Functional profile of CD4+ and CD8+ T cells in latently infected individuals and patients with active TB.

Tuberculosis (TB) is one of the most important infectious diseases around the world. Several studies have focused on the identification of correlates of protection against TB. Most of them have concentrated on the study of IFN-γ due to its robust association with protection against TB. However, given the complexity of the immune response elicited after Mtb infection, other cytokines should also be considered. In the present study, we evaluated Th1 and Th17 responses and their association with the protection or development of active disease. Therefore, non infected individuals (nonTBi), latently infected individuals (LTBi) and patients with active TB (ATB) were studied. The evaluation of the number of cytokine producing cells by ELISPOT showed a higher number of IFN-γ-producing cells in ATB patients, but no differences were found regarding the number of IL-17 producing cells among studied groups. The evaluation of IFN-γ, IL-2, TNF-α and IL-17 producing CD4+ and CD8+ T cells at 1 day and 6 days of stimulation with mycobacterial antigens suggests the presence of functional signatures associated with latency or active TB. The results presented herein suggest the possible use of the evaluation of Th1-type cytokines, such as IFN-γ and/or TNF-α, as a correlate of protection against TB; however, these results need to be validated for other groups.

Reduced frequency of memory T cells and increased Th17 responses in patients with active tuberculosis.

Phenotypic and functional alterations in Mycobacterium tuberculosis T cell subsets have been reported in patients with active tuberculosis. A better understanding of these alterations will increase the knowledge about immunopathogenesis and also may contribute to the development of new diagnostics and prophylactic strategies. Here, the ex vivo phenotype of CD4(+) and CD8(+) T cells and the frequency and phenotype of gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-producing cells elicited in short-term and long-term cultures following CFP-10 and purified protein derivative (PPD) stimulation were determined in noninfected persons (non-TBi), latently infected persons (LTBi), and patients with active tuberculosis (ATB). Phenotypic characterization of T cells was done based on the expression of CD45RO and CD27. Results show that ATB had a reduced frequency of circulating CD4(+) CD45RO(+) CD27(+) T cells and an increased frequency of CD4(+) CD45RO(-) CD27(+) T cells. ATB also had a higher frequency of circulating IL-17-producing CD4(+) T cells than did LTBi after PPD stimulation, whereas LTBi had more IFN-γ-producing CD4(+) T cells than did non-TBi. The phenotype of IFN-γ-producing cells at 24 h differs from the phenotype of IL-17-producing cells with no differences between LTBi and ATB. At 144 h, IFN-γ- and IL-17-producing cells were mainly CD45RO(+) CD27(+) T cells and they were more frequent in ATB. These results suggest that M. tuberculosis infection induces alterations in T cells which interfere with an adequate specific immune response.

Regulatory T cell frequency and modulation of IFN-gamma and IL-17 in active and latent tuberculosis.

Regulatory T cells (Tregs) play an essential role in immune homeostasis. In infectious diseases Tregs may inhibit protective responses facilitating pathogen multiplication and dissemination, but they may also limit the inflammatory response diminishing tissue damage. Although there is experimental and clinical evidence that Tregs are induced during Mycobacterium tuberculosis infection, their role in the immunopathogenesis of tuberculosis is still not completely understood. In this study, the phenotype, frequency and activity of circulating Tregs in active and latent tuberculosis were evaluated. Phenotypic analysis showed that Tregs were CD4(+)CD25(high)FOXP3(+)CD45RO(+)CD127(-). High levels of circulating Tregs were found in patients with active pulmonary tuberculosis, compared to individuals with latent infection. Treg activity was evaluated by ELISPOT by determining the effect of CD25(+) cell depletion on the frequency of IFN-gamma and IL-17 producing cells after in vitro stimulation with ESAT-6, CFP-10 and PPD. Treg depletion increased the frequency of IFN-gamma producing cells, without affecting the frequency of IL-17 producing cells, in both active and latent tuberculosis, irrespective of the antigen used. Neutralization of IL-10 did not have any effect on the frequency of IFN-gamma and IL-17 producing cells. Altogether, these results suggest that during active tuberculosis Tregs inhibit protective Th1 responses, but not the proinflammatory Th17 responses, facilitating mycobacterial replication and tissue damage.

IFNgamma response to Mycobacterium tuberculosis, risk of infection and disease in household contacts of tuberculosis patients in Colombia.

OBJECTIVES: Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNgamma produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNgamma levels in HHCs correlate with tuberculosis development. METHODS: A cohort of 2060 HHCs was followed for 2-3 years after exposure to a tuberculosis case. Besides TST, IFNgamma responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination. RESULTS: Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNgamma response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNgamma responders to CFP-10 (HR 1.82 95% CI 0.79-4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNgamma producers was observed (trend Log rank p = 0.007). DISCUSSION: CFP-10-induced IFNgamma production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprophylaxis to child contacts regardless of BCG vaccination.

Kidney graft recipients with pretransplantation HLA CLASS I antibodies and high soluble CD30 are at high risk for graft loss.

In the present study, we investigated whether pretransplantation HLA class I and class II antibodies and pretransplantation levels of soluble CD30 (sCD30) and IgA anti-Fab autoantibodies are predictive of kidney allograft survival. Pretransplantation sera of 504 deceased-donor kidney recipients were tested for IgG HLA class I and class II antibodies, sCD30, and IgA anti-Fab levels using the CTS 4 ELISA kit. Kidney graft survival was estimated by Kaplan-Meier method and multivariate Cox regression. Regardless of the presence of HLA class II antibodies, recipients with high HLA class I reactivity had lower 1-year graft survival than recipients with low reactivity (p < 0.01). Recipients with high sCD30 had lower 5-year graft survival rate than those with low sCD30 (p < 0.01). The sCD30 effect was observed in presensitized and nonsensitized recipients, demonstrated a synergistic effect with HLA class I antibodies (p < 0.001), and appeared to be neutralized in recipients with no HLA class II mismatches. IgA anti-Fab did not influence kidney graft survival. Our results indicate that high pretransplantation sCD30 levels and HLA class I positivity increase the risk of kidney graft loss regardless of other factors. Consequently, such determinations should be routinely performed to estimate recipients' risks of graft rejection before transplantation.

Interleuquina-17: características, vías de diferenciación, señalización y funciones biológicas / Interleukin-17: characteristics, differentiation pathways, signaling and biological functions

La Interleuquina 17 (IL-17) es una citoquina proinflamatoria con diversas funciones biológicas secretada por varios subtipos de células T activadas.Su receptor se encuentra en los distintos tipos celulares de un amplio rango de tejidos. La IL-17 se ha relacionado con el desarrollo de enfermedades autoinmunes, rechazo de aloinjertos, cáncer, respuestas de hipersensibilidad inmediatas y tardías y control de infecciones, entre ellas la respuesta inmune contra Mycobacterium tuberculosis. Esta revisión pretende abarcar los aspectos hasta ahora elucidados sobre las características, las vías de diferenciación de las células productoras de IL-17, así como la señalización y funciones de ésta.

Chemokine/cytokine production by mononuclear cells from human lymphoid tissues and their modulation by Mycobacterium tuberculosis antigens.

The majority of knowledge about the role of cytokines and chemokines in controlling Mycobacterium tuberculosis infection mainly derives from animal models. In humans, this knowledge is still mainly limited to the blood compartment or accessible lymphoid organs, such as tonsils. Here, we studied cytokine and chemokine production and their modulation by M. tuberculosis antigens in mononuclear cells from human blood, spleen and hilar lung lymph nodes. Results show that the kinetics and magnitude of cytokine and chemokine production varied according to the tissue of cell origin. Mycobacterium tuberculosis antigens enhanced cytokine and chemokine production in blood, but the enhancement was restricted in spleen and hilar lung lymph node cells. We show, for the first time in humans, differences in cytokine and chemokine microenvironments according to lymphoid tissues, and suggest that these differences may affect the way cells respond to M. tuberculosis infection.

[Human leucocyte antigen gene (HLA-A, HLA-B, HLA-DRB1) frequencies in deceased organ donors]. / Frecuencias alélicas, genotípicas y haplotípicas HLA-A, HLA-B, HLA-DRB1 en donantes fallecidos, Medellín, Colombia.

INTRODUCTION: Genetic characterization of the human leucocyte antigen (HLA) system has provided insights into mechanisms of susceptibility to diverse diseases and immunological phenomena during pregnancy, as well as providing evidence for compatibility in the selection of organ transplant donors and recipients. OBJECTIVE: The HLA-A,-B,-DRB1 allele, genotype and haplotype frequencies were determined in deceased organ donors in Medellín, Colombia. MATERIALS AND METHODS: The genotypes of 926 deceased donors were evaluated over a 17-year period (1989- 2006). HLA-A, HLA-B and HLA-DRB1 typing was performed by sequence specific primer-polymerase chain reaction (SSP-PCR). Maximum likelihood frequencies were estimated by the zipper version of expectation maximation algorithm. Hardy-Weinberg equilibrium were determined by an exact test analogous to Fishers test by using Markovs chain, and linkage disequilibrium between pairs of loci. RESULTS: Twenty-two, 43 and 14 alleles were identified for HLA-A, -B and -DRB loci, respectively. The most frequent were A*02, A*24, B*35, and DRB1*04. A deficiency in the proportion of heterozygotes in HLA-A and B loci (p<0.01 and p<0.00001, respectively). The most frequent haplotypes were as follows: HLA-A*24, B*35 (7.7%) for HLA-A,-B; HLA-B*35, DRB1*04 (6.4%) for HLA-B,-DRB1 and HLA-A*24, DRB1*04 (8.9%) for HLA-A,-DRB1. For the 3 loci HLA-A,-B,-DRB1, the most frequent haplotypes were A*24, B*35, DRB1*04 (4.6%) and A*24, B*61, DRB1*04 (2.0%). CONCLUSIONS: These results confirm the three-ethnic ancestry of the Medellin population. The predominance of Caucasian admixture differs from many other Latin-American populations and can serve as a reference for comparative studies of these populations as well as applications within the Medellin population.

Functional and phenotypic changes in monocytes from patients with tuberculosis are reversed with treatment.

Alterations of monocyte/macrophages have been reported in patients with tuberculosis (TB), but their significance is poorly understood. Blood mononuclear cells from patients with different clinical forms of TB, at various times of anti-TB treatment, and healthy tuberculin positive individuals, were double-stained for CD14 plus CD206, TLR-2, IFN-gammaR1, CD40, HLA-DR, CD36 and CD163, and analyzed by flow cytometry. Monocytes were infected with Mycobacterium tuberculosis H37Rv and 24h later the phenotype, induction of necrosis and apoptosis and production of tumor necrosis factor TNFalpha, interleukin (IL)-10 and IL-12p40 were determined. TB patients presented higher percentage of CD14+ cells but lower percentage of CD14+DR+ and CD14+CD36+ cells. Expression of CD14, HLA-DR and CD36 was decreased in TB patients. Normal percentages and expression were restored during anti-TB treatment. Monocytes from TB patients underwent necrosis and apoptosis after M. tuberculosis infection, whereas monocytes from healthy controls exhibited only apoptosis. Anti-TB treatment reverted necrosis. There were no differences between the various clinical forms of TB. In vitro M. tuberculosis infection decreased expression of the membrane molecules studied. HLA-DR and CD36 inhibition correlated with induction of apoptosis. Restoration of monocyte alterations during anti-TB treatment suggests that such alterations may be caused by the high M. tuberculosis load present during active disease.

Chemokine receptor expression and modulation by Mycobacterium tuberculosis antigens on mononuclear cells from human lymphoid tissues.

Chemokine receptor switching on lymphoid cells is an important factor regulating migration and homing, but little is known about the expression of such molecules during Mycobacterium tuberculosis infection in humans. We describe CCR2, CCR5 and CCR7 expression on human cells from blood, spleen and pulmonary hilar lymph nodes (PHLN) stimulated by M. tuberculosis antigens. CCR2 was not expressed by CD3+ cells regardless of the presence of antigen, but was highly expressed on CD14+ CD63+ monocytes/macrophages. CCR2 decreased on splenic monocytes/macrophages by nearly 50% in culture, independent of antigen, but remained high in blood and PHLN. CCR5 was low in CD3+ cells and was down-regulated by M. tuberculosis antigens on blood and splenic cells but not in PHLN. CCR5 was highly expressed on monocytes/macrophages and was down-regulated by M. tuberculosis antigens at 48 hr only in blood. Less than 15% of CD3+ cells from spleen and PHLN were CCR7+, whereas nearly 40% from blood expressed this receptor on primary isolation. However, CCR7 in PHLN increased in culture, independent of antigen. Monocytes/macrophages did not express CCR7. Thus, we characterize, for the first time, chemokine receptor expression and differential modulation by M. tuberculosis antigens on human mononuclear cells from spleen, blood and PHLN. Knowledge of chemokine receptor switching in human lymphoid tissue provides novel insight into mechanisms of the immune response to M. tuberculosis with potential effects on directing cell trafficking.

Cytokine gene polymorphisms in Colombian patients with different clinical presentations of tuberculosis.

Tuberculosis (TB) has different clinical presentations. Pulmonary TB affects only the lungs and exhibits variable anti-mycobacterial immune responses. Pleural TB is a localized disease with a strong immune response. Miliary TB is a disseminated form with poor immune response. Cytokines play a pivotal role in anti-mycobacterial response and may determine the type of TB. Thus, gene polymorphisms associated with cytokine production may be associated with clinical presentations of TB. In this study, 54 tuberculin-negative healthy controls, 81 tuberculin-positive healthy controls, 140 patients with pulmonary TB, 30 with pleural TB and 20 with miliary TB were studied. Single nucleotide polymorphisms were typed for tumour necrosis factor-alpha, interferon-gamma (IFN-gamma), transforming growth factor-beta1, interleukin-10 (IL-10) and interleukin-6 by sequence-specific primer polymerase chain reaction (SSP-PCR). Allelic, genotypic and haplotypic associations with clinical forms of TB were evaluated. IL-10 -1082 A/A genotype and IFNgamma+874 T allele were associated with pleural TB. Seventy-five extended genotypes were found; two differed between patients and controls, and two between groups of patients. Results suggest that IL-10 low-producer polymorphism and IFN-gamma high-producer polymorphism are associated with pleural TB.

Kidney transplant patients with long-term graft survival have altered expression of molecules associated with T-cell activation.

BACKGROUND: Patients with long-term functioning organ allografts may have developed different mechanisms that explain the lack of graft rejection. However, it is not known how these mechanisms interplay or whether one of them predominates in such situations. METHODS: The authors analyzed the expression of T-cell surface molecules involved in alloantigen recognition, signal transduction, co-stimulation, and activation markers on circulating T cells from patients with normal kidney function 10 or more years after transplantation, short-term survival (1-4 years), and chronic rejection and from healthy adults. The percentage and the median fluorescent intensity of each marker were determined by flow cytometry. Proliferative response against specific and third-party donors and mitogenic stimulation were also determined. RESULTS: Peripheral blood lymphocytes from patients with long-term surviving kidneys had decreased expression of T-cell receptor (TCR)-alphabeta (P < 0.01), CD3epsilon (P < 0.05), and CD3 zeta-chains (P < 0.001); diminished percentages of CD4(+)CD28(+) (P < 0.001) cells; and increased expression of CTLA-4(+) (P < 0.01) on CD3(+) cells. CD4(+)CD25(+)CD69(+) cells were also increased in long-term surviving patients. Long-term surviving patients had decreased donor-specific proliferative responses. CONCLUSIONS: The decreased expression of TCR-alphabeta and epsilon- and zeta-chains on circulating T cells of long-term surviving patients suggests that these cells may have defects in alloantigen recognition or signal transduction that may result in decreased numbers of T cells expressing co-stimulatory molecules and activation markers as well as a decreased specific proliferative response. The decrease in the percentage of CD28(+) cells and the increase in CD4(+)CD25(+)CD69(+) cells suggest that regulatory mechanisms of the immune response are still active in such patients.

Differential induction of apoptosis and necrosis in monocytes from patients with tuberculosis and healthy control subjects.

BACKGROUND: Mycobacterium tuberculosis and purified protein derivative (PPD) induce apoptosis in murine macrophages and apoptosis and necrosis in human monocytes and alveolar epithelial cells. Macrophages from bronchoalveolar lavages and granulomas from patients with tuberculosis (TB) present both types of cell death; however, the significance of the type of cell death in TB remains uncertain. METHODS: Monocytes from PPD-positive control subjects and from patients with TB were exposed to PPD or M. tuberculosis. Apoptosis, necrosis, and the percentage of tumor necrosis factor (TNF)-alpha -positive and interleukin (IL)-10-positive cells were determined cytofluorometrically. Levels of lactate dehydrogenase, TNF-alpha, and IL-10 were measured in culture supernatants. The role of TNF-alpha and IL-10 was tested by blockade experiments. RESULTS: PPD and M. tuberculosis induced apoptosis in monocytes from PPD-positive control subjects, whereas cells from patients with TB presented apoptosis and necrosis. Cells from PPD-positive control subjects produced mainly TNF-alpha, whereas cells from patients with TB produced mainly IL-10. Blockade experiments suggest that TNF-alpha and IL-10 regulate the type of cell death occurring in response to M. tuberculosis. CONCLUSIONS: Results suggest that apoptosis of monocytes exposed to mycobacteria may partly explain the protective immune response found in PPD-positive control subjects, whereas necrosis may be determinant of the bacterial dissemination and tissue damage that occur in patients with active TB.

Determinación de anticuerpos y aloanticuerpos linfocitoxicos en pacientes candidatos a trasplante renal

Las pruebas cruzadas para detectar la presencia de anticuerpos linfocitotóxicos en el suero de candidatos a trasplante renal, son decisivas en el estudio pretrasplante. En el presente estudio se reportan los hallazgos en 97 pacientes con insificiencia renal crónica, candidatos a trasplantes y 127 posibles donantes vivos relacionados. Las pruebas incluyerón detección de anticuerpos antilifocitos T y B separadamente a 4,20 y 37 grados contra linfocitos del posible donante y contra las células autólogas. Se incluye además el tratamiento con ditiotreitol (DTT) para diferenciar entre los isotopos IgM e IgG. Los resultados muestran que 40.2 por ciento de los pacientes y 16.5 por ciento (p<=0.04) de los donantes clínicamente sanos tiene anticuerpos que pueden reaccionar con células alogénicas. Los aloanticuerpos se dectectaron en 38 por ciento de los pacientes. La mayoria de los anticuerpos estaban dirigidos contra los linfocitos B (71.7 por ciento) y correspondieron al isotipo IgM (66.7 por ciento), aunque tambien se detectaron tanto auto como aloanticuerpos IgG.No se detectó un efecto significativo de las trasfuciones o embarazos previos en el desarrollo de anticuerpos ni de aloanticuerpos. De otro lado se observó una frecuencia significativamente mayor (p=0.0009) de donantes intrafamiliares con autoanticuerpos positivos, en pacientes tambien positivos para autoanticuerpos que de receptores negativos para autoanticuerpos positivos que fueron trasplantados con riñones provenientes de sus donantes intrafamiliares o de cadáver, tienen sus injertos funcionantes hasta un año después. Los pacientes sin autoanticuerpos presentaron una menor sobrevida de sus injertos especialmente en el caso de los injertos de cadáver, de los cuales soló sobrevivió la tercera parte después de un año. Los resultados hacen énfasis en la necesidad de realizar las pruebas cruzadas previas al trasplante en condiciones que permitan obtener la mayor información posible sobre los anticuerpos linfocitotóxicos presentes en el suero de los pacientes candidatos a trasplante renal.