HIV INFECTION OF THE CD8+ T CELLS: CONTROVERSY AND CLARIFICATION
HIV is able to infect diverse blood leukocytes, but controversy has persisted regarding the infection of CD8+ T cells for quite sometime. Recent work has shown that HIV is capable of productively infecting CD8+ T cells.
All initial descriptions of CD8+ T cells came from in vitro experiments [4-7], which were followed by evidence that CD8+ T cells can harbour and express HIV-1 in vivo [8, 9], and may also express small amounts of CD4 RNA [10]. It has since been confirmed that CD8+ T cells harbor considerable amounts of HIV provirus [8, 11], suggesting their susceptibility to HIV.So far, the HIV infection of CD8+ T cells has mainly focused on the mechanism of viral entry, and the origins of CD8+ cell infection [1]. It is believed that the CD8+ T cells become infected through a conventional CD4- dependent mechanism during their maturation in the thymus. The most plausible explanation could be the infection at the double-positive (DP) stage, where CD4 is co-expressed on the CD8 cell surface. Implants of human thymic tissue containing infected DP thymocytes in SCID mice have been shown to produce infected single positive (SP) CD8+ T lymphocytes in the peripheral circulation [12, 13]. HIV-1 proviral DNA is also preferentially distributed in the naive (CD45RA+) subset of CD8+ T cells, further supporting the thymus as a source of CD8+ T cell infection [14].
In addition to possible intrathymic mechanisms, stimulation of highly purified CD8+ T cells with mitogens, allogeneic dendritic cells or anti-CD3 and anti-CD28 antibodies in vitro leads to de novo synthesis of CD4 and susceptibility to HIV-1 infection [14, 15]. Activated subsets of circulating CD8+ T lymphocytes express high frequencies of CD4 in vivo, rendering these cells vulnerable to virus-mediated destruction [9, 16-18].
Reports of CD8+ T cell-mediated, CD4 independent entry into CD8+ T cells have also been provided by Saha and colleagues, who showed specifically the role of CXCR4 in CD8+ T cell infection by a peculiar CD8- tropic HIV isolate. Multiple CD8+ clones generated from patients with AIDS were characterized and it was found that several singly positive CD8 were endogenously infected by HIV [19]. Subsequent biological characterization of these isolates demonstrated CD8-mediated cell entry without the requirement for any known chemokine co-receptor [19]. Recent work by this group has led to the further characterization of CD4-independent entry of HIV-1 in CD8+ T cells [20].
In addition to clarification of CD8+ T cell entry mechanisms, several studies have focused on quantitative aspects of CD8+ T cell infection by HIV. Several reports have sought to compare the abundance of HIV provirus in CD8+ T cells with those of other leukocytes. These reports have concluded that CD8+ T cells harbor substantial amounts of provirus. Livingstone and colleagues found that in late stage disease, infection in CD8+ T cells accounted for between 66% and 97% of total proviral DNA, and observed a strong inverse relationship between CD8+ T cell count and the frequency of CD8+ T cell infection [8]. Similarly, more recent reports have further confirmed that CD8+ T cells do contain significant amounts of provirus [21]. Additional studies have also attempted to clarify the distribution of proviral DNA in specific CD8+ T cell subsets. One report demonstrated a preferential distribution of HIV in the naive (CD45RA+) subset of CD8+ T cells compared to the memory/effector (CD45RO+) population. In contrast to all previous findings, it has been suggested that naive and memory CD8+ T cells are rarely infected by HIV [22].
There is debate over whether the infection of CD8+ T cells contributes significantly to the immunodeficiency observed in AIDS.
This stems from the existence of a number of other possible mechanisms that could account for the numerical decline and functional impairment of CD8+ T cells observed on disease progression. These include increased susceptibility to apoptosis from alterations in the cytokine milieu in lymphoid tissue, bystander effects from neighbouring productively infected CD4+ T cells, or toxicity from the release of HIV derived proteins such as gp120 or Tat [23, 24]. Loss of CD4+ T cell helper function leading to impaired clonal expansion and function of CD8+ T lymphocytes on antigenic pressure also contributes [25]. Thymic destruction of precursor CD8+ T cells has been proposed as an explanation for the eventual failure of CD8+ T cell homeostasis, the decline in circulating numbers of first naive and then memory CD8+ T cells upon disease progression [26], and the recovery in naive CD8+ T cell numbers on commencement of antiretroviral therapy [27].Viral Evolutionary Processes in HIV-Infected CD8+ T cells
Previous analyses have shown the compartmentalization of HIV in CD4+ T cells, CD8+ T cells and monocytes in patients receiving HAART [28-30]. To investigate whether this compartmentalization impacts significantly at the functional level during therapy, Potter et al. [28], conducted a detailed phylogenetic and evolutionary analysis of viral populations derived from multiple blood leukocytes. Mutations in the protease (PR) and reverse transcriptase (RT) genes from the virus in each compartment were analyzed to identify cellular reservoirs of HIV-1 drug resistance, and to clarify the trafficking of viral strains between cell-free and cell- associated compartments. Their analyses revealed that HIV populations from individual blood cell compartments were in many cases genetically distinct. This was evident in most patients, particularly those who displayed cellular compartmentalization of drug resistance mutations.
Most notably, virus isolated from CD8+ T cells was phylogenetically distinct in a number of subjects, and branched separately from other blood cell types. In addition, they also demonstrated reduction in HIV drug resistant HIV strains uniquely in CD8 compartment. Interestingly, in some patients, the PCR amplification of virus from CD8+ T cells and monocytes was unsuccessful despite repeated attempts. This phenomenon was unique to patients who showed low to undetectable plasma viremia with moderate to high CD4+ and CD8+ T cell counts. This suggests that the infection of CD8+ T cells and monocytes may be less prominent during low plasma viremia phase and elevated T cell counts. There appears to be some correlation between compartmentalization of HIV variants with lower levels of drug resistance in CD8+ T cells and the contribution of this compartment to plasma virus. It was also interesting to note that in patients with extremely low CD4+ T cell counts, CD8+ T cells and monocytes were more closely related to the plasma population than HIV clones from CD4+ T cells. This suggested that additional leukocyte reservoirs might play a more significant role in the absence of viable numbers of CD4+ T cells. Overall, the data of Potter et al. [29, 30] has demonstrated that in most cases HIV variants isolated from CD8+ T cells and monocytes are often genetically distinct from those found in CD4+ T cells and other cellular compartments.At present, there is no clear explanation for the notable absence of drug resistance mutations in virus derived from CD8+ T cells observed in some HIV patients receiving HAART. One possibility is that antiretroviral drugs were less efficient in targeting CD8+ T cell compartment in some individuals leading to a decrease in the selective pressure for the development of drug resistant variants. However, if this were the case, it would also be likely that drug resistant viral strains could arise due to replication in suboptimal drug concentrations. An alternative explanation could be that drug resistant strains were not tropic for CD8+ T cell entry.
It is also conceivable that these strains represent ‘archival species’ established in memory CD8+ T cell subsets soon after primary seroconversion. Several studies have demonstrated that latently infected memory CD4+ T cell reservoirs are established soon after infection [31] and can be activated by pro-inflammatory cytokines in vitro and potentially in vivo [32]. Viral strains derived from latent memory CD4+ T cells have been shown to contain few antiretroviral drug resistance mutations compared to the predominant viral population suggesting the absence of ongoing replication [33, 34]. The marked absence of drug resistance mutations in the CD8+ T cell viral population of a number subjects was also indicative of reduced HIV turnover and evolution. This raises the possibility that wild type HIV variants isolated from the CD8+ compartment of these patients may derive from memory CD8+ T cell subsets. However, this was not confirmed in the study by [28], as the individual contribution of the memory CD8+ T cell subset was not assessed. Other factors that are likely to contribute to the lack of HIV drug resistance in CD8+ T cells include tissue distribution, persistence and/or slower HIV replication. CD8+ T cell infection appeared to be productive in a number of other patients further complicating this issue. Nonetheless, these findings on the infection of total CD8+ T cell populations are of significant biological relevance to the clinical management of HIV patients. The protection of CD8+ T cells from HIV-1 infection during HAART should lead to better T-cell responses and is crucial for long lasting CD8-mediated antiviral activity and natural immunity in infected patients.Role of CD8+ T Cells in Acute HIV/SIV Infection
Vigorous immune responses accompanied by the proliferation of selected T cell receptor V beta (BV)- expressing CD8+ T cells characterize acute HIV infection.
Such ‘expansions’ can persist during chronic HIV infection and are likely to result in the dominance of some selected clones. The oligoclonal expansion of CD8+ T cells in vivo is HIV specific during chronic HIV infection and the clonal population is usually consistent with antigen-driven expansions of CD8+ T cells [35].In greater number of HIV-infected individuals the acute HIV infection is usually presented as a transient symptomatic illness, together with elevated levels of HIV-1 replication and an expansive HIV-specific immune responses. Understanding the true role of CD+8 T cells in vivo is vital for understanding HIV pathogenesis and for the designing of future vaccine candidates for HIV control. A plethora of clinical evidence suggests the potential role of cellular immune responses in the control of HIV replication in infected individuals. Recently, evidence attesting the role of CD8+ T cells in controlling SIV replication in macaques was shown [36, 37]. Here, the CD8+ T lymphocytes were depleted in rhesus macaques infected with SIV- mac251 and the effect of CD8+ T cell depletion was assessed on plasma viral load and p24 antigenemia. The animals that were depleted of CD8+ lymphocytes for more than 4 weeks failed to control viral RNA and p24 antigenemia and developed disease. These data imply that acute viremia is a consequence of response from SIV-specific CD8+ T cells and not a consequence of waning CD4+ T cell numbers. Incomplete CD8+ T cell depletion in vivo, due to variability in plasma viremia, has hampered interpretation of the exact role of CD8+ T cells in acute SIV infection [38]. In contrast, Matano et al. [39] depleted CD8+ T cells from a SIV-infected animal during early infection and showed a role for CD8+ T cells in plasma viremia clearance.
A number of factors, such viral fitness, host genetics, and host immune responses are some factors that modulate acute infection and are known to influence viral replication and defining the viral set point during acute infection. Although the induction of HIV-1-specific CD8(+) T cells during acute infection is associated with a decline in viremia, the role CD8+ T effectors in subsequently establishing the viral set point remains unknown. Cao et al. [40] analyzed two acutely infected HIV patients with the same initial Tat-specific CD8+ T cell response. They analyzed their CD8+ T cell responses over time in relation to viral load and sequence evolution. In one patient following the initiation of treatment during acute infection, the frequencies of Tat- specific CD8+ T cells gradually diminished but persisted, and the Tat epitope sequence was largely unaltered. Interestingly, in the second patient, who declined treatment, Tat-specific CD8+ T cells also declined to below detection levels, in tandem with Gag-specific CD4+ T cell loss, as plasma viremia reached a set point. This in vivo event temporally coincided with the emergence of an escape variant within the Tat epitope with an additional Vpr epitope. As a consequence, new CD8+ T cell responses emerged with no further decline in plasma viremia. These data suggest that, in the absence of treatment, the initial CD8+ T cell responses have the greatest impact on reducing viremia, and that later, continuously evolving responses are less efficient in further reducing viral load. These results also imply that T cell help is one of the principal contributors to the antiviral efficiency of the acute CD8+ T cell response.
Further, there is supporting evidence showing a massive, oligoclonal expansion of CD8+ T cells during the acute phase of infection [41], which coincides with the appearance of HIV-1-specific CD8+ T and the initial decline in plasma viremia [42]. These HIV-specific CD8+ T cells can potentially eliminate HIV-1-infected cells directly by MHC class I-restricted cytolysis or by a non-cytotoxic mechanism through the secretion of soluble factors [43]. Thus, the biological relevance of CD8+T cell immune responses is evident from both human and non-human primate studies in acute HIV/SIV infection.
CD8+ T cell Responses and their Significance in Chronic HIV/SIV Infection
HIV-infected, therapy naive, long-term non-progressors (LTNP) usually display strong CD4+ T-cell lymphoproliferative responses to HIV-1 gag antigens [7, 44, 45]. In addition, these patients also maintain a strong lymphoproliferative capacity of HIV-specific CD8+ T-cells [46]. This is known to enhance the function of effector CTL [47]. Disappointingly, the large majority of HIV patients with chronic HIV-1 infection show signs of functional impairments in both CD4+ and CD8+ HIV-reactive T-cells, which is attributed to the continued low-level viral replication and possibly a simultaneous slow decline in the number of CD4+ T cells. Animal model studies have also demonstrated that although HIV-specific CD8+ T cell numbers are maintained by sustained viral replication, the maturation of CTL becomes skewed [48]. At present the functional aspects of HIV-specific CTL in vivo are poorly understood. Considerable evidence exists to show that HIV-1, through mutation of viral sequences recognized by CTL epitopes, can evade host immune responses [49], and these molecular changes provide HIV with an arsenal to counteract the host immune system at various stages of HIV disease and therapy. Both CD4 and CD8+ T cell responses are crucial to the success of HAART, but as the HIV-specific CD4+ T cell responses seem to improve with HAART over time, the CD8+ T cell responses show a decline due to decreasing antigenemia and possibly due to the deleterious effects of some drugs on CNAR activity. As a consequence the defenses of these cells weaken and are not sufficient and potent enough to prevent surges in viral replication in the absence of HAART [50].