To characterize the pulmonary immune response during the course of HIV treatment, we investigated viral infection, T-cell functionality, CD4 T-cell depletion, and CD4 T-cell reconstitution following HAART from PB and BAL. Five major points emerged: (i) BAL CD4 T cells are not massively depleted or preferentially infected by HIV compared to levels for PB; (ii) CD4 T cells reconstitute following HAART, coincidently with decreases in the frequencies of memory CD4 T cells infected by HIV in vivo; (iii) in PB and BAL, the reconstitution of CD4 T cells in BAL involves the compartmentalized proliferation of memory CD4 T cells; (iv) mitogenically stimulated T cells are more polyfunctional in BAL than in PB, and functionality can improve moderately with long-term HAART; and (v) CMV-specific T cells, on the other hand, are significantly more polyfunctional in PB than in BAL, and functionality remains fairly constant longitudinally after the administration of HAART.
There are a number of reasons for studying the pulmonary immune response during the course of HAART. In HIV-infected subjects, pulmonary infections are common and likely due to the failure of local immunity during the course of disease (14
). Additionally, the ability to repeatedly access the lung compartment by BAL permits a relatively noninvasive way to monitor mucosal responses, which has been shown to have prognostic value in early HIV studies (1
). Lymphocytes obtained from BAL are analogous to lymphocytes obtained from other noninductive mucosal sites, such as the GI lamina propria, as they lack a naïve population (unlike PB). In pathogenic SIV infections of Asian macaques, CD4 T cells are depleted rapidly from the lung, much like the GI tract, and suggest a common theme of mucosal T-cell depletion in these animals (23
In contrast to SIV models of infection, our recent cross-sectional human study showed that BAL CCR5+
CD4 memory T lymphocytes, unlike lymphocytes at other mucosal sites, were maintained in HIV-infected subjects (4
). This difference in the lung compartment between SIV- and HIV-mediated CD4 T-cell depletion during infection could be due to differences in the kinetics of virus replication, pulmonary host factors such as local concentrations of CCR5-binding chemokines, or the diverse nature of the cohorts enrolled in human studies. Indeed, we found a significant correlation between the frequencies of BAL CCR5+
CD4 memory T cells and plasma viral replication. These data indicate that CCR5+
CD4 memory T cells in BAL support viral replication and are a source of plasma viral load. However, after these cells are depleted, possibly by direct viral infection, plasma viral loads subsequently decrease. Regardless, these data highlight the anatomically restricted immunological occurrences of chronically HIV-infected individuals and suggest that, unlike SIV-infected rhesus macaques, BAL cannot be used as a reflection of occurrences at all mucosal sites.
Our current study suggests that both CCR5−
and, to a lesser extent, CCR5+
CD4 T cells are reconstituting the BAL compartment, an event not likely to occur in the GI tract. The differential reconstitution after the initiation of HAART within the GI tract (11
) compared to that of BAL might be explained by fibrosis within lymphoid tissue (10
). The lung has a paucity of locally organized lymphoid tissue. However, fibrosis described within peripheral lymph nodes (27
) and, to a greater extent, within the GI tract (10
) clearly correlates with the inability to restore CD4 T-cell pools. Indeed, this hypothesis is consistent with the requirement for the local proliferation of mucosal CD4 T cells upon immune reconstitution within BAL. On the other hand, the contraction of the virally expanded CD8 T-cell pool in BAL may be partially responsible for the increased frequencies of BAL CD4 T cells we observe after the initiation of HAART (29
). Nevertheless, increases in BAL CD4 T-cell frequency in combination with proliferating Ki67+
memory CD4 T cells strongly suggests a component of local pulmonary T-cell reconstitution. However, it is possible that these cells actually were proliferating in lymph nodes and then homed to BAL, leading to reconstitution.
The presence of T cells capable of simultaneously producing several effector cytokines has been associated with better protection against HIV (2
) and leishmania (7
). The increased functionality of memory T cells we observe within the BAL compared to that of PB may help explain the lack of pulmonary opportunistic infections within HIV-infected individuals during the long, asymptomatic chronic phase of infection. Interestingly, CMV-specific (in contrast to mitogenically stimulated and HIV-specific) CD8 and CD4 memory T cells are more polyfunctional in blood than lung, suggesting a hierarchy of antigenic responses in various compartments. Moreover, the decreased T-cell polyfunctionality observed within PB of HIV-infected individuals is maintained even after 1 year of HAART. Further studies are required to examine the impact of the early initiation of HAART on local immune reconstitution to pulmonary pathogens.
There are several confounding issues that require further study and analysis. First, half of our volunteers were smokers at the time of enrollment, which may decrease the breadth of local T-cell responses. Second, Ki67+ memory CD4 T cells frequently are increased at 1 month. It is unclear if this proliferation represents pathological proliferative responses to antigens present in the HIV-infected lung in some subjects. Third, although all subjects were on HAART, occasional increases in viral load were noted in blood throughout the 1-year course of follow-up. The impact of low-level intermittent viremia cannot be completely assessed. Finally, given the small numbers of T cells in some samples, not all analyses could be performed on all subjects at every time point. Increased numbers of samples and longitudinal analysis beyond 1 year may be useful in this regard.
In summary, while causal relationships between viral replication, T-cell functionality, CD4 T-cell reconstitution, and local immunological phenomena often are difficult to delineate, our findings show an anatomically distinct mucosal site that contains ample cellular targets for HIV infection that nevertheless remains relatively intact, is capable of compartmentalized reconstitution after HAART, and maintains polyfunctional T cells. These findings provide a mechanism underlying the differential depletion and subsequent reconstitution after HAART of mucosal CD4 T cells.