Immunological AspectsCharacterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis
Introduction
Tuberculosis (TB) remains as one of the major health problems worldwide.1 The failure of the epidemiological surveillance programs, the increase of the human immunodeficiency virus (HIV)-TB co-infection, and the emergence of multidrug resistant strains have contributed to its persistence and increased prevalence in many countries.2 Most infected individuals develop an immune response able to contain the microorganism, although the bacteria are not completely eliminated, leading to a latent infection (LTBi).3 Latency is considered a dynamic event in which an active immune response keeps the persistent mycobacteria under control.4, 5 Reactivation of the mycobacteria may occur in 5–10% of LTBi individuals during their life time, leading to active TB.
Two to four weeks after the infection, specific CD4+ and CD8+ T cell responses are evident. Among other cytokines, these cells produce IFN-γ and Tumor Necrosis Factor (TNF)-α6 that enhances the effector mechanisms of innate immunity, and IL-2 which is critical for the clonal expansion and differentiation of specific T cells.7 The adaptive immune response against Mycobacterium tuberculosis, encompasses the expansion of CD4+ and CD8+ effector cells and the generation of long-lived memory T cells.8, 9, 10 CD4+ T cells are considered the main source of IFN-γ, which is a critical cytokine for anti-TB response in both humans and mice that plays an important role in the anti-mycobacterial protective immune response.11, 12, 13 Mice deficient in CD4+ T cells are highly susceptible to M. tuberculosis infection although their IFN-γ production is only transiently diminished.14 Compelling evidence for the role of CD4+ cells in humans is provided by HIV+ individuals who are highly susceptible to TB reactivation and re-infection.15, 16 CD8+ T cells are also an important source of IFN-γ during in vivo M. tuberculosis infection17, 18, 19 and in vitro in response to mycobacterial antigens.20, 21, 22, 23 However, the CD4/CD8 ratio of IFN-γ production under different experimental or clinical conditions is not fully elucidated.
Decreased lymphocyte proliferation and production of IFN-γ, IL-2 by peripheral blood mononuclear cells in TB patients, compared to healthy controls, have been reported in cultures stimulated with different mycobacterial preparations.24, 25, 26, 27 The diminished IFN-γ production correlates with the severity of the disease and the development of extra pulmonary TB.28 On the other hand, it has been proposed that increased levels of IFN-γ production in individuals recently exposed to an active TB case are indicative of actively replicating mycobacteria and is thus a biomarker of high risk susceptibility.29 This hypothesis is supported by our findings in a cohort of household contacts of patients with active TB, in which incident cases were more frequent among high-IFN-γ producers.30 However, the type of T cells involved in these contrasting conditions, as well as the antigen-specific T cells in LTBi individuals have not been completely identified.
Measurement of T cell proliferation and IFN-γ production have been traditionally carried out following Purified Protein Derivative (PPD) stimulation, which do not differentiate among M. tuberculosis infection, BCG vaccination and exposure to environmental mycobacteria. However, the discovery of the region of differentiation 1 (RD-1), present in the genome of M. tuberculosis and few environmental mycobacteria, but absent in most non-tuberculous mycobacteria strains and Mycobacterium bovis BCG31, 32, 33, 34 led to the development of more specific diagnostic assays.35 The RD-1 genes encoded the 6 kDa early secretory antigenic target of 6 (ESAT-6) and the 10 kDa secreted protein from culture filtrated (CFP-10).36 These proteins are highly immunogenic and induce T cell proliferation and production of IFN-γ.37 Thus ESAT-6 and CFP-10 have been used to identify individuals infected with M. tuberculosis, independently of active or latent infection, from those BCG vaccinated or exposed to environmental mycobacteria.35, 38
Another protein used to identify latent infection is Rv2031c, also known as α-crystalline, HSpX or 16 kDa antigen, which is a cytosolic heat shock protein that is expressed as a result of an induced state of metabolic persistence by the intracellular environmental stress conditions of macrophages.39, 40 HSpX is recognized by T cells from household contacts of smear-positive PTB patients and patients with pulmonary TB and its induction of IFN-γ production correlates with M. tuberculosis infection.41, 42
Since the events underlying TB reactivation are still not completely understood, the comparison of the anti-mycobacterial immune response in patients with PTB and LTBi could help to elucidate these events. This may also lead to the development of early diagnostic procedures for individuals at risk of TB reactivation and the development of new strategies for TB control. In this study we quantified the percentage of CD4+ and CD8+ T cell precursors producing IFN-γ in response to PPD, CPF-10, ESAT-6 and HSpX antigens in long-term PBMC cultures of pulmonary tuberculosis patients (PTB), healthy household contacts (HHC) highly exposed to M. tuberculosis that had not developed active disease, and healthy tuberculin positive individuals (TST+) with no demonstrated recent exposure to TB patients. We also determined the memory phenotype of CD4 and CD8 IFN-γ-producing cells in these individuals. Our results show that in TB patients cell cultures stimulated with PPD and RD1 antigens, there are reduced frequencies of precursor CD4+IFN-γ+ and CD8+IFN-γ+ cells, compared with HHCs and TST+ healthy individuals. In HHC proliferation and production of IFN-γ was mediated by both T cell subpopulations, but mainly by CD4+ T cells. We also show that HSpX poorly stimulated IFN-γ production in all of the groups studied. Finally, under our culture conditions CD4 and CD8 cells producing IFN-γ exhibited a central memory phenotype.
Section snippets
Reagents and media
RPMI-1640 was obtained from Gibco-BRL (Grand Island, NY, USA), penicillin–streptomycin and the Limulus amebocyte Lysate (LAL) kit from Cambrex-BioWhittaker (Walkersville, MD, USA), Dulbeco’s PBS, Histopaque, bovine serum albumin (BSA) and Brefeldin A (BFA) from Sigma–Aldrich (St Louis, MO, USA). Bicinchoninic acid (BCA™) was purchased from PIERCE (Rockford, IL, USA). Carboxyfluorescein succinimidyl ester (CFSE) was purchased from Invitrogen (Eugene, OR, USA), monoclonal anti-CD4-PE-Cy5 (clone
Demographic and clinical characteristics of subjects studied
Table 1 shows the demographic and clinical characteristics of the subjects studied. There were not significant differences among the groups regarding age or gender. There were no differences in the age of TST+ individuals, HHCs, and PTB patients. BCG scar was found in 87% TST+ individuals, 83% HHCs and 72% PTB (ns). In TST+ individuals and HHCs, the TST induration diameter was ≥10 mm in all of them as defined in the inclusion criteria. Among PTB patients, the indurations were variable; 9 had
Discussion
The results presented herein show that both CD4+ and CD8+ precursor T cells proliferate and produce IFN-γ, but their proportion is different among the groups studied depending upon the antigen used. The percentage of CD4+ precursor cells and CD4+ producing IFN-γ in response to the antigens used was higher in HHCs, intermediate in TST+ subjects and lower in PTB patients. The percentage of CD8+ precursor cells was also higher in HHCs than in TST+ subjects and PTB patients, but the difference
Acknowledgements
The authors thank the Tuberculosis Control Programs of the Servicio Seccional de Salud de Antioquia and the Secretaria de Salud de Medellín for allowing us to have access to the patients and their clinical records. The authors recognize the valuable help of Sara C. París and Blanca L. Ortiz, and Luis F. Barrera for their critical reading of the manuscript. We also acknowledge the patients, their household contacts and the volunteers that kindly participated in this study.
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Evaluation of profile and functionality of memory T cells in pulmonary tuberculosis
2017, Immunology LettersCitation Excerpt :When reporting the result in index it is possible to perceive more clearly the response of PBMC by the presence of specific stimulus and eliminate the interference of memory cells present on the occasion of other stimuli in vivo. In addition, some studies have used the cell culture for a short period of time (16–24 h) [18,19,22,44], while others carried out cultures with long-term stimulation (5–9 days) [7,8,17], as well as this study. Long-term stimulation assays ensure more time for cells expansion, which corroborates for a greater sensitivity of results; allow the assessment of specific T antigen-specific cells, regarding the presence and the ability to produce functional markers such as cytokines; they are highly recommended to evaluate subpopulations of lymphocytes that require longer periods of antigen presentation to activate and expand (as in the case of the evaluation of memory cells, in which it was proposed a better measurement in long term assays); the results are less affected by manipulation of the cells during isolation or cryopreservation and the incubation time of PBMC, after blood collection, It is less important than in short-term tests [45].
- d
CMR was supported by the “Programa de Sostenibilidad”, Vicerrectoría de Investigaciones, Universidad de Antioquia, Medellín, Colombia.
- e
NDM is recipient of a predoctoral scholarship from Colciencias, Bogotá, Colombia.
- f
LFG and MR share senior authorship.