A distinct double positive IL-17A+/F+ T helper 17 cells induced inflammation leads to IL17 producing neutrophils in Type 1 reaction of leprosy patients.

Type 1 reactions (T1R) an inflammatory condition, of local skin patches in 30-40% leprosy patients during the course of MDT. IL-17A and IL-17F play an important role in regulating skin inflammation through neutrophils. In the present study, we have analyzed 18 of each T1R and Non-reactions (NR) patients through flow cytometry and qPCR. Interestingly we found that, CD3+CD4+ gated IL-17A+IL-17F+ cells were significantly high in T1R in both MLSA stimulated PBMCs and skin lesions as compared to NR leprosy patients. Hierarchical clustering analysis of gene expression showed that CXCL6, CXCL5, CCL20, CCL7, MMP13 and IL-17RB expression were significantly associated with IL-17A and IL-17F expression (Spearman r2 = 0.77 to 0.98), neutrophils and monocyte markers respectively. In this study, the inflammation noted in lesions of T1R is a different phenotype of Th17 which produce double positive IL-17A+IL17F+ and also contributes IL-17 producing neutrophils and thus would be useful for monitoring, diagnosis and treatment response before reactions episodes.

Leprosy in a patient infected with HIV.

A 60-year-old Nigerian man, who had lived in Europe for 30 years but had returned home frequently, presented with right frontalis muscle weakness and right ulnar nerve palsy, without skin lesions. Neurophysiology showed a generalised neuropathy with demyelinating features. Blood tests were positive for HIV, with a normal CD4 count. There was nerve thickening both clinically and on MRI. Nerve biopsy showed chronic endoneuritis and perineuritis (indicating leprosy) without visible mycobacteria. His neuropathy continued to deteriorate (lepra reaction) before starting treatment with WHO multidrug therapy, highly active antiretroviral therapy and corticosteroids. There are 10 new cases of leprosy diagnosed annually in the UK. Coinfection with HIV is rare but paradoxically does not usually adversely affect the outcome of leprosy or change treatment. However, permanent nerve damage in leprosy is common despite optimal therapy. Leprosy should be considered in patients from endemic areas who present with mononeuritis multiplex.

CD4+, CD8+, CD3+ cell counts and CD4+/CD8+ ratio among patients with mycobacterial diseases (leprosy, tuberculosis), HIV infections, and normal healthy adults: a comparative analysis of studies in different regions of India.

In this study, we estimated the CD4+, CD8+, CD3+ cell counts and the CD4/CD8 ratio among normal healthy controls (adults and children), leprosy patients (without any complications and during reactional states), TB patients (with and without HIV), and HIV-positive patients (early infection and full-blown AIDS) and correlated the changes with disease progression. In our study, it was observed that among adults, CD4+ cell counts ranged from 518-1098, CD8+ from 312-952, whereas CD4/CD8 ratio from 0.75-2.30. Among children, both CD4+ and CD8+ cells were more and the CD4/CD8 ratio varied from 0.91-3.17. With regard to leprosy patients, we observed that CD4+ and CD8+ cell counts were lower among PB (pauci-bacillary) and MB (multi-bacillary) patients. CD4/CD8 ratio was 0.99 ± 0.28 among PB patients while the ratio was lower, 0.78 ± 0.20, among MB patients. CD4+ cell counts were raised during RR (reversal reactions) and ENL (erythema nodosum leprosum) among the PB and MB patients whereas the CD8+ cell counts were lower among PB and MB patients. CD4/CD8 ratio doubled during reactional episodes of RR and ENL. Among the HIV-negative tuberculosis (TB) patients, both the CD4+ and CD8+ cell counts were found to be less and the CD4/CD8 ratio varied between 0.53-1.75. Among the HIV-positive TB patients and HIV-positive patients, both the CD4+ and CD8+ cells were very less and ratio drops significantly. In the initial stages of infection, as CD4+ counts drop, an increase in the CD8+ cell counts was observed and the ratio declines. In full-blown cases, CD4+ cell counts were very low, 3-4 to 54 cells, CD8+ cells from 12-211 and the ratio drops too low. This study is the first of its kind in this region of the country and assumes importance since no other study has reported the values of CD4+ and CD8+ T-lymphocyte counts among patients with mycobacterial diseases (leprosy and TB), HIV infections along with normal healthy individuals of the region, and correlation with clinical presentations of patients.

Decreased expression of CD3zeta and nuclear transcription factor kappa B in patients with pulmonary tuberculosis: potential mechanisms and reversibility with treatment.

BACKGROUND: The protective immune response against Mycobacterium tuberculosis relies both on antigen-presenting cells and on T lymphocytes. In patients with different forms of tuberculosis, varying degrees of T cell function--ranging from positive delayed-type hypersensitivity, in asymptomatic infected healthy individuals, to the absence of the response, in patients with miliary or pulmonary tuberculosis (PTB)--have been reported. The decreased expression of CD3zeta reported in T cells from patients with either cancer or leprosy has provided possible explanations for the altered immune response observed in these diseases. METHODS: The present study aimed to compare the expression of CD3zeta , nuclear transcription factor- kappa B (NF- kappa B), arginase activity, and cytokine production in 20 patients with PTB, in 20 tuberculin-positive asymptomatic subjects, and in 14 tuberculin-negative control subjects. RESULTS: Compared with those in tuberculin (purified protein derivative)-negative control subjects, peripheral-blood T lymphocytes from patients with active PTB had significantly (P < .001) decreased expression of CD3zeta and absence of the p65/p50 heterodimer of NF- kappa B. These alterations were reversed only in patients who responded to treatment. Also reported here for the first time is that the presence of arginase activity in peripheral-blood mononuclear-cell lysates of patients with PTB parallels high production of interleukin-10. CONCLUSIONS: The presence of arginase could, in part, explain the decreased expression of CD3zeta . These findings provide a novel mechanism that may explain the T cell dysfunction observed in patients with PTB.

CD-3 positive extranodal T-cell lymphoma of nasal type with skin involvement.

A 40-year-old previously healthy lady presented with nasal obstruction and localized plaques over the right arm. She developed complete nasal obstruction due to a mass in the right nasal cavity and skin lesions that ulcerated to present as ecthyma gangrenosum like lesions. Patient's condition deteriorated fast and she developed icterus with fatal outcome within 4 weeks of developing skin lesions. Nasal and skin biopsy revealed angiocentric T-cell lymphoma, which on immuno-phenotyping revealed CD-3 positive; and CD-20, CD-30, ALK and EMA negativity. She was seronegative for HIV. Final diagnosis of CD-3 positive extranodal T-cell lymphoma of nasal type was made. Extranodal T-cell lymphomas are very aggressive NHLs with poor prognosis. Prognosis depends on histology, stage of the disease and sites of involvement. NK/T cell lymphoma of nasal type is common with EBV association. Skin involvement is rare and is also an indicator of poor prognosis.

Local nerve damage in leprosy does not lead to an impaired cellular immune response or decreased wound healing in the skin.

This study investigated whether peripheral nerve damage in patients with leprosy impairs local cellular immune responses, thereby reducing wound healing and leading to chronic skin ulceration. Anesthetic and contralateral sensitive skin sites in 42 patients with leprosy were compared for delayed-type hypersensitivity responses to purified protein derivative (PPD) of tuberculin. Leukocyte recruitment, epidermal activation, keratinocyte proliferation, and rates of wound healing after skin biopsy were compared. No significant differences in PPD-induced induration, epidermal activation and thickening or numbers of total T cells, CD8+ T cells, CD1a+ Langerhans cells, and proliferating Ki67+ keratinocytes were observed between anesthetic and sensitive skin sites. Similarly, rates of wound healing over 5 days after skin biopsy did not differ significantly. Thus, local leprosy-associated anesthesia does not appear to contribute to local immune compromise or impaired wound healing. Rather, chronic cutaneous ulceration in leprosy most likely results from repeated trauma associated with loss of sensation.

Reactive oxygen species differentially affect T cell receptor-signaling pathways.

Oxidative stress plays an important role in the induction of T lymphocyte hyporesponsiveness observed in several human pathologies including cancer, rheumatoid arthritis, leprosy, and AIDS. To investigate the molecular basis of oxidative stress-induced T cell hyporesponsiveness, we have developed an in vitro system in which T lymphocytes are rendered hyporesponsive by co-culture with oxygen radical-producing activated neutrophils. We have observed a direct correlation between the level of T cell hyporesponsiveness induced and the concentration of reactive oxygen species produced. Moreover, induction of T cell hyporesponsiveness is blocked by addition of N-acetyl cysteine, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, and catalase, confirming the critical role of oxidative stress in this system. The pattern of tyrosine-phosphorylated proteins was profoundly altered in hyporesponsive as compared with normal T cells. In hyporesponsive T cells, T cell receptor (TCR) ligation no longer induced phospholipase C-gamma1 activation and caused reduced Ca(2+) flux. In contrast, despite increased levels of ERK1/2 phosphorylation, TCR-dependent activation of mitogen-activated protein kinase ERK1/2 was unaltered in hyporesponsive T lymphocytes. A late TCR-signaling event such as caspase 3 activation was as well unaffected in hyporesponsive T lymphocytes. Our data indicate that TCR-signaling pathways are differentially affected by physiological levels of oxidative stress and would suggest that although "hyporesponsive" T cells have lost certain effector functions, they may have maintained or gained others.

Reduced T-cell receptor CD3zeta-chain protein and sustained CD3epsilon expression at the site of mycobacterial infection.

Control of mycobacterial infection by the cellular immune system relies both on antigen-presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T-cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T-cell receptor signalling, we examined the protein expression of T-cell signal transduction molecules (CD3zeta, ZAP-70, p59fyn, p56lck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3zeta-chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3epsilon and CD3zeta expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3epsilon and CD3zeta expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3zeta compared to CD3epsilon. Using double immunofluorescence analysis, virtually no CD3zeta expression could be detected in comparison to the CD3epsilon expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3zeta-chain expression, which may be restored by cytokines. IL-2-enhanced zeta-chain expression and T-cell effector functions, defined by interferon-gamma release, in M. tuberculosis-specific and human leucocyte antigen-DR restricted CD4+ T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3zeta is essential for CD3 signalling and for eliciting T-cell effector functions, reduced CD3zeta protein expression could result in altered signal transduction and inefficient T-cell effector functions. Alternatively, reduced CD3zeta-chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.

T-cell release of granulysin contributes to host defense in leprosy.

A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

Thalidomide costimulates primary human T lymphocytes, preferentially inducing proliferation, cytokine production, and cytotoxic responses in the CD8+ subset.

The efficacy of thalidomide (alpha-phthalimido-glutarimide) therapy in leprosy patients with erythema nodosum leprosum is thought to be due to inhibition of tumor necrosis factor alpha. In other diseases reported to respond to thalidomide, the mechanism of action of the drug is unclear. We show that thalidomide is a potent costimulator of primary human T cells in vitro, synergizing with stimulation via the T cell receptor complex to increase interleukin 2-mediated T cell proliferation and interferon gamma production. The costimulatory effect is greater on the CD8+ than the CD4+ T cell subset. The drug also increases the primary CD8+ cytotoxic T cell response induced by allogeneic dendritic cells in the absence of CD4+ T cells. Therefore, human T cell costimulation can be achieved pharmacologically with thalidomide, and preferentially in the CD8+ T cell subset.

Proliferative responses of T cells from the skin and nerve lesions of leprosy patients.

In the current study we compared the mitogenic responses of T cells from skin and nerve biopsies of leprosy patients with those of peripheral blood mononuclear cells (PBMC). Lymphocytes from these sources were cultured at < or = 100 cells/well in the presence of PHA, irradiated autologous feeder cells, and IL-2, and proliferation was assessed after 6 to 12 days. Whereas PBMC were capable of vigorous responses, the growth of cells from skin and nerve was markedly reduced. The diminished response was independent of the clinical status of leprosy patients and was also observed in skin-infiltrating lymphocytes from patients suffering from other disorders. Analysis of proliferative responses at 1 cell/well suggested both a reduction in precursor frequency and a decrease in mean burst size. Analysis of lymphokine production suggested that cultured cells from skin lesions had reduced IL-w and IL-4 production relative to PBMC generated under similar conditions. Equal numbers of CD3+ cells were present in each source, but lesion cells were enriched in CD45RA- "memory" T cells, as well as CD3+CD28+ T cells. However, these alterations in subpopulation distribution could not account for the substantial differences in proliferative potential. We conclude that significant differences exist in the activation potential of cells from different tissue sources.

Functional heterogeneity among CD4+ T-cell clones from blood and skin lesions of leprosy patients. Identification of T-cell clones distinct from Th0, Th1 and Th2.

In the present study we examined the functional properties of T-cell clones reactive with Mycobacterium leprae and other mycobacterial antigens. Clones isolated from the skin lesions and blood of leprosy patients across the spectrum were exclusively CD4+CD8- and expressed the alpha beta T-cell receptor. Substantial heterogeneity in the production of cytokines, in particular interleukin-4 (IL-4), was observed, although no striking correlation with clinical status was apparent. A variety of patterns of cytokine secretion distinct from those of T-helper type-1 (Th1) Th2 or Th0, as defined in murine studies, was evident. Most noteworthy was a large number of clones from skin which secreted neither IL-2 nor IL-4, but large amounts of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Clones isolated from the blood of leprosy patients had a more restricted cytokine secretion profile, and appeared to resemble more closely previously described patterns, including those of high level production of IL-2 and/or IL-4. Virtually all clones, from either skin or blood, produced high levels of IFN-gamma, and thus many clones were IL-4 and IFN-gamma co-producers. The pattern of cytokine production by skin-derived T-cell clones was significantly affected by the in vitro activation status of the cells. Cells enriched in activated blasts tended to produce more IL-4 than small resting cells. In addition, the production of IFN-gamma by skin T-cell clones after < or = 10 weeks of culture was strikingly distinct from that of these clones after 5 months of culture. IL-4 and IFN-gamma co-producing clones shifted to a Th2-like pattern with much less IFN-gamma secretion, whereas non-IL-4-producing clones secreted much higher levels of IFN-gamma after prolonged culture, and became much more Th1-like. However, there was still no correlation between clinical status and pattern of cytokines produced. These results imply that a high fraction of T cells exists in leprosy lesions that is distinct from or that has not yet fully matured into Th1 or Th2 cells.

Molecular evidence for two forms of Crohn disease.

Recent epidemiological evidence suggests that there are two forms of Crohn disease (CD): perforating and nonperforating. We hypothesized that, just as with tuberculoid and lepromatous leprosy, differences in the two forms of CD would be both identified and determined by differences in the host immune response. Resected intestinal tissue from control patients as well as perforating and nonperforating CD patients was evaluated for mRNA levels. We employed 32P PCR amplification with published or custom-designed primers of a housekeeping gene (beta-actin); a human T-cell marker (CD3-delta); and the cytokines tumor necrosis factor alpha, transforming growth factor beta, granulocyte/macrophage colony-stimulating factor, interleukin (IL) 1 beta, IL-1ra, and IL-6. Differences were identified with IL-1 beta (control = 162 +/- 57 vs. perforating = 464 +/- 154 vs. nonperforating = 12,582 +/- 4733; P < or = 0.02) and IL-1ra (control = 1337 +/- 622 vs. perforating = 2194 +/- 775 vs. nonperforating = 9715 +/- 2988; P < or = 0.02). These data corroborate the epidemiological observation that there are two forms of CD. Nonperforating CD, the more benign form, is associated with increased IL-1 beta and IL-1ra mRNA expression. We conclude that it is the host immune response that determines which form of CD becomes manifest in any given individual and discuss the investigative, diagnostic, and therapeutic implications of these observations.

Chronic mucocutaneous candidiasis with deficient CD2 (E receptor) but normal CD3 mononuclear cells.

A case of chronic mucocutaneous candidiasis in a Malaysian child who subsequently developed disseminated tuberculosis and toxoplasmosis is described. The phenotype of her peripheral blood mononuclear cells showed discordance for her T cell markers. The presence of a subpopulation of CD2-/CD3+ mononuclear cells leading to an immunodeficiency state is consistent with failure of activation of CD2-mediated alternative pathway resulting in immunodeficiency. Such abnormal CD2-/CD3+ subpopulations have been described in lepromatous leprosy and foetal abortuses.

Human suppressor T cell clones lack CD28.

Previously we showed that certain T cell lines and clones from a lepromatous leprosy patient displayed a dose-dependent suppression of the proliferation of autologous T cells to Mycobacterium leprae (M. leprae) but not mitogen or an unrelated antigen. The latter cells were also cloned and did not display this suppressive activity, were CD4+ and proliferated vigorously to M. leprae presented by autologous HLA-DR molecules. We shall refer to these cells as T helper (Th) cells. Most of the suppressive T cell clones (Ts) were also CD4+ and also proliferated to M. leprae presented by HLA-DR, but much less strongly than Th cells. In this study we report on our search for (a) the mechanism of this apparently antigen-specific suppression by T cells, and (b) a possible phenotypic difference between Th and Ts clones. The two main conclusions are that Ts clones possess a lytic machinery, but that M. leprae-specific suppression and cytotoxicity can be clearly dissociated, and that the only phenotypic difference between Th and Ts is the presence of the CD28 marker on Th and its absence on Ts clones.

Suppression of T-cell proliferation by Mycobacterium leprae and its products: the role of lipopolysaccharide.

Addition of soluble molecules obtained from sonicated Mycobacterium leprae markedly suppressed the proliferative response to the mitogen anti-CD3 of peripheral blood mononuclear cells and isolated T cells. Suppression was nonspecific and occurred with cells from lepromatous and tuberculoid leprosy patients as well as control donors. The purified lipoarabinomannans from M. leprae and Mycobacterium tuberculosis had a similar spectrum of inhibition whereas their deacylated derivatives were without effect. All mycobacterial preparations of either a crude or purified state, which suppressed cellular responses, contained appreciable quantities of bacterial lipopolysaccharide by the Limulus amebocyte assay. Contamination with lipopolysaccharide could account for the extent and nonselectivity of the T-cell suppression. Suppression was also monocyte-dependent and in part due to the release of arachidonate metabolites of the cyclooxygenase pathway.

On the mechanism of human T cell suppression.

Previous evidence from several laboratories suggests that CD8+ T suppressor cells may be important regulatory elements governing specific unresponsiveness of lepromatous lepromatous leprosy patients to M.leprae. To analyse the mechanism of suppression, CD8+ Ts clones were established from lesions and peripheral blood of lepromatous patients and tested for ability to suppress antigen-responsive CD4+. Th clones or PBL. Suppression required induction by specific M.leprae antigen, but was effected in an antigen-non-specific fashion. The Ts clones failed to exhibit cytotoxicity of four antigen-exposed MHC-matched target cells: (i) an ori-SV40 transformed macrophage line; (ii) EBV transformed B cell lines; (iii) primary macrophages; and (iv) M.leprae responsive CD4+ cells. The possibility that Ts clones induce functional inactivation of CD4+ clones in vitro was investigated. M.leprae-responsive CD4+ clones were preincubated with Ts CD8+ clones, APC, and antigen for 16 h, after which the CD8+ cells were removed. The CD4+ clones with M.leprae and APC remained unresponsive to restimulation with APC and antigen for at least 10 days, although they responded to IL-2. Addition of IL-2 to the pre- or post-incubation cultures neither prevented the induction of unresponsiveness, nor reversed it. Earlier models of tolerance have suggested that receptor occupancy in the absence of second signals induces tolerance in B and T cells. Under conditions in which antigen responses of Th clones were HLA-DR-restricted, the Ts clones were able to suppress the response of DR mismatched Th clones. Thus, the effect of the Ts cells, like mechanisms requiring antigen presentation without a second signal, appears to be induction of clonal anergy in Th cells, perhaps by a novel mechanism.

The classical and alternate pathways of T cell activation are impaired in leprosy.

The proliferative response of circulating T lymphocytes from bacterial index-positive lepromatous patients to mitogenic anti-CD3 and pairs of anti-CD2 monoclonal antibodies was significantly reduced. In these patients, the CD2 but not CD3 receptor expression was down-regulated. Further, the CD2 modulation and the associated suppression of proliferative response to monoclonals was brought about in T cells of healthy subjects by prior incubation of mononuclear cells in vitro with Mycobacterium leprae. Thus, the T cell activation pathways through the CD3 and CD2 receptors are impaired in lepromatous leprosy patients and the impairment appears to be due to the modulation of the CD2 receptor specifically by M. leprae.