.     

HIV Infection of Monocytes-Derived Dendritic Cells Inhibits Vγ9Vδ2 T Cells Functions

DCs act as sentinel cells against incoming pathogens and represent the most potent antigen presenting cells, having the unique capability to prime naïve T cells. In addition to their role in induction of adaptive immune responses, DC are also able to activate innate cells as γδ T cells; in particular, a reciprocal crosstalk between DC and γδ T cells was demonstrated. However, whether HIV infection may alter DC-Vγ9Vδ2 T cells cross-talk was not yet described. To clarify this issue, we cultured activated Vγ9Vδ2 T cells with HIV infected monocyte derived DC (MoDC). After 5 days we evaluated MoDC phenotype, and Vγ9Vδ2 T cells activation and proliferation. In our model, Vγ9Vδ2 T cells were not able to proliferate in response to HIV-infected MoDC, although an up-regulation of CD69 was observed. Upon phosphoantigens stimulation, Vγ9Vδ2 T cells proliferation and cytokine production were inhibited when cultured with HIV-infected MoDC in a cell-contact dependent way. Moreover, HIV-infected MoDC are not able to up-regulate CD86 molecules when cultured with activated Vγ9Vδ2 T cells, compared with uninfected MoDC. Further, activated Vγ9Vδ2 T cells are not able to induce HLA DR up-regulation and CCR5 down-regulation on HIV-infected MoDC. These data indicate that HIV-infected DC alter the capacity of Vγ9Vδ2 T cells to respond to their antigens, pointing out a new mechanisms of induction of Vγ9Vδ2 T cells anergy carried out by HIV, that could contribute to immune evasion.     

Immunodominant HIV-Specific CD8+ T-Cell Responses Are Common to Blood and Gastrointestinal Mucosa,and Gag-Specific Responses Dominate in Rectal Mucosa of HIV Controllers

Previous studies have suggested that polyfunctional mucosal CD8⁺ T-cell responses may be a correlate of protection in HIV controllers. Mucosal T-cell breadth and/or specificity may also contribute to defining protective responses. In this study, rectal CD8⁺ T-cell responses to HIV Gag, Env, and Nef were mapped at the peptide level in four subject groups: elite controllers (n = 16; viral load [VL], <75 copies/ml), viremic controllers (n = 14; VL, 75 to 2,000 copies/ml), noncontrollers (n = 14; VL, >10,000 copies/ml), and antiretroviral-drug-treated subjects (n = 8; VL, <75 copies/ml). In all subject groups, immunodominant CD8⁺ T-cell responses were generally shared by blood and mucosa, although there were exceptions. In HIV controllers, responses to HLA-B27- and HLA-B57-restricted epitopes were common to both tissues, and their magnitude (in spot-forming cells [SFC] per million) was significantly greater than those of responses restricted by other alleles. Furthermore, peptides recognized by T cells in both blood and rectal mucosa, termed “concordant,” elicited higher median numbers of SFC than discordant responses. In magnitude as well as breadth, HIV Gag-specific responses, particularly those targeting p24 and p7, dominated in controllers. Responses in noncontrollers were more evenly distributed among epitopes in Gag, Env, and Nef. Viremic controllers showed significantly broader mucosal Gag-specific responses than other groups. Taken together, these findings demonstrate that (i) Gag-specific responses dominate in mucosal tissues of HIV controllers; (ii) there is extensive overlap between CD8⁺ T cells in blood and mucosal tissues, with responses to immunodominant epitopes generally shared by both sites; and (iii) mucosal T-cell response breadth alone cannot account for immune control.Despite more than two decades of intensive research, the immunologic correlates of protection from human immunodeficiency virus (HIV) infection and disease progression remain incompletely understood. To date, the majority of studies of HIV-specific T-cell responses have focused on the measurement of such responses in peripheral blood lymphocytes. Nevertheless, the majority of the body''s lymphocytes are housed in mucosal tissues, notably the gastrointestinal (GI) tract (18, 33, 40). The gastrointestinal mucosa also serves as a major target of HIV infection and CD4⁺ T-cell depletion (7, 25, 36), as well as an important site of transmission (18, 33, 40). Antigen-experienced T cells may preferentially traffic to tissue sites of infection (50), where they may also expand in an antigen-driven manner. Because of the unique role of the gastrointestinal mucosa in HIV pathogenesis, detailed studies of HIV-specific immune responses in this compartment may contribute important insights to our understanding of the disease process.An important question is the degree to which T-cell responses in mucosal tissues are “compartmentalized” and distinct in specificity and/or clonality from those found elsewhere in the body, including in peripheral blood. Because of the technical challenges associated with obtaining large numbers of viable lymphocytes from mucosal biopsy specimen tissue, comprehensive mapping of the fine specificity of mucosal HIV-specific T-cell responses has been difficult. Relying on a polyclonal expansion approach, Ibarrondo and colleagues successfully mapped HIV-specific CD8⁺ T-cell responses in blood and rectal mucosa of chronically infected persons to the level of peptide pools but not to individual epitopes (29). Their studies revealed a similar pattern of responses, and nearly identical immunodominance hierarchies, in the two tissue sites.We have focused our recent studies of mucosal immunity on a group of individuals who control HIV infection in the absence of antiretroviral therapy. These are often called “long-term nonprogressors” (LTNP) (14), referring to their ability to maintain normal CD4⁺ T-cell counts for more than 10 years without medication. LTNP are believed to account for 5 to 15% of the HIV-infected population. Several recent studies have used the term “HIV controllers,” defined as those who maintain undetectable plasma HIV RNA levels (“elite controllers”) and those who have persistently detectable but low plasma HIV RNA levels (“viremic controllers”). Elite controllers represent less than 1% of the HIV-infected population (14). In contrast, individuals with viral loads of >10,000 copies/ml in the absence of therapy are termed “noncontrollers.” Recently, we found that “polyfunctional” HIV-specific T cells, producing multiple antiviral factors, were significantly more abundant in gastrointestinal mucosa of HIV controllers than in those of noncontrollers or subjects on highly active antiretroviral therapy (HAART) (20). Furthermore, in many cases these strong, polyfunctional mucosal T-cell responses were not mirrored in peripheral blood, suggesting that HIV-specific T cells either preferentially traffic to or undergo expansion within mucosal tissues.Because of these findings, we undertook a follow-up study to determine the breadth and fine specificity, to the peptide level, of mucosal CD8⁺ T-cell responses to HIV Gag, Env, and Nef among HIV controllers, noncontrollers, and individuals on HAART. We hypothesized that controllers might harbor an unusually broad repertoire of HIV-specific CD8⁺ T cells in mucosal tissues. We found a similar response breadth in mucosal tissues of all three subject groups, arguing against a critical role for mucosal T-cell response breadth in determining the extent of HIV control. In contrast, we found that high-magnitude mucosal responses directed at well-conserved regions in Gag were a strong and consistent correlate of control. Finally, concordant responses, defined as those common to blood and mucosa, were generally stronger than discordant responses, underscoring the observation that T cells responding to immunodominant epitopes are broadly distributed throughout the body in both controllers and noncontrollers.     

Protective Efficacy of Cross-Reactive CD8+ T Cells Recognising Mutant Viral Epitopes Depends on Peptide-MHC-I Structural Interactions and T Cell Activation Threshold

Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8⁺ T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP_336–374 peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8⁺ T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vβ regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2D^bNPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D^b, including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8⁺ responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8⁺ T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A.     

Peripheral Vγ9Vδ2 T Cells Are a Novel Reservoir of Latent HIV Infection

Eradication of HIV infection will require the identification of all cellular reservoirs that harbor latent infection. Despite low or lack of CD4 receptor expression on Vδ2 T cells, infection of these cells has previously been reported. We found that upregulation of the CD4 receptor may render primary Vδ2 cells target for HIV infection in vitro and we propose that HIV-induced immune activation may allow infection of γδ T cells in vivo. We assessed the presence of latent HIV infection by measurements of DNA and outgrowth assays within Vδ2 cells in 18 aviremic patients on long-standing antiretroviral therapy. In 14 patients we recovered latent but replication-competent HIV from highly purified Vδ2 cells demonstrating that peripheral Vδ2 T cells are a previously unrecognized reservoir in which latent HIV infection is unexpectedly frequent.     

Polyfunctional Type-1, -2, and -17 CD8+ T Cell Responses to Apoptotic Self-Antigens Correlate with the Chronic Evolution of Hepatitis C Virus Infection

Caspase-dependent cleavage of antigens associated with apoptotic cells plays a prominent role in the generation of CD8⁺ T cell responses in various infectious diseases. We found that the emergence of a large population of autoreactive CD8⁺ T effector cells specific for apoptotic T cell-associated self-epitopes exceeds the antiviral responses in patients with acute hepatitis C virus infection. Importantly, they endow mixed polyfunctional type-1, type-2 and type-17 responses and correlate with the chronic progression of infection. This evolution is related to the selection of autoreactive CD8⁺ T cells with higher T cell receptor avidity, whereas those with lower avidity undergo prompt contraction in patients who clear infection. These findings demonstrate a previously undescribed strict link between the emergence of high frequencies of mixed autoreactive CD8⁺ T cells producing a broad array of cytokines (IFN-γ, IL-17, IL-4, IL-2…) and the progression toward chronic disease in a human model of acute infection.     

Efficacious Early Antiviral Activity of HIV Gag- and Pol-Specific HLA-B*2705-Restricted CD8+ T Cells

The association between HLA-B*2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B*2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8⁺ T cells. In order to better define the mechanisms of the HLA-B*2705 immune control of HIV, we first characterized the CD8⁺ T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B*2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B*2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8⁺ T-cell response. By comparing inhibitions of viral replication by CD8⁺ T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B*2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8⁺ T cells but not until 18 h postinfection by VL9-specific CD8⁺ T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B*2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B*2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8⁺ T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.Current human immunodeficiency virus (HIV) vaccine strategies are focused on emulating the protective effect observed for HIV-infected individuals carrying alleles such as B*2705 by inducing the virus-specific CD8⁺ T-cell responses that are thought to be responsible for delaying or preventing disease progression. Understanding why such alleles confer protection facilitates a rational approach to vaccine design. It has been hypothesized that the slow progression to AIDS exhibited by HLA-B*2705-positive (HLA-B*2705⁺) HIV-infected individuals is due to the immunodominant B*27-restricted CD8⁺ T-cell response toward the p24 Gag epitope KRWIILGLNK (KK10) (Gag residues 263 to 272). Escape from this epitope typically occurs late in infection and is associated with rapid progression to AIDS (14, 16). The commonly selected mutation R264K abrogates CD8⁺ T-cell recognition but also confers a substantial fitness cost to the virus, and the selection of compensatory mutations is required to restore viral replicative capacity (19, 29, 30). This has prompted the hypothesis that CD8⁺ T-cell responses that can drive escape mutations that reduce viral fitness are a contributing factor in the immune control of HIV, either by promoting the outgrowth of a viral quasispecies with a lower replicative capacity or by delaying the selection of escape mutations, both of which may slow the onset of AIDS (11, 21, 25).To better understand how CD8⁺ T cells can be most effective against HIV, recent studies have directly assessed the antiviral activity of CD8⁺ T cells via the viral suppression of HIV-infected CD4⁺ T cells during coculture. Such studies indicated that Gag-specific CD8⁺ T cells have a higher potency for viral suppression than Env-specific CD8⁺ T cells (10), supporting previous data indicating that broad CD8⁺ T-cell targeting of Gag epitopes was associated a with lower viral set point and, hence, slower progression to AIDS (20). A recent study of simian immunodeficiency virus (SIV) suggested that the protective effect of Gag-specific CD8⁺ T cells is mediated by the early presentation of Gag epitopes, processed from the viral Gag protein from incoming virions during infection, which can sensitize target cells for lysis by Gag-specific CD8⁺ T cells within 6 h of infection (26, 27). In addition, it was proposed previously that the ability of CD8⁺ T cells to secrete multiple cytokines may also be an important correlate of immune protection (6), and a further recent study demonstrated a more polyfunctional cytokine profile of Gag-specific B*2705-KK10 CD8⁺ T-cell responses than those of other HIV-specific CD8⁺ T-cell responses (1). The ability of CD8⁺ T cells to proliferate in response to the cognate epitope peptide has also been associated with immune control (1, 12). Other studies demonstrated the importance of lytic granule loading of CD8⁺ T cells for the effective elimination of HIV-infected cells (6, 22). However, the induction of a Gag KK10-specific CD8⁺ T-cell vaccine response in a B*2705-positive vaccinee did not protect against rapid progression following subsequent HIV-1 infection (5). This anecdotal case suggests the possibility that HLA-B*2705-associated immune control of HIV-1 may not be dependent on the Gag KK10-specific CD8⁺ T-cell response alone.Since current vaccine strategies hope to induce a protective effect, such as that observed for HLA-B*2705⁺ HIV-infected individuals, the study of the functional and phenotypic characteristics of B*2705-specific CD8⁺ T cells provides an opportunity to redefine the proposed correlates of immune protection essential for rational vaccine design. In this study we analyze three different specificities of HLA-B*2705-restricted CD8⁺ T cells from chronically HIV-infected individuals in order to directly compare antiviral activity with potential correlates of immune protection, including the kinetics of viral inhibition, cytokine profile, granzyme production, proliferative capacity, and cytotoxicity.     

Clinical Outcome of HIV Viraemic Controllers and Noncontrollers with Normal CD4 Counts Is Exclusively Determined by Antigen-Specific CD8+ T-Cell-Mediated HIV Suppression

In this cross-sectional study we evaluated T-cell responses using several assays to determine immune correlates of HIV control that distinguish untreated viraemic controllers (VC) from noncontrollers (NC) with similar CD4 counts. Samples were taken from 65 ART-naïve chronically HIV-infected VC and NC from Thailand with matching CD4 counts in the normal range (>450 cells/μl). We determined HIVp24-specific T-cell responses using standard Interferon-gamma (IFNγ) ELISpot assays, and compared the functional quality of HIVp24-specific CD8⁺ T-cell responses using polychromatic flow cytometry. Finally, in vitro HIV suppression assays were performed to evaluate directly the activity of CD8⁺ T cells in HIV control. Autologous CD4⁺ T cells were infected with primary patient-derived HIV isolates and the HIV suppressive activity of CD8⁺ T cells was determined after co-culture, measuring production of HIVp24 Ag by ELISA. The HIVp24-specific T-cell responses of VC and NC could not completely be differentiated through measurement of IFNγ-producing cells using ELISpot assays, nor by the absolute cell numbers of polyfunctional HIVp24-specific CD8⁺ T cells. However, in vitro HIV suppression assays showed clear differences between VC and NC. HIV suppressive activity, mediated by either ex vivo unstimulated CD8⁺ T cells or HIVp24-specific T-cell lines, was significantly greater using cells from VC than NC cells. Additionally, we were able to demonstrate a significant correlation between the level of HIV suppressive activity mediated by ex vivo unstimulated CD8⁺ T cells and plasma viral load (pVL) (Spearman r = -0.7345, p = 0.0003). This study provides evidence that in vitro HIV suppression assays are the most informative in the functional evaluation of CD8⁺ T-cell responses and can distinguish between VC and NC.     

Dendritic cells are less susceptible to human immunodeficiency virus type 2 (HIV-2) infection than to HIV-1 infection

Human immunodeficiency virus type 1 (HIV-1) infection of dendritic cells (DCs) has been documented in vivo and may be an important contributor to HIV-1 transmission and pathogenesis. HIV-1-specific CD4⁺ T cells respond to HIV antigens presented by HIV-1-infected DCs and in this process become infected, thereby providing a mechanism through which HIV-1-specific CD4⁺ T cells could become preferentially infected in vivo. HIV-2 disease is attenuated with respect to HIV-1 disease, and host immune responses are thought to be contributory. Here we investigated the susceptibility of primary myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) to infection by HIV-2. We found that neither CCR5-tropic primary HIV-2 isolates nor a lab-adapted CXCR4-tropic HIV-2 strain could efficiently infect mDCs or pDCs, though these viruses could infect primary CD4⁺ T cells in vitro. HIV-2-exposed mDCs were also incapable of transferring virus to autologous CD4⁺ T cells. Despite this, we found that HIV-2-specific CD4⁺ T cells contained more viral DNA than memory CD4⁺ T cells of other specificities in vivo. These data suggest that either infection of DCs is not an important contributor to infection of HIV-2-specific CD4⁺ T cells in vivo or that infection of DCs by HIV-2 occurs at a level that is undetectable in vitro. The frequent carriage of HIV-2 DNA within HIV-2-specific CD4⁺ T cells, however, does not appear to be incompatible with preserved numbers and functionality of HIV-2-specific CD4⁺ T cells in vivo, suggesting that additional mechanisms contribute to maintenance of HIV-2-specific CD4⁺ T-cell help in vivo.     

Cross-Reactivity of Herpesvirus-Specific CD8 T Cell Lines Toward Allogeneic Class I MHC Molecules

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65_495–503/A*0201, BMLF1_259–267/A*0201, or BZLF1_54–64/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-α production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-α production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection.     

Role of human immunodeficiency virus (HIV)-specific T-cell immunity in control of dual HIV-1 and HIV-2 infection

Progressive immune dysfunction and AIDS develop in most cases of human immunodeficiency virus type 1 (HIV-1) infection but in only 25 to 30% of persons with HIV-2 infection. However, the natural history and immunologic responses of individuals with dual HIV-1 and HIV-2 infection are largely undefined. Based on our previous findings, we hypothesized that among patients with dual infection the control of HIV-1 is associated with the ability to respond to HIV-2 Gag epitopes and to maintain HIV-specific CD4⁺ T-cell responses. To test this, we compared the HIV-specific ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay responses of 19 dually infected individuals to those of persons infected with HIV-1 or HIV-2 only. Further, we assessed the functional profile of HIV Gag-specific CD4⁺ and CD8⁺ T cells from nine HIV dually infected patients by using a multicolor intracellular cytokine staining assay. As determined by ELISPOT assay, the magnitude and frequency of IFN-γ-secreting T-cell responses to gene products of HIV-1 were higher than those to gene products of HIV-2 (2.64 versus 1.53 log₁₀ IFN-γ spot-forming cells/10⁶ cells [90% versus 63%, respectively].) Further, HIV-1 Env-, Gag-, and Nef- and HIV-2 Gag-specific responses were common; HIV-2 Nef-specific responses were rare. HIV-specific CD4⁺ T helper responses were detected in nine of nine dually infected subjects, with the majority of these T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and, to a lesser extent, interleukin-2. The HIV-1 plasma viral load was inversely correlated with HIV-2 Gag-specific IFN-γ-/TNF-α-secreting CD4⁺ and HIV-2 Gag-specific IFN-γ-secreting CD8⁺ T cells. In conclusion, the T-cell memory responses associated with containment of single HIV-1 and HIV-2 infection play a similar significant role in the immune control of dual HIV-1 and HIV-2 infection.     

GagCM9-specific CD8+ T cells expressing limited public TCR clonotypes do not suppress SIV replication in vivo

Several lines of evidence suggest that HIV/SIV-specific CD8(+) T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag(45-269)) that were subsequently infected with SIVsmE660. These seven Mamu-A*01(+) animals developed CD8(+) T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8(+) T cells could not control virus replication in vivo. GagCM9-specific CD8(+) T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8(+) T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20-250 GagCM9-specific CD8(+) T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8(+) T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8(+) T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8(+) T cell population elicited by vaccination and infection.     

Maturation and Mip-1β Production of Cytomegalovirus-Specific T Cell Responses in Tanzanian Children,Adolescents and Adults: Impact by HIV and Mycobacterium tuberculosis Co-Infections

It is well accepted that aging and HIV infection are associated with quantitative and functional changes of CMV-specific T cell responses. We studied here the expression of Mip-1β and the T cell maturation marker CD27 within CMVpp65-specific CD4⁺ and CD8⁺ T cells in relation to age, HIV and active Tuberculosis (TB) co-infection in a cohort of Tanzanian volunteers (≤16 years of age, n = 108 and ≥18 years, n = 79). Independent of HIV co-infection, IFNγ⁺ CMVpp65-specific CD4⁺ T cell frequencies increased with age. In adults, HIV co-infection further increased the frequencies of these cells. A high capacity for Mip-1β production together with a CD27^low phenotype was characteristic for these cells in children and adults. Interestingly, in addition to HIV co-infection active TB disease was linked to further down regulation of CD27 and increased capacity of Mip-1β production in CMVpp65-specific CD4⁺ T cells. These phenotypic and functional changes of CMVpp65-specific CD4 T cells observed during HIV infection and active TB could be associated with increased CMV reactivation rates.     

Regulatory T Cell Suppression of Gag-Specific CD8+ T Cell Polyfunctional Response After Therapeutic Vaccination of HIV-1-Infected Patients on ART

We tested the hypothesis that therapeutic vaccination against HIV-1 can increase the frequency and suppressive function of regulatory, CD4⁺ T cells (Treg), thereby masking enhancement of HIV-1-specific CD8⁺ T cell response. HIV-1-infected subjects on antiretroviral therapy (N = 17) enrolled in a phase I therapeutic vaccine trial received 2 doses of autologous dendritic cells (DC) loaded with HIV-1 peptides. The frequency of CD4⁺CD25^hiFOXP3⁺ Treg in blood was determined prior to and after vaccination in subjects and normal controls. Polyfunctional CD8⁺ T cell responses were determined pre- and post-vaccine (N = 7) for 5 immune mediators after in vitro stimulation with Gag peptide, staphylococcal enterotoxin B (SEB), or medium alone. Total vaccine response (post-vaccine–pre-vaccine) was compared in the Treg(+) and Treg-depleted (Treg-) sets. After vaccination, 12/17 subjects showed a trend of increased Treg frequency (P = 0.06) from 0.74% to 1.2%. The increased frequency did not correlate with CD8⁺ T cell vaccine response by enzyme linked immunosorbent assay for interferon γ production. Although there was no significant change in CD8⁺ T cell polyfunctional response after vaccination, Treg depletion increased the polyfunctionality of the total vaccine response (P = 0.029), with a >2-fold increase in the percentage of CD8⁺ T cells producing multiple immune mediators. In contrast, depletion of Treg did not enhance polyfunctional T cell response to SEB, implying specificity of suppression to HIV-1 Gag. Therapeutic immunization with a DC-based vaccine against HIV-1 caused a modest increase in Treg frequency and a significant increase in HIV-1-specific, Treg suppressive function. The Treg suppressive effect masked an increase in the vaccine-induced anti-HIV-1-specific polyfunctional response. The role of Treg should be considered in immunotherapeutic trials of HIV-1 infection.     

TGF-β and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

Immune dysregulation in HIV-1 infection is associated with increased expression of inhibitory molecules such as CTLA-4, TGF-β, and IL-10. In this study we examined one potential mechanism for regulating TGF-β and IL-10 expression by HIV-specific suppressor CD8+ T cells. No overlap between TGF-β, IL-10, and IFN-γ cytokine production by HIV-specific CD8+ T cells was observed. TGF-β positive and IL-10 positive cells were FOXP3 negative, CD25 negative, and displayed a heterogeneous surface expression of CD127. TGF-β and IL-10 positive CD8+ T cells did not express CTLA-4. Nevertheless, CTLA-4 blockade resulted in a significant decrease in HIV-specific TGF-β positive and IL-10 positive CD8+ T cell responses, and a concomitant increase in HIV-specific IFN-γ positive CD8+ T cell responses. Depletion of CD4+ T cells abrogated the impact of CTLA-4 on HIV-specific TGF-β positive and IL-10 positive CD8+ T cells. Our study suggests that CTLA-4 Signaling on CD4+ T cells regulates the inhibitory functions of the HIV-specific suppressor CD8+ T cells.     

Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4⁺ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4⁺ T cells and diminished the response to FV infection. Surprisingly, however, CD4⁺ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4⁺ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4⁺ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4⁺ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity.