Study of T-cell Receptor Variable  (TCRV), Macrophages MigrationInhibitory Factor (MIF) and Natu

Abstract

Background: Leprosy exhibits a wide range of cell mediated response in disease status ranging from resistant form, tuberculoid leprosy (TT), to low resistant form, lepromatous leprosy (LL). T cells are important in the immune response to M-Leprae, the antigen recognition occurs via T cell receptor (TCR V b) that is embedded in the membrane of T cell.

Aim of Work: This work aimed to study some of the T cell receptor variable beta (TCR Vb) regions, Macrophages migration inhibitory factor (MIF) activity and natural killer cytotoxicity (NK) and their role in the pathogenesis of leprosy for better understanding of immune-pathogenesis of leprosy.

Methods: The study was conducted on thirty three patients with leprosy and diagnosed on the basis of clinical and bacteriological criteria, ten lepromatous leprosy (LL), eight tuberculoid Leprosy (TT), eight reversal reaction (RR) and seven erythrma nodosum leprosum (ENL), they were fifteen females and eighteen males with age ranged from 19-76 years. They were all subjected to slit skin smear for bacteriological examination and assessment of each case. Ten normal healthy subjects were the control group. Skin biopsies were taken for determination of TCR and MIF in skin by RT-PCR, serum samples for MIF estimation by ELISA, EDTA blood for detection of TCR in peripheral blood, heparinized blood samples for assessment of CD₁₆, CD₅₆ by flow cytometry and NK cytotoxicity using 4 hours chromium 51 release assay.

Results: Results of the present study showed expression of TCR Vb₆ and Vb₁₈ in skin lesion of 7/8 and 6/8 of TT patients respectively. Vb₆ was expressed in the skin and peripheral blood in 1/10 and 1/7 LL and ENL patients, respectively. Vb_6, Vb₁₂, Vb₁₄, Vb₁₈ were expressed in 3/8, 4/8, 2/8 and 3/8 RR patients respectively, while in the control group Vb₂ was expressed in only two subjects. As regards MIF, a significant increase in serum level of MIF as well as MIF m RNA in tissue of TT, RR and ENL patients was detected. Meanwhile, significant decrease of both serum level and tissue MIF m RNA was detected in LL patients. There was no significant difference in the percentage of CD₁₆, CD₅₆ NK cells in peripheral blood of TT or RR patients compared to controls. On the contrary, NK cytotoxicity showed significant higher level in TT and RR patients compared to controls. In contrast to LL and ENL patients there was significant decrease in the percentage of CD₁₆, CD₅₆ and NK cytotoxicity compared to control group.

Conclusion: In conclusion studies on TCR variable beta, NK cell number and function, MIF may reflect their important role in the pathogenesis of leprosy. Immunomodulation is of great importance and can serve an important and promising role for immunotherapy of leprosy.

Received: 12 May 2009

Accepted: 1^st referee: 14 June 2009

2^nd referee: 9 July 2009

Study of T-cell Receptor Variable b (TCRVb), Macrophages Migration
Inhibitory Factor (MIF) and Natural Killer Cytotoxicity in Leprosy

Elham Ragab Abd El Samee^*, Farha El Chennawy^*, Hala Ismail^**, Amal El Bendary^***, Said Abdou^***
and Zinab Abdel-Samad^****

Departments of Clinical Pathology^*, Mansoura University, Microbiology^**, Clinical Pathology^***
and Dermatology^****, Tanta University

Abstract

Background: Leprosy exhibits a wide range of cell mediated response in disease status ranging from resistant form, tuberculoid leprosy (TT), to low resistant form, lepromatous leprosy (LL). T cells are important in the immune response to M-Leprae, the antigen recognition occurs via T cell receptor (TCR V b) that is embedded in the membrane of T cell.

Aim of Work: This work aimed to study some of the T cell receptor variable beta (TCR Vb) regions, Macrophages migration inhibitory factor (MIF) activity and natural killer cytotoxicity (NK) and their role in the pathogenesis of leprosy for better understanding of immune-pathogenesis of leprosy.

Methods: The study was conducted on thirty three patients with leprosy and diagnosed on the basis of clinical and bacteriological criteria, ten lepromatous leprosy (LL), eight tuberculoid Leprosy (TT), eight reversal reaction (RR) and seven erythrma nodosum leprosum (ENL), they were fifteen females and eighteen males with age ranged from 19-76 years. They were all subjected to slit skin smear for bacteriological examination and assessment of each case. Ten normal healthy subjects were the control group. Skin biopsies were taken for determination of TCR and MIF in skin by RT-PCR, serum samples for MIF estimation by ELISA, EDTA blood for detection of TCR in peripheral blood, heparinized blood samples for assessment of CD₁₆, CD₅₆ by flow cytometry and NK cytotoxicity using 4 hours chromium 51 release assay.

Results: Results of the present study showed expression of TCR Vb₆ and Vb₁₈ in skin lesion of 7/8 and 6/8 of TT patients respectively. Vb₆ was expressed in the skin and peripheral blood in 1/10 and 1/7 LL and ENL patients, respectively. Vb_6, Vb₁₂, Vb₁₄, Vb₁₈ were expressed in 3/8, 4/8, 2/8 and 3/8 RR patients respectively, while in the control group Vb₂ was expressed in only two subjects. As regards MIF, a significant increase in serum level of MIF as well as MIF m RNA in tissue of TT, RR and ENL patients was detected. Meanwhile, significant decrease of both serum level and tissue MIF m RNA was detected in LL patients. There was no significant difference in the percentage of CD₁₆, CD₅₆ NK cells in peripheral blood of TT or RR patients compared to controls. On the contrary, NK cytotoxicity showed significant higher level in TT and RR patients compared to controls. In contrast to LL and ENL patients there was significant decrease in the percentage of CD₁₆, CD₅₆ and NK cytotoxicity compared to control group.

Conclusion: In conclusion studies on TCR variable beta, NK cell number and function, MIF may reflect their important role in the pathogenesis of leprosy. Immunomodulation is of great importance and can serve an important and promising role for immunotherapy of leprosy.

Introduction

Leprosy is an infectious disease caused by Mycobacterium leprae in which the clinical manifestations correlate with the cell-mediated immunity (CMI). At one pole, tuberculoid leprosy is characterized by strong CMI and a Th1 cytokine pattern has been documented in the lesions. On the other pole, lepromatous leprosy patients are weak responders to M. Leprae antigens and their lesions are characterized by a Th2 cytokine pattern. Arrest of mycobacterial growth is thought to be mediated in part by cytotoxic T lymphocytes, multibacillary (MB) patients are unable to generate a cytotoxic response to M. leprae heat shock protein 65 (hsp65). Considering that protection against intracellular pathogens is critically dependent on the function of NK cells at early stages of the immune response and on Th1 cells at later stages⁽¹⁾. IL-18 and IL-13, two cytokines that are able to influence NK cell activity, in the hsp65-CTL generation in PBMC from leprosy patients. The production of proinflammatory cytokines by phagocytic cells during infections starts a cascade of cellular reactions resulting in inflammation and activation of effector cells of innate immunity. Depending on the cytokine milieu, these early events will lead the immune response towards type 1 or type 2 activities which can reciprocally regulate each other. Upon infection with bacteria or intracellular parasites, monocytes/ macrophages produce interleukin (IL)-12 which is modulated by positive and negative feedback signals. IL-12 is required for proliferation and for IFNg production by Natural killer (NK) cells and differentiated T cells. It has been reported that IL-1, IL-2, IL-12, IL-18 and TNFa are potent inductors or coinductors of IFNg in NK and T cell. In addition, IFNg regulates a variety of immunological responses in both innate and acquired immunity⁽²⁾. In mycobacterial infections release of IL-2 and IFNg is generally associated with resistance to intracellular infections, whereas release of IL-4 and IL-10 is associated with progressive disease. As IL-12, IL-18 is a macrophages/monocyte derived cytokine that participates in the induction of IFNg production, in the increase of NK activity and in the stimulation of Th1 cell differentiation. NK cells were initially defined for their ability to spontaneously lyse certain tumour cells and virally infected cells. However, they do not lyse normal cells from the same host. NK cells recognize their target cells by different receptors that determine their function. Beyond their capacity to kill specific target cells, NK cells are also capable of producing type 1 or type 2 cytokines⁽³⁾. The early migration of IFNg-producing NK cells into the inflammatory sites is important in the generation of a Th1 response. On the other hand, enhancement of NK cytotoxicity by IL-18 does not require endogenous IL-12 production probably because the IL-18 receptor (IL-18R) is constitutively expressed on the surface of NK cells. Recent studies have revealed that IL-18 does not require help from IL-12 to induce Th2 cytokines production in T and NK cells while it induces IL-13 production by the same cells acting synergistically with IL-2 when IFNg is lacking. IL-13 is a pleiotropic cytokine derived from activated T cells that acts on monoctyes, macrophages, NK cells and normal and malignant B cells and is also involved in the development of humoral immunity. Although IL-13 has only 30% sequence homologuey with IL-4, both share biological functions by inducing the activation of STAT6 and JAK3 but differentially regulate functional activities in fresh primary T and NK cells. IL-13 strongly inhibits the production of inflammatory cytokines and chemokines by LPS-activated monocytes⁽⁴⁾. Mycobaterium leprae is an obligate intracellular bacterium that resides and replicates within macrophages and Schwann cells. It is the causative agent of leprosy, a potentially debilitating disease that is still prevalent worldwide^(5,6). Cellular immune response leading to elimination of the intracellular pathogen is considered to have an important role in determining either the development of protective immunity in healthy individuals or the evolution of symptoms and complications driving to the outcome of clinical disease⁽⁷⁾. M leprae specific cytotoxicity T cells with capacity to lyse mycobacterial antigen pulsed macrophages have been demonstrated in leprosy^(8,9). These cells may participate in the elimination of mycobacteria and at the same time in adverse responses observed in leprosy patients⁽¹⁰⁾. M. leprae specific T cell response is elicited by recognition of different mycobacterial antigens. Several M. leprae antigens have been identified and include proteins implicated in bacterial response to stress-heat shock protein (hsp)⁽¹¹⁾. Leprosy is characterized by a close relationship between the cellular immune reaction of the host and outcome of infection. It is thought that in most cases, infection with M. leprae results in an adequate cellular immune reaction and hence no disease manifestation⁽¹²⁾. In a minority of individuals, the cellular immune reaction is inappropriate or absent, resulting in either the tubercoloid (TT) or lepromatous (LL) form of leprosy, respectively^(13,14). T-cells are important in the immune response to M. leprae. They are stimulated by foreign protein antigen on target cells only when presented by restricting element that is provided by the major histocompatibility complex (MHC) class I or II molecules expressed on the antigen presenting cells. The antigen recognition occurs via T-cell receptor (TCR) that is embedded in the membrane of T-cell. This multi chain receptor complex has been well studied and shown to be responsible for both antigen and MHC recognition via two chains of the TCR complex termed a and b^(12,15). TCR genes are attractive candidates because they confer specificity on T cell recognition and must physically interact with HLA molecules in immune response. A functional b chain is formed by variables (v), diversity (D) and joining (j) region segments. Several clusters of complementarity determining region CDR1/CDR2/CDR3 can be recognized on the TCR^(12,16). In humans, evidence exists for limited TCR heterogeneity in autoimmune disease such as multiple sclerosis. However no evidence exist to date for limited TCR heterogeneity in the recognition of a small peptide in the context of a single HLA^(17,18). As regards Macrophage migration inhibitory factor (MIF), it was described as T-cell derived lymphokine with the potential to inhibit the random migration of macrophages. Previous studies on the inhibitory activity in leprosy showed difference in the ability of lymphocytes to elaborate MIF in different types of leprosy and in reactional states and was thought to have a role in the pathogenesis of leprosy⁽¹⁹⁾. Therefore, the aim of this study was to estimate the extent of T-cell receptor repertoire and MIF in lesional skin and peripheral blood as well as to estimate CD₁₆ & CD₅₆ natural killer cells and NK cytotoxicity in the peripheral blood of different types of leprosy in a trial to understand some immunological aspects of leprosy.

Subjects and Methods

Thirty three leprosy patients, diagnosed on the basis of clinical and bacteriological criteria and classified according to Ridley and Jopling⁽²⁰⁾, were studied: ten lepromatous leprosy (LL), eight tuberculoid leprosy (TT), eight reversal reaction (RR) and seven erythema nodosum leprosum (ENL). They were 15 females and 18 males. Their age ranged from 19-76 years. Slit skin smears were made routinely. All the patients included in this study were free of other infectious diseases and were not receiving any treatment for at least 3 weeks before beginning the study. All the patients were subjected to the following laboratory investigations: skin biopsies to detect the expression of TCR Vb and MIF using RT-PCR; serum samples for MIF estimation by ELISA and heparinized blood samples for determination of CD₁₆, CD₅₆ by flow cytometry and assessment of NK cytotoxicity using the standard four hour Cr¹⁵ release assay.

Total RNA extraction: after excision, skin biopsies were immediately homogenized using a rotor-stator homogenizer and total RNA was extracted from both homogenized skin sample and blood using lysis RLT buffer from RNA purification kit (QIA amp Mini Blood Kit-QIAGEN Inc. USA) according to manufacturer's instructions.

Reverse transcription polymerase chain reaction (RT-PCR): TCR Vb cDNA was synthesized from leucocytes and skin using reverse transcription primer CBR, 5'GCT CTA CCC CAG GCC TCG GCG C3', which recognizes bases common to Vb chain sequences and superscript II reverse transcriptase (GIBCO, Paisley UK). PCR reactions were carried out in a total volume of 50 ml containing 10 pmol of each primer, 50 mmol/L dNTPs (Pharmacia. LKB. Uppsala. Sweden) and 0.8 units of Taq polymerase (Perkin-Elmer Foster City, CA) in 1 X PCR buffer (50 mmol/L KCL. 20 mmol/L) Tris pH 8.4, 2.5 mmol/L MgCl₂ and 0.005% bovine serum albumin (Boehringer-Mannheim, Germany).

The parameters used for amplification of Vb 2, 5, 6, 12, 14, 18 were: 94°C for 30 sec (denaturation), 60°C for 30 sec (annealing) and 72°C for 60 sec (extension) for 35 cycles.

For MIF mRNA, template RNA was subjected to RT-PCR using QIAGEN. An initial step of RT at 50°C for 45 min. was followed by amplification reaction of PCR for 23 cycles of denaturation (94°C, 1 min) annealing (53°C for 2 min) and extension (72°C, 1 min) in Eppendorf-Netheler GmbH, Germany, thermal cycler. The sequences of primers were numbers according to Arden et al⁽²¹⁾. The used primer generated bands at 527 bp for Vb 2, 424 bp for Vb 5, 582 bp for Vb 6, 434 bp for Vb 12, 529 bp for Vb 14 and 455 bp for Vb18.

The sequences of available TCRVb primers, used through the study were:

Vb 2: 5'-CCACATACGAGCAAGGCGTCGA-3'

Vb 5: 5'-CTGATCAAAACGAGAGGACAGCA-3'

Vb 6: 5'-CAG GTG CTG GAG TCT CCC AG-3'

Vb 12: 5'-GAC AGA GGA TTT CCT CCT CAT-3'

Vb 14: 5'-GGGCTCGGCTAAGGCAGACCTAC-3'

Vb 18: 5'-AGCCCAATGAAAGGACACAGTCAT-3'

The human MIF primer⁽¹²⁾:

5' CGT TCC GAG CTC ACC CAG CAG 3'

5' GCA ACT AAG TCA TAG TCC GC 3'

They generated bands of 255 bp and those for b-actin were:

5' CGT TCT GGC GGC ACC ACC AT 3'

5' GCA ACT AAG TCA TAG TCC GC 3'

They generated bands of 254 bp.

PCR products were separated by electrophoresis on 2% agarose gel and visualized by UV light illumination using ethidium bromide staining. DNA ladder (Fx 174, Hae III, Sigma) was used as DNA size markers. Gel documentation system (Gel-pro Analyser version 3 Medica cybernetics, 1995-1997, USA) was used to analyze the band pattern, optical density: molecular size and copy number. The relative amount of the target gene was estimated according to gene copy number standard curve. The relative amount of gene expression = copy number of target gene/copy number of b-actin⁽²²⁾. The result was expressed as relative amount x 100.

ELISA: MIF was measured in the serum of patients by ELISA kit according to manufacturer's protocol (R & D system, Abingdom, UK)⁽²³⁾.

NK cytotoxicity: The cytotoxic activity of NK cells against K₅₆₂ (human erythroleukemia cell line) is assessed by the standard 4 hour Cr51 release assay according to Matsuzuki et al⁽²⁴⁾. The cell line is maintained in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, 2ml L-Glutamine and 1 mg/ml gentamycin and Fungizone (Sigma, Saint-Quentin, France) and cultured in 37°C, 5% CO₂ humidified incubator. The cytotoxicity was assayed as follows: The K₅₆₂ target cell (10⁶ cell) were labeled with 100 ml ⁵¹Cr (100 uci of ⁵¹Cr sodium chromate. Amersham, UK) for two hours at 37°C, then washed three times and 5 x 10³ (100ml) ⁵¹Cr Labelled target cells were mixed with effector's cells 100 ml at different effector/target ratios. After 4 hours at 37°C in humidified 5% CO₂ incubator, 100 ml of the supernatant was collected for liquid scintillation counting the radioactivity measured using gamma counter.

The percentage of specific lysis was calculated as follows:

% cytotoxicity =                                                                                              x 100

Experimental release was determined by measuring Cr⁵¹ release from labeled target cells with patients freshly separated PBMncs. Spontaneous release was determined by incubation of labeled target cells with medium. Maximum release was determined by solubilizing target cells using triton X 100, the spontaneous release was <10% of maximum release. Results are presented as the means of triplicate samples. The K₅₆₂ was kindly provided by Carosella, Saint Louis, France which was obtained from American Tissue culture collection (ATCC).

CD16, CD56 measurements: Heparinized blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation⁽²⁵⁾. The presence of lymphocytes expressing CD₁₆ and CD₅₆ were measured by coulter XL (R-E pics flow cytometry) using specific monoclonal antibodies (Diaclone, France). 50 ml of separated lymphocytes were added to 5 ml of CD₁₆ or CD₅₆ monoclonal antibody, then incubated for 30 min on ice. Non-specific binding was analyzed by incubation of cell suspension with FITC labeled F (ab) anti-mouse IgG (Diaclone, France). 2 ml of lysis buffer were added, vortex and after centrifugation for 5 min the supernatant was removed then 2 ml phosphate buffer saline (PBS) were added and centrifuged. The supernatant was decanted followed by addition of 300 ml phosphate buffer were added. The stained cells were analyzed using coulter XL (R-E pics flow cytometry). Results are expressed as percentage of positive cells.

Results

TCR: In TT (tuberculoid leprosy) group of patients (N=8). TCR Vb 6 was found to be markedly expressed in both skin lesion (21.5 ± 2.3) and peripheral blood PB (7.3 ± 2.3) in 7 out of 8 patients. Also, Vb 18 was expressed, though not markedly but in most of the patients, in skin (9.5 ± 4.5) and PB (8.3 ± 2.1) in 6 out 8 TT patients. TCR Vb 5 was expressed in skin samples (19.05 ± 11.01) and PB (7.7 ± 1.3) in 2 out of the 8 patients, while Vb 14 was expressed in skin sample (15.1 ± 2.4) and PB (6.3 ± 1.5) of 3 out of 8 of the same patients. It was also noticed that the percentage of expression of Vb 5, 6 and 14 was almost more than double fold in skin lesion than in peripheral blood.

On the other hand, patients with lepromatous leprosy (LL) (N=10) and those with erythema nodosum leprosum (ENL) (N=7), showed expression of Vb 6 in only one out of 10 and one out of 7 patients, respectively (table 1).

Patients with reversal reaction (RR) (N=8), showed expression of Vb 6, Vb 12, Vb 14 and Vb 18 in 3, 4, 2 and 3 out of 8 patients respectively. Meanwhile, Vb 2 was expressed in only 2 out of 10 controls.

MIF: From the results obtained, it was noticed that serum level of MIF was significantly increased in patients with TT (mean=32.4, SD ± 11.3), RR (mean =35.1, SD ± 12.2) and ENL (mean=36.2, SD ± 15.5) compared to controls (mean=10.2, SD ± 1.1). In addition, MIF mRNA was significantly expressed in TT (mean=0.38, SD ± 0.16), RR (mean=0.39, SD ± 0.17) and ENL (mean=0.41, SD ± 0.14), compared to controls (mean=0.26, SD ± 0.02). However LL patient, showed significant decreased both serum level (mean=5.7, SD ± 2.1) and tissue MIF mRNA (mean=0.16, SD ± 0.07) (tables 2 and 3).

CD₁₆, CD₅₆, Natural killer cells and NK cytotoxicity: There was no significant difference in the percentage of CD₁₆₊/CD₅₆₊ and NK cells in peripheral blood of TT patients or RR patients as compared to control. There was significantly higher NK cytotoxicity in TT patients and RR patients compared to control. In contrast to LL and ENL patients, there was significant decrease in the percentage of CD₁₆, CD₅₆ as well as NK cytotoxicity when compared to control group (Table 4).

Table 1.    TCR-variable Beta *gene expression in all studied patients and controls.

Table 2.    Serum macrophage migration inhibitory factor levels (ng/ml) in patients with leprosy and control

P<0.05 is significant     LL= lepromatous leprosy    TT= tubercoloid leprosy    RR= reversal reaction    ENL= erythema nodosum leprosum

Table 3.    MIF mRNA expression in leprosy patients and control

  *P< 0.05 is significant       *expressed as mean % relative to expression of b actin

Table 4.    Mean percentage of CD₁₆+ and CD₅₆+ and their cytotoxicity in leprosy patients and controls

                                              M       1           2            3          4          5          6          7                

Fig. 1.            Agarose gel electrophoresis for PCR products stained with ethidium bromide, detecting Vb expression in skin biopsy detected by RT-PCR. M=marker, lane (1) negative, lane (2) Vb2, lane (3) Vb5, lane (4) Vb6, lane (5) Vb12, lane (6)Vb14, lane (7)Vb18

                                                      M    1     2     3      4     5    6     7     8     9   10    11

                                                M    1       2     3    4     5     6     7     8     9   10   11

Fig. 2.            Agarose gel electrophorsis for PCR products stained with ethidium bromide, detecting MIF expression in skin biopsies, detected by RT-PCR. Above: M=marker, lane 1, 3, 4, 6, 8, 9, 10=skin biopsies positive for MIF mRNA, lane 2, 5, 7, 11 = negative samples for MIF expression. Below: M=marker, lane 2, 3, 4, 6, 7, 9 and 11 = positive samples while lane 1, 5, 8, 10 = negative samples for MIF.

Discussion

Leprosy continues to be an important public health problem for the developing world and an estimated 2-3 million people currently live with deformity brought by their disease⁽²⁶⁾. Though significant progress has been made in reducing new case presentation⁽²⁷⁾, improved diagnostic tests and therapeutic regimen are still needed. Two major obstacles impending the progress of research in these areas have been our inability to cultivate Mycobacterium leprae (the etiologic agent for leprosy) on artifical media in the laboratory and the lack of a robust animal model for studying this infection⁽²⁸⁾. Resistance to M. Leprae is mediated through cellular immune processes and involves a complex interplay of cytokines and chemokines. Prominent among these is interferon gamma (IFN-g), which stimulates macrophages to up-regulate antimicrobial, anti-tumour, and antigen processing and presentation pathways⁽²⁹⁾. In rodent immune system, activation of macrophages by IFN-g results in effective growth restriction and clearance of mycobacteria with production of reactive nitrogen intermediates as effector molecules. However this potent antimicrobial mechanism varies from species to species. Human IFN-g-activated peripheral blood macrophages demonstrate little or no production of nitric oxide and are unable to kill several different mycobacterial species⁽²⁾. Leprosy is a disease characterized by a close relationship between the cellular immune reaction of the host and outcome of infection^(30,31). The skin lesions of leprosy provide a window into the immuno regulatory events involved in the human immune response infection. T-cells are thought to play a role in the pathogenesis of different forms of the disease^(3,28). Reversal reaction represents naturally occurring delayed type hypersensitivity which is a classic measure of T-cell responsiveness to a foreign antigen^(32,33,34). Our study showed expression of some T-cell reactive variable B repertoire in TT and reversal reactions with a strong bias towards the use of the Vb₆, Vb₁₈ in TT and Vb₆, Vb₁₂, Vb₁₄, Vb₁₈ in reversal reactions. Wim et al⁽¹²⁾., and Swann et al⁽³⁵⁾., showed that the majority of M.leprae reactive clones from a single patients contain mRNA that employs V-region gene segments belonging to Vb₅ or Vb₁₈ family. Most DR₃ restricted clones from the tuberculoid leprosy patients use Vb5.1 gene segment and recognize the 65 heat shock protein (hsp) peptide from residue 2-12 (65 hsp 2-12) whereas most DR₂ restricted clones from the same patient express the Vb₁₈ gene segment and recognize the 65 hsp peptide from residues 418-427 (65hsp 418-427). Wang et al⁽³⁴⁾., and Sabet et al⁽¹⁷⁾., found that gene subfamilies V beta 6.1 through V beta 6.4 (Vb 6.1.4) were strongly over expressed in lesions versus PBMC of tuberculoid patients and lepromatous. Furthermore, Struyk, et al⁽¹⁸⁾, revealed the exclusion usage of TCR. BV₅ gene segment and a predominance of one V-D-J gene segment rearrangement which is indicative of clonal expansion of these T-lymphocytes in TT patients. In contrast, most LL and ENL patients in the present study showed a diverse usage of Vb chains. This was in agreement with previous studies⁽³⁴⁾, in which only few LL patients showed restricted TCR Vb⁶ usage. Wim et al⁽¹²⁾, showed that HLA DR₃ T cell clones preferentially use Vb5 while HLA DR₂ T cell clones preferentially use Vb₁₈ genes in TT patients, moreover, it was found that there was also a restricted T cell reactive V alpha repertoire in granulomatous leprosy lesion which suggest distinct T-cell recruiting mechanism^(36). The limited TCR repertoire expressed by the infiltrating T-cells may be due to a set of antigens which are recognized in TT and in delayed type hypersensitivity response to M. leprae^(1,37). It was found that mycobacterial components that are able to stimulate T-cell can be broadly divided into somatic and secreted antigens. Somatic antigens belonging to the heat shock protein (hsp) group are mainly derived from destroyed bacilli during the late phase of infection. While secreted antigens are produced by metabolically active organisms and are related to the early or active phase of infection. Furthermore, secreted antigens are readily accessible for MHC processing as long as the bacteria survive inside macrophages⁽³⁸⁾. On the other hand, somatic proteins are only released and processed by MHC after bacteria are killed by macrophages or by specific chemotherapy. Besides, it is well known that LL patients are responder to M. leprae antigens. This may be due to absence of free hsp 10 or incapacity of stressed antigen present in cells to process hsp 10 antigen, leading to impaired T-cell stimulation. It was previously found that immuno suppression in LL is produced by both macrophage suppression to M. leprae (early event) and T lymphocytes suppression (late event)^(2,39). The limited TCR repertoire in TT and RR accounts for a restricted immune response to M. leprae, and in particular to M. leprae 65 hsp, with trimming and preferential outgrowth of a certain TCR⁽⁴⁾. It was suggested that the MHC haplotype influences DTH by presentation of specific peptides with subsequent selection of specific TCRs followed by local oligocloncal expansion^(17,34). Therefore it is tempting to speculate that regulation of the disease through antigens TCR intervention is feasible in leprosy. This approach requires that the disease related T-cell use a restricted set of TCR genes^(1,12). As regard MIF, there was significant increase in its serum level as well as its mRNA relative amount in tissues of TT, RR and ENL patients, while it was significantly decreased in LL. In agreement with our results, Thomas et al.⁽⁴⁰⁾, found macrophage activity inhibition in serum of leprosy patients and reactional states. However, they found this activity also in serum of LL, but previous studies reported failure of LL lymphocytes to elaborate MIF. Similarly, Se Jong et al.⁽⁴¹⁾, found a defect in the production of leukocyte inhibitory factor in LL patients as compared to TT patients, also they proved that circulating immune complexes were higher among LL patients that TT patients. Defective production of MIF in LL patients might be attributed to the deficit in other lymphokines such as IL^(42,43) & INF-g^(44,45) in LL. The preponderance of CD8 T cells in skin lesions might be associated with subsequent defective expansion of specifically sensitized T cells, which account for decreased MIF activity⁽⁴⁶⁾. Recently, it was reported that MIF has a direct role in immune response as well as in delayed hypersensitivity reaction⁽⁴⁷⁾. Regarding NK cells, the present study showed significant increased NK cytotoxicity in TT and RR patients while this activity as well as CD₁₆⁺, CD₅₆⁺ were significantly lowers in LL and ENL patients. Silvia et al.⁽³⁹⁾, Yuko et al.⁽⁴⁸⁾, and Meyer et al.⁽⁴⁹⁾, suggested that the difference in cytotoxic activity in leprosy patients could be attributed to difference in the epitopes in the 10k Da antigen of M. leprae, while TT patients recognize both common and species specific epitopes. The impaired cytotoxic activity observed in LL patients could be ascribed either to imbalance in cytokine production and/or to poor antigen-presenting capability of stressed macrophages. Moreover, the low cytotoxic lymphocyte activity could be due to a low NK and/or gamma delta cell (which express CD₅₆⁺) biological function and/or presence of IL4 that down modulate INF-g production. It was found that the addition of INF-g, TNF and IL-12 could be key factors in the generation of cytotoxic lymphocytic activity in LL and ENL patients^(4,45). Moreover, IL-18 also enhances type 1 cytokine for mycobacteria reactive T cell clones with improvement of cytotoxic activity^(9,35,50). Finally, it could be concluded that although LL and ENL patients showed lower spontaneous cytotoxicity, it can be modulated favorably by lymphokines which indicate a promising role of immuno therapy in leprosy in the future. The studies related to the T-cell response in leprosy will allow for the development of strategies to control this disease. One obvious target for regulation of the disease would be a TCR idiotype. This approach requires the use of a restricted set of TCR genes by the disease-related T cells.

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