Tuberculosis is an important infectious disease in humans accounting for nearly 1.7 million deaths, annually . The disease is caused by Mycobacterium tuberculosis, which is the leading cause of morbidity and mortality in developing countries among patients immunocompromised by infection with HIV .
In healthy individuals, M. tuberculosis establishes a latent infection that is tightly controlled by M. tuberculosis-specific cellular immune responses . In HIV-infected individuals, the ongoing viral replication and virus-induced cell death during acute HIV-infection results in massive depletion of memory CD4 T cells at mucosal sites that can be partially restored, in particular under antiretroviral therapy (ART) [3,4]. However, if CD4 T-cell loss and functional impairment of cellular immunity continues in the chronic phase of HIV-infection, this may contribute to reactivation of tuberculosis from latency. In addition, development of tuberculosis may further be favoured by reinfection with the pathogen. Obviously, this is more frequently observed in countries with a high tuberculosis burden as compared with low-prevalence countries [1,2].
The factors that determine the antigen-specificity of CD4 T cells restored after the initial massive T-cell loss during primary HIV-infection are not completely understood. It may depend on the individual history of prior infections and vaccinations or continuous exposure to environmental antigens. Both possibilities seem to occur in other immunocompromised individuals such as solid organ transplant recipients or patients after stem cell transplantation. In solid organ transplant recipients, nondepleting immunosuppressive drugs mediate a generalized suppression of T-cell functionality with only minor measurable changes in the specificity of the individual T-cell repertoire that is established. This is illustrated by the fact that long-term transplant recipients in low tuberculosis endemic regions retain M. tuberculosis-specific T-cell immunity despite the absence of continuous rechallenge [5,6]. In contrast, the T-cell specificities emerging after severe lymphocyte-depletion in stem cell transplant recipients do not necessarily resemble those of the stem-cell source, but rather seem to be driven by environmental antigens or persistent pathogens of the recipient .
This study was based on the hypothesis that continuous antigen encounter determines the specificity of the T-cell repertoire emerging in HIV-infected individuals. This was investigated by a comparative analysis of M. tuberculosis-specific CD4 T-cell responses in HIV-positive individuals in tuberculosis high and low-endemic countries. Immunity toward cytomegalovirus (CMV) was analyzed as control for a persistent pathogen that poses a constant antigenic challenge to the immune system. We provide evidence that the specificity of the T-cell repertoire is antigen-driven, as HIV-infected patients from tuberculosis low-endemic regions show a lower prevalence and frequency of specific immunity toward M. tuberculosis, whereas constant exogenous exposure in high-endemic regions contributes to its maintenance. Together with preserved CMV-specific immune responses in both groups, this indicates that the T-cell repertoire in HIV-infected individuals is restricted to specificities that are urgently needed for pathogen control.
The study was conducted among 60 HIV-infected individuals from Homburg, Germany (low-tuberculosis-endemic country), and 39 HIV-infected patients from Mbeya, Tanzania (high-tuberculosis-endemic country). Immunocompetent HIV-seronegative individuals from both countries served as controls (Table 1)  and were recruited from the same geographic regions as HIV-positive patients. All individuals from Germany were German whites except for two HIV-infected individuals of African origin. All individuals from Tanzania were of African ethnicity. Study participants had no signs or symptoms of active tuberculosis during the study period. Furthermore, a group of 34 HIV-infected patients with active tuberculosis from Tanzania were recruited. Active disease was diagnosed by clinical symptoms and sputum smear testing. Bacillus Calmette-Guerin vaccination status, CD4 cell counts and ART status was not consistently available for all study participants (Table 1). The study was approved by the local ethics committees and all individuals gave informed consent.
Quantitation of antigen-specific CD4 T cells
Quantitation of antigen-specific CD4 T cells was performed using intracellular cytokine-staining from heparinized whole-blood samples as described before . Briefly, samples were stimulated for a total of 6 h using purified protein derivative (PPD, 222 IU/ml, Tuberkulin-GT1000; Chiron-Behring, Marburg, Germany; diluent as negative control), CMV antigen (32 μl/ml; Virion, Würzburg, Germany; control-antigen as negative control) and Staphylococcus aureus Enterotoxin B (SEB, 2.5 μg/ml; Sigma, Deisenhofen, Germany) in the presence of 1 μg/ml αCD28 and αCD49d (BD, Heidelberg, Germany), respectively. The percentage of antigen-specific CD4 T cells was determined as activated CD69-positive/IFNγ-coexpressing cells (Fig. 1a) and calculated by subtraction of the frequency obtained by the respective control stimulations. The lower limit of detection is 0.05%.
Statistical analysis was performed using Prism V5.01-software (Graphpad, San Diego, California, USA). Significant differences were determined using the Mann–Whitney test for comparison of two groups or Kruskall–Wallis test for comparison of three groups. Fisher's test was used to qualitatively compare the percentage of individuals reactive toward PPD, and the Spearman test was used to analyze correlations between PPD-specific T-cell frequencies and viral load or CD4 T-cell counts.
A low prevalence and frequency of purified protein derivative-specific immunity is a particular feature of HIV-related immunosuppression in tuberculosis low-endemic regions
To test if HIV-related immunodeficiency may affect cellular immunity toward mycobacterial antigens, PPD-reactive CD4 T cells were assessed in 60 HIV-infected individuals and 144 age-matched HIV-seronegative healthy controls from Germany. Typical dot plots of antigen-specific CD4 T cells are shown in Figure 1a. The percentage of individuals with detectable T-cell immunity toward PPD was significantly lower in HIV-positive patients (20%) as compared to healthy controls (52.7%, P < 0.0001, Fig. 1b). In line with these results, HIV-positive patients showed a significantly lower frequency of PPD-specific CD4 T cells (median = 0.008%, ≤0.001–2.08%) as compared to immunocompetent individuals (median = 0.05%, ≤0.001–3.76%; P < 0.001). Interestingly, this was not due to a general loss of immunity toward recall antigens, as both groups did not differ in CMV-reactive or SEB-reactive T-cell frequencies (P = 0.20 and P = 0.30, respectively, Fig. 1b).
A high antigen prevalence is associated with a sustained purified protein derivative-specific immunity in HIV-infected individuals from a tuberculosis high-prevalence region
The observed loss in PPD-specific immunity in HIV-positive individuals may be due to a considerably low antigenic challenge in a low-endemic region such as Germany. To assess the effect of a higher extent of exogenous challenge with M. tuberculosis, PPD-reactive T cells were analyzed in a group of 39 HIV-infected patients from a high-prevalence country. Notably, in this setting, the percentage of patients with detectable PPD-specific immunity was higher (64.1%) as compared to HIV-positive patients in a low-endemic region and indistinguishable to HIV-seronegative healthy controls (51.6%, P = 0.34). Likewise, median frequencies of PPD-reactive CD4 T cells did not differ between HIV-positive individuals and controls from endemic regions (0.09%, ≤0.001–1.91% and 0.06%, ≤0.001–0.99%, respectively, P = 0.29, Fig. 1c). Moreover, the two groups did not differ in their frequencies of reactive T cells toward CMV (P = 0.22) and SEB (P = 0.17, Fig. 1c). Together this indicates that an increased extent of mycobacterial exposure in a high-endemic area may contribute to the maintenance of specific immunity in HIV-infected individuals. Of note, differences in sex did not have any confounding effect on our results observed in low and high endemic regions. In Germany, the percentages of both male and female HIV-infected individuals with positive PPD responses was lower as respective controls (45.7% female controls vs. 14.3% female HIV-infected individuals, P = 0.04; 66.0% male controls vs. 21.7% male HIV-infected individuals, P < 0.0001). Likewise, overall results in Tanzania were not confounded by sex (60% female controls vs. 63.2% female HIV-infected individuals, P = 1.00; 47.6% male controls vs. 77.8% male HIV-infected individuals, P = 0.23). Moreover, there was no significant difference in the percentage of PPD-positive responses in patients with and without ART (Germany: P = 1.00, Tanzania: P = 0.74).
To assess the effect of active tuberculosis on PPD-specific immunity in HIV-infected individuals, a total of 34 patients with active tuberculosis were recruited from the same high-endemic region. In this group, both the prevalence of a specific response (88.2%) as well as their median frequencies (0.9%, ≤0.001–5.2%) were significantly higher as compared to HIV-infected patients or healthy controls without active tuberculosis (P < 0.001, Fig. 1d). PPD-specific T-cell frequencies did not show any correlation with CD4 T-cell counts/μl (r = 0.03, P = 0.89) or viral load (r = 0.02, P = 0.96). Likewise, although most patients with tuberculosis were treatment naive, their PPD-specific T-cell frequencies were still higher as compared to respective HIV-infected patients without tuberculosis (P = 0.017). Thus, differences in these parameters are unlikely to contribute to the increased frequency of PPD-reactive T cells in patients with active tuberculosis. As expected, tuberculosis in HIV-infected individuals did not have any effect on the frequencies of CMV-reactive or SEB-reactive CD4 T cells (P = 0.10). Notably, however, CMV-reactive T-cell levels in patients with active tuberculosis were slightly higher as compared to HIV-seronegative controls (P = 0.03).
This study demonstrates that the maintenance of antigen-specific T-cell responses in HIV-infected individuals is determined by continuous antigen exposure, which may result from either exogenous rechallenge with highly prevalent environmental microbes or periodic reactivation events by persistent pathogens. The effect of exogenous rechallenge is supported by our findings that PPD-specific immunity was largely lost in countries with low tuberculosis endemicity, whereas respective T-cell responses were preserved in a country with high tuberculosis prevalence. In line with this evidence and with results from immunocompetent individuals , an even stronger mycobacterial challenge during active tuberculosis was associated with a further increase in PPD-specific CD4 T-cell frequencies. Consequently, immunity toward a persistent pathogen such as CMV that poses a constant antigenic challenge to the immune system should not differ in magnitude between the two groups. Indeed, T-cell immunity toward CMV or the bacterial superantigen SEB was detectable in patients and controls from both low and high tuberculosis endemic areas. Together this suggests a constant skewing and restriction of specific T-cell immunity toward environmental antigens in HIV-infected individuals.
In general, PPD-specific immunity wanes in the absence of re-exposure, but this is rather a slow process that occurs over decades . Hence, the relative maintenance of PPD-specific immunity in healthy controls is a common observation even in low endemic countries such as Germany, where frequent exposure to M. tuberculosis is unlikely. Measurable PPD-specific immunity is observed not only in immunocompetent controls, but also in patients with other types of immunodeficiency such as chronic renal failure or renal transplant recipients [6,9]. Thus, given that HIV-infection is usually acquired later in life and that HIV-infected individuals were recruited from the same geographic region as controls, one would primarily expect similar levels of PPD-specific T cells as in controls or other immunocompromised groups. As there were no differences in ethnicity and countries of origin between controls and HIV-infected individuals in Germany, the lower frequency of PPD-specific T cells is a particular feature of HIV-related immunosuppression in low-prevalence regions and generally indicates a loss of T-cell specificities that are not urgently needed to control environmental or persistent pathogens. In line with this evidence, results from HIV-infected patients in Tanzania show that PPD-reactive T cells are maintained in areas of higher M. tuberculosis endemicity. Our observations are compatible with current views of HIV-pathogenesis and maintenance of antigen-specific memory CD4 T cells in long-term HIV-infected patients under ART . Although HIV-infection leads to a profound depletion of CD4 T cells already during the primary infection phase [12,13], subsequent treatment restores antigen-specific responses that mediate protection against opportunistic agents . The trigger that determines the antigen specificity of the recovering immunity seems to be environmental exposure to the antigen itself or other cross-reacting antigens. In support of this, ART in vaccinia-vaccinated patients did not lead to a full recovery of memory T cells specific for smallpox, a now eradicated virus , whereas T-cell responses against CMV, a persistent viral pathogen, were readily restored [14,15]. A similar dependence on antigen has been shown for specific T-cell responses recovering after profound lymphopenia in allogeneic stem cell transplant recipients, wherein early expansion of donor-derived CMV-specific T-cell clones in a recipient was observed only when the recipient was CMV seropositive . Thus, exposure to antigen is the key determinant of a measurable T-cell response in HIV-seropositive individuals.
Assessment of PPD-specific immunity using tuberculin skin testing is a widely applied diagnostic test to assess evidence of latent M. tuberculosis infection. It is well known that skin testing has a generally lower sensitivity in immunocompromised individuals . Although low skin-test sensitivity in patients on non-T-cell-depleting immunosuppressive medication may primarily be due to a drug-induced decrease in T-cell functionality , the sensitivity in chronically HIV-infected individuals may rather be directly linked to the net frequency of PPD-reactive T cells that are maintained in response to pathogen prevalence. In support of this, recent studies have made apparent that skin-test sensitivity is generally higher in individuals from high-prevalence countries as compared to that of patients from low-prevalence regions [16,17]. Although we did not perform skin testing in our study, HIV-infected patients from Tanzania would seem more likely to have positive skin-test results, as the percentage of individuals with detectable PPD-reactive T-cell frequencies was higher and median T-cell frequencies were more than six-fold higher as compared to those in patients from a low-prevalence country. Thus, our results not only may contribute to clarifying the apparent dilemma between differences in skin-test sensitivities in high and low-prevalence countries, but also improve our understanding on the use of T-cell-based tests in regions with different pathogen prevalence in general. Given the low prevalence of PPD-specific immunity, it is tempting to speculate whether HIV-infected patients from low prevalence regions are more prone to develop disease after exposure as compared to HIV-infected patients from high prevalence regions that have higher levels of measurable immunity. In addition, a positive PPD-specific immune response in an HIV-infected individual from a low-prevalence region may indicate re-exposure, and hence a higher positive predictive value for progression as compared to a positive PPD-specific immune response in HIV-negative individuals.
Our study has limitations in that both patients and controls showed some differences in sex, use of ART, CD4 cell counts and HIV-load. Nevertheless, we did not find any confounding effect of these parameters on the results described here.
In conclusion, the antigen-specific T-cell repertoire in chronically HIV-infected individuals seems to be skewed toward environmentally present antigens. Thus, pathogen prevalence may critically influence the specificity of this recovering repertoire in HIV-infected individuals. Whether the observed differences in the levels of PPD-specific immunity in HIV-infected individuals from low and high endemic regions may translate into differences in the control of M. tuberculosis after re-exposure merits further investigation.
We thank Candida Guckelmus, Karin Schindler and Vera Kleinfeldt for excellent technical assistance, and Dr Steffen Geis for the logistic support of the study in Tanzania. The participation of all patients and control persons is acknowledged.
Author contributions are as follows: conception and design: M.S., U.S., A.M.; experiments performed by J.D., A.S., A.H., N.E., C.G., E.S., L.M., K.S., M.H.; analysis and interpretation: A.S., J.D., M.S., U.S., A.M.; drafting the manuscript for important intellectual content: J.D., M.S., A.M.; all authors revised and approved the final version.
Financial support was given in part by grants from the Else Kröner Fresenius Stiftung and from ‘HOMFOR’ to M.S., from the Spanish Ministry of Science and Innovation (SAF2010-21336) to A.M. The work in Tanzania was supported by grants from the European Commission (DG XII, INCO-DC, ERBICA4-CT-2002-10035 and DG X, EuropeAID SANTE/2004/078-545/130) to M.H.
Conflicts of interest
The authors do not declare any conflict of interest.
2. Sester M, Giehl C, McNerney R, Kampmann B, Walzl G, Cuchi P, et al. Challenges and perspectives for improved management of HIV/Mycobacterium tuberculosis co-infection
. Eur Respir J
3. Geldmacher C, Schuetz A, Ngwenyama N, Casazza JP, Sanga E, Saathoff E, et al. Early depletion of Mycobacterium tuberculosis-specific T helper 1 cell responses after HIV-1 infection
. J Infect Dis
4. Guihot A, Bourgarit A, Carcelain G, Autran B. Immune reconstitution after a decade of combined antiretroviral therapies for human immunodeficiency virus
. Trends Immunol
5. Sester U, Wilkens H, van Bentum K, Singh M, Sybrecht GW, Schäfers HJ, Sester M. Impaired detection of Mycobacterium tuberculosis immunity in patients using high levels of immunosuppressive drugs
. Eur Respir J
6. Sester U, Junker H, Hodapp T, Schütz A, Thiele B, Meyerhans A, et al. Improved efficiency in detecting cellular immunity towards M. tuberculosis in patients receiving immunosuppressive drug therapy
. Nephrol Dial Transplant
7. Gandhi MK, Wills MR, Okecha G, Day EK, Hicks R, Marcus RE, et al. Late diversification in the clonal composition of human cytomegalovirus-specific CD8+ T cells following allogeneic hemopoietic stem cell transplantation
9. Sester M, Sester U, Clauer P, Heine GH, Mack U, Moll T, et al. Tuberculin skin testing underestimates a high prevalence of latent tuberculosis infection in hemodialysis patients
. Kidney Int
10. Sester U, Fousse M, Dirks J, Mack U, Prasse A, Singh M, et al. Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from nonactive States
. PLoS One
11. Mack U, Migliori GB, Sester M, Rieder HL, Ehlers S, Goletti D, et al. LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement
. Eur Respir J
12. Brenchley JM, Hill BJ, Ambrozak DR, Price DA, Guenaga FJ, Casazza JP, et al. T-cell subsets that harbor human immunodeficiency virus (HIV) in vivo: implications for HIV pathogenesis
. J Virol
13. Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, et al. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract
. J Exp Med
14. Puissant-Lubrano B, Combadiere B, Duffy D, Wincker N, Frachette MJ, Ait-Mohand H, et al. Influence of antigen exposure on the loss of long-term memory to childhood vaccines in HIV-infected patients
15. Sester U, Sester M, Köhler H, Pees HW, Gärtner BC, Wain-Hobson S, et al. Maintenance of HIV-specific central and effector memory CD4 and CD8 T cells requires antigen persistence
. AIDS Res Hum Retroviruses
16. Dheda K, van Zyl Smit R, Badri M, Pai M. T-cell interferon-gamma release assays for the rapid immunodiagnosis of tuberculosis: clinical utility in high-burden vs. low-burden settings
. Curr Opin Pulm Med
17. Elzi L, Schlegel M, Weber R, Hirschel B, Cavassini M, Schmid P, et al. Reducing tuberculosis incidence by tuberculin skin testing, preventive treatment, and antiretroviral therapy in an area of low tuberculosis transmission
. Clin Infect Dis