Our study is the first one to prospectively evaluate the IFN-γ production by T cells in response to Mtb antigens in cART-naïve TB-HIV patients before and after cART introduction using the ELISpot assay. An increase in the magnitude of response to Mtb antigens was observed after 30 days of cART and was higher for the PPD antigen, followed by 38 kDa/CFP-10 and ESAT-6, and then stabilized or declined later on. The proportion of responders was higher for PPD and 38 kDa/CFP-10 than for the ESAT-6 antigen. PPD was the antigen associated with the strongest response in our study and in other studies including HIV-negative TB patients [17,18,19,20,21,22]. A hypothesis to explain the higher response to PPD antigen stimulation is that it contains not only Mtb-specific antigens but also other cross-reactive antigens, increasing its antigenicity.
In our work, in addition to PPD, we evaluated the response to the ESAT-6 antigens separately and to the antigen CFP-10 combined with the 38 kDa. The 38-kDa protein is highly immunogenic and is antigenic for human T cells and presented a response in TB patients andindividuals exposed to Mtb (TST positive) [22, 23]. Tavares et al. (2007) demonstrated that the CFP-10 and 38 kDa antigen separately present a power of response, but when combined, this power increases in individuals with TB, which was the reference data for the choice of these combined antigens for our study .
The analysis of risk factors associated with an increased immune response to both antigens showed a different pattern between the PPD and 38 kDa/CFP-10 antigens. Previous studies covering Brazilian TB patients and individuals latently infected with Mtb showed good responses to the ESAT-6 Mtb-specific antigen [22, 24], which was not observed in our study. Such differences might be due to the antigenicity of this molecule and the restricted T cell repertoire of the TB-HIV patients included in our study, who were in most cases severely immunosuppressed.
In general, TB-HIV patients are immunosuppressed, and most of them do not respond well to these mycobacterial antigens even if they have TB . Our interest was to study the response to these antigens after combined antiretroviral therapy in cART-naïve TB-HIV patients, assuming that immune reconstitution could improve the immune response to them. However, for ESAT-6, which is an antigen included in commercial IFN-gamma release assays to assess TB infection , such improvement of the immune response was not achieved, suggesting that this antigen, at least in our experience, does not seem to be adequate to evaluate the immune response in severely immunosuppressed TB-HIV patients.
However, a previous study indicated that severely immunosuppressed individuals (<200 cells/mm3) have a better response to PPD, ESAT-6 and CFP-10 antigens than less immunosuppressed ones (≥200 cells/mm3) . However, our study showed that patients with CD4+ T cell counts <200 cells/mm3 at baseline exhibited an increased response to PPD but not to 38 kDa/CFP-10 compared to ones with CD4+ T cell counts ≥200 cells/mm3. Similarly, during the follow-up, increases in CD4+ T cell counts were associated with increased IFN-γ production in response to PPD with a steeper slope seen among those with absolute CD4+ T cell counts <200 cells/mm3 compared with those above this range, which probably reflected the unspecific and complex immune activation seen in these highly immunosuppressed patients However, IFN-γ production in response to 38 kDa/CFP-10 was associated with an increase in CD4+ T cell counts only among those with CD4+ T cell counts ≥200 cells/mm3, suggesting a profile of specific immune restoration in response to cART.
We have more men than women in our study, but this is an expected result since men are significantly more at risk of contracting and dying from TB than women . We did not observe any association of gender with increased IFN-γ production in response to the PPD and 38 kDa/CFP-10 antigens.
Younger age (18 to 32 vs. 38 to 44 years) was associated with higher IFN-γ production in response to PPD but not 38 kDa/CFP-10. The impact of age on immune restoration stimulated by mycobacterial antigens has not yet been consistently described. Neilsen et al. (2013) observed that cells from healthy adults show an increased production of cytokines (IFN-γ, TNF, and IL-10) after stimulation with PPD when compared with children . These findings highlight the potential influence of age in T cell capacity to respond to Mtb antigens with a possible biphasic profile since young adults apparently have a better response than both children and older adults.
In our study, the extrapulmonary forms of TB in immunosuppressed individuals were associated with a higher production of IFN-γ when T cells are stimulated with PPD antigen compared with other forms of TB. A previous study evaluating IFN-γ production in immune-compromised vs. immune-competent individuals with extrapulmonary TB failed to detect differences between the groups for ESAT-6 and CFP-10 antigens . The response to the PPD antigen was not evaluated in this context, and the ESAT-6 response in our study was too low to be analyzed.
Production of IFN-γ by cells stimulated with the 38 kDa/CFP-10 antigen was associated with VL control and higher CD4+ T cell counts in response to cART at the follow-up. Our results might suggest that with viral control and immune reconstitution due to cART in immunosuppressed patients (TB-HIV), it is possible to restore the immune response to Mtb-specific antigens with increased levels of IFN-γ. This is in accordance with a previous study in TB-HIV negative patients .
IRIS is expected to occur in immunosuppressed individuals after cART introduction, as the immune reconstitution is more intense at the beginning of the HIV treatment to the rapid control of VL, although it is not clear why this phenomenon affects only a subset of TB-HIV patients with lower CD4+ T cell counts [30,31,32]. The low incidence of paradoxical IRIS cases in the studied population precluded the analysis of the risk factors for IRIS. However, a distinct pattern of immune response was observed in these cases, which could help in the IRIS diagnosis and contribute to the understanding of this distinct phenomenon.
Immune reconstitution is an important factor to consider when choosing the best regimen to treat TB-HIV patients. Our study was nested in a clinical trial comparing the efficacy and safety of two different doses of efavirenz (600 mg and 800 mg) in TB-HIV, cART-naïve patients. Our results showed that the patients treated with 800 mg of efavirenz had a significantly greater production of IFN-γ in response to PPD and production with borderline significance in response to 38 kDa/CFP-10. These patients showed a stronger immune restoration compared to those who received the 600 mg dose, independent of the effect on CD4+ T cell counts or HIV VL. Studies have been conducted to compare the antiretroviral efficacy of these two different doses, and they have both been shown to be potent in terms of viral control [33, 34]. Our findings could contribute to a better understanding of immune reconstitution and be considered in the future when choosing the best strategy to treat TB-HIV patients with efavirenz-based regimens.
The present study has some potential limitations. A larger study population would have been necessary to more accurately demonstrate, by means of more significant power, whether the association between demographic, clinical and laboratory data with IFN-γ production was significant. The low proportion of IRIS cases also prevented the exploration of risk factors for this syndrome.
These findings highlight the differences in immune response according to the specificity of the Mtb antigen, which contributes to a better understanding of TB-HIV immunopathogenesis.