tcr paper of solomon

Int. J. Medical Engineering and Informatics, Vol. 4, No. 1, 2012 25

Copyright © 2012 Inderscience Enterprises Ltd.

T-cell receptor variable beta (1–24) gene repertoire in patients with Wuchereria bancrofti infections

M.S. Sudhakar Centre for Biotechnology, Anna University, Chennai 600 025, India E-mail: [email protected]

K.V. Alala Sundaram Department of Plastic Surgery, Royapettah General Hospital, Chennai 600 014, India E-mail: [email protected]

R.B. Narayanan* Centre for Biotechnology, Anna University, Chennai-600025, India Fax: 91-44-22350299 E-mail: [email protected] *Corresponding author

Abstract: T-cell receptor V beta (TCRVβ) gene repertoire (Vβ1–Vβ24) was evaluated in the peripheral blood mononuclear cells (PBMCs) from asymptomatic and amicrofilaremic normal individuals (EN), and patients with chronic pathology (CP) harbouring Wuchereria bancrofti. TCRVβ gene expression in phytohemagglutinin (PHA) stimulated PBMC cultures from EN and CP individuals was in the order EN > CP while in Brugia malayi adult antigen (BmA) or purified protein derivative from mycobacterium tuberculosis (PPD) stimulation or the unstimulated conditions, the order was CP>EN. Thus, the PBMCs of the CP patients showed elevated levels of TCRVβ gene expression both in the unstimulated and stimulated conditions compared to EN.

Keywords: Filariasis; T-cell receptor V beta; TCRVβ; Purified Protein Derivative; PPD; Brugia malayi adult antigen; BmA.

Reference to this paper should be made as follows: Sudhakar, M.S., Sundaram, K.V.A. and Narayanan, R.B. (2012) ‘T-cell receptor variable beta (1–24) gene repertoire in patients with Wuchereria bancrofti infections’, Int. J. Medical Engineering and Informatics, Vol. 4, No. 1, pp.25–35.

Biographical notes: M.S. Sudhakar completed his Masters in Biotechnology from Andhra University, Vizagapatnam and did his PhD in Biotechnology from Anna University, Chennai at Dr. Narayanan’s laboratory. At present he is working as an Assistant Professor, Department of Biotechnology, Rajalakshmi Engineering College, Thandalam.

26 M.S. Sudhakar et al.

K.V. Alala Sundaram is a well known surgeon handling patients with lymphoedema and performs corrective surgery. At present he is the Head, Department of Plastic Surgery, Government Royapettah Hospital, and Chennai.

R.B. Narayanan has a PhD in Immunology from University of London, UK. Subsequently, he has worked in immunology of leprosy at AIIMS, New Delhi, India and National JALMA Institute for Leprosy and Mycobacterial Diseases, Agra, UP. At present he is a Professor at Centre for Biotechnology, Anna University working in immunology of human filarial infections and shrimp viral infections. He has published several papers in refereed international and national journals. He has received research grants from various national and international agencies for his immunology work.

1 Introduction

Human lymphatic filariasis is a chronic infectious disease of humans caused by nematode parasites Wuchereria bancrofti, Brugia malayi, and Brugia timori. A striking feature of this disease is, different clinical manifestations exhibited by the endemic inhabitants (Ottesen, 1984). They include individuals who are clinically asymptomatic and characterised by persistent microfilaraemia (MF); followed by symptomatic patients with chronic lymphatic pathology (CP) presented as elephantiasis, recurrent episodes of filarial fevers (FF) or adenolymphangitis (ADL), and tropical pulmonary eosinophilia (TPE). Apart from these individuals, there are putatively immune endemic normals (EN) who are asymptomatic and amicrofilaremic. These differing clinical manifestations of infection were associated with varied immune responses to parasite antigens (Ottesen, 1984).

An effective immune response to parasite antigen depends on the interaction between APCs with Ag-MHC-TCR complex, accompanied by the expression of co-stimulatory molecules (secondary signals) like CD40, CD80, and CD86 etc. Affinity and specificity of TCR binding to MHC, is of primary concern as weak or inappropriate TCR binding results in impaired immune response. In this regard nature and function of clonally expanded T-cell subsets within disease lesions has been an important focus of research in the pathogenesis of a number of diseases. PCR-based methods (Duchmann et al., 1993; Rosenberg et al., 1992; Roth et al., 1989) or flow cytometry, using TCRVβ – specific monoclonal antibodies (Beck et al., 2003; Chini et al., 2002; Faint et al., 1999; Mancia et al., 1998) were employed to assess the expression of TCR α and β variable genes. Studies on TCRVβ repertoire have shown the role played by microbial toxins or super antigens in activating human immune system (Choi et al., 1990; Davies et al., 1991; Mancia et al., 1998; McCormick et al., 2001).

With respect to Bancroftian filariasis, the role of TCR in T-cell responses was examined in infiltrating T-cell lesions and blood of patients, where an over representation of Vβ14 and Vβ24 was observed (Freedman, 1998). Similarly in Brugia malayi infected population, a selective appearance of several Vβ genes in antigen stimulated PBMC cultures compared to unstimulated controls (Sartono et al., 1997) was noticed. In addition, experimental studies with Schistosoma mansoni, suggested the differential expression of TCRVβ genes and its relevance to pathogenesis (Secor and Freeman, 2001).

T-cell receptor variable beta (1–24) gene repertoire in patients 27

There are no reports on TCR gene expression profiles in filarial patients especially from Indian sub-continent. Hence, the present manuscript describes differential TCR gene expression profiles (Vβ1–Vβ24) in Indian population from Chennai, an endemic area for Bancroftian filariasis.

2 Materials and methods

2.1 Study population

Standardised histories were obtained and physical examinations were done on all the participant residents, during epidemiological surveys in and around Chennai in India, an area endemic for Wuchereria bancrofti infections. Parasitological examination of all individuals was done by detection of MF in blood smears taken from endemic individuals after 10 pm. Patients were recruited through the National Filaria Control Units under the Department of Public Health and Preventive Medicine (Chennai, India) after informed consent was obtained with protocols approved by the Institutional Review Board of Anna University. The study population was divided into three categories, asymptomatic amicrofilaremic individuals (EN (n = 5)), symptomatic individuals with chronic lymphedema and/or lymphatic obstruction and no microfilaria in their blood (CP, n = 5) and those with MF, based on clinical and parasitological evaluations (Table 1). All the individuals were screened for the presence of circulating microfilaria in the blood during night blood survey that lasted from 10.00 PM–2.00 AM.

2.2 Antigens and mitogens

Saline extract of adult B. malayi filarial antigen (BmA) was a kind gift from Dr. N. Shailja Bhattacharya, CDRI, Lucknow, U.P, India. BmA along with Phytohemagglutinin (PHA, Sigma Chemical Co., St. Louis, MO) and Purified Protein Derivative (PPD, Span Diagnostics, Surat, India) were used at concentrations of 10µg/ml each respectively.

2.3 T-cell receptor variable beta primers

26 TCRVβ family specific forward primers (Vβ1–Vβ24) and one constant region CβR specific primer (Monteiro et al., 1995) were synthesised by Sigma-Aldrich (Sigma-Genosys, Bangalore, India) (Table 2).

2.4 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

PBMCs were obtained by density gradient centrifugation from heparinised blood diluted using Ficoll-Diatrizoate gradient (ICN Biomedicals, Inc., Ohio, USA, as per standard procedures (Boyum, 1968). The cells at the interface were collected and washed thrice in RPMI-1640 (Invitrogen, San Diego, USA) at 400 g for 10 min at 4°C. Finally, the cell yield and viability was assessed using tryphan blue exclusion assay.


Table 1 Primer sequences for TCRVβ (1–24) genes

Primers Sequence Base

Vβ1 5’ CAA CAG TTC CCT GAC TTG CAC 3’ 21 Vβ2 5’ TCA ACC ATG CAA GCC TGA CCT 3’ 21 Vβ3 5’ TCT AGA GAG AAG AAG GAG CGC 3’ 21 Vβ4 5’ CAT ATG AGA GTG GAT TTG TCA TT 3’ 23 Vβ5.1 5’ TTC AGT GAG ACA CAG AGA AAC 3’ 21 Vβ5.2 5’ CCT AAC TAT AGC TCT GAG CTG 3’ 21 Vβ6 5’ AGG CCT GAG GGA TCC GTC TC 3’ 20 Vβ7 5’ CTG AAT GCC CCA ACA GCT CTC 3’ 21 Vβ8 5’ TAC TTT AAC AAC AAC GTT CCG 3’ 21 Vβ9 5’ AAA TCT CCA GAC AAA GCT CAC 3’ 21 Vβ10 5’ CAA AAA CTC ATC CTG TAC CTT 3’ 21 Vβ11 5’ ACA GTC TCC AGA ATA AGG ACG 3’ 21 Vβ12 5’ GAC AAA GGA GAA GTC TCA GAT 3’ 21 Vβ13.1 5’ GAC CAA GGA GAA GTC CCC AAT 3’ 21 Vβ13.2 5’ GTT GGT GAG GGT ACA ACT GCC 3’ 21 Vβ14 5’ TCT CGA AAA GAG AAG AGG AAT 3’ 21 Vβ15 5’ GTC TCT CGA CAG GCA CAG GCT 3’ 21 Vβ16 5’ GAG TCT AAA CAG GAT GAG TCC 3’ 21 Vβ17 5’ CAC AGA TAG TAA ATG ACT TTC AG 3’ 23 Vβ18 5’ GAG TCA GGA ATC CCA AAG GAA 3’ 21 Vβ19 5’ CCC CAA GAA CGC ACC CTG C 3’ 19 Vβ20 5’ TCT GAG GTG CCC CAG AAT CTC 3’ 21 Vβ21 5’ GAT ATG AGA ATG AGC AAG CAG 3’ 21 Vβ22 5’ CAG AGA AGT CTG AAA TAT TCG A 3’ 22 Vβ23 5’ TCA TTT CGT TTT ATG AAA AGA TGC 3’ 24 Vβ24 5’ AAA GAT TTT AAC AAT GAA GCA GAC 3’ 24 Cβ-R 5’ CTT CTG ATG GCT CAA ACA C 3’ 19

Table 2 Study population (demographic details)

Clinical group No. Age in years

(Range)

mf/ml Blood Range

Clinical Manifestations Treatment

CP 5 28–80 Nil IV-Grade Lymphoedema, of legs and hands in female patients. Males had Scrotal swelling and lymphoedema of leg.

Heat therapy, Compression therapy, and Surgical Size Reduction. Hetrazan, Amoxicillin, and B-Complex

MF 2 22–40 5–30 Frequent fever with bulging of lymph nodes in arm pits

No Treatment

EN 5 25–29 Nil Nil Nil

Notes: CP-Chronic pathology; MF-Microfilaraemics; EN-EndemicNormals; No-Number of subjects and mf- microfilariae


2.5 Lymphocyte cultures for T-cell receptor repertoire expressions

Lymphocytes (PBMCs) isolated from heparinised venous blood were cultured in 6-well flat bottom tissue culture plates (Costar Cambridge, MA, USA) at a density of 5 × 106 cells/well in cRPMI 1,640. The cells were stimulated with 10 µg/ml of BmA, PPD and PHA for 96 hours in a humidified atmosphere of 5% CO2 at 37ºC. The cells were harvested after the incubation time and used for total RNA extraction.

2.6 RNA extraction and reverse transcriptase PCR

Total cellular RNA was extracted from PBMCs as described previously (Chomczynski and Sacchi, 1987) using TRI Reagent® (MRC, Inc., Ohio, USA) and CHISAM (24 parts chloroform (Qualigens, India) + 1 part Iso-amyl alcohol (Qualigens, India). RNA pellet was air dried and dissolved in appropriate volume of DEPC (Diethyl pyrocarbonate, Amersham Pharmacia) – treated sterile MilliQ water and quantified at 260nm using a spectrophotometer (Eppendorf BioPhotometer).

The extracted RNA (5 µg/ml) was reverse transcribed using Super Script™ first-strand cDNA synthesis RT-PCR kit (Invitrogen life technologies, California USA) as per manufacturer’s protocol.

2.7 PCR amplifications of cDNAs

2 µl of the obtained cDNA was used for PCR amplification of T-cell receptor genes (Vβ1–Vβ24), in a PCR thermal cycler (MJ research, USA). Prior to this, all the cDNAs were confirmed for the expression of house keeping gene β-Actin (202bp) by using gene specific primers (Forward Primer-5’ CCTTCCTGGGCATGGAGTCC TG 3’; Reverse Primer -5’GGAGCAA TGATCTTGATCTTC 3’).

PCR master mix (50 μl ) was prepared using 10X PCR buffer, 10 mM dNTP (Eppendorf, Germany), 20 pM of forward and reverse primers and 5 units of Taq DNA polymerase (Invitrogen Life Technologies, USA). All the PCR amplifications were carried out in an MJ Research thermal cycler under the following conditions: 5 min pre-dwell at 94°C followed by 35 cycles of 1 min at 94°C, 1 min annealing (58–60oC), 1 min extension at 72°C and a post-dwell period of 10 min at 72°C.

Finally, the amplicons were confirmed by resolving them on a 1% Agarose gel along with a standard 100 bp ladder (Fermentas, USA). The differential expression of the TCRVβ genes among the study population was assessed by densitometric scanning (IDV- Integrated Density Values, calculated using the QUANTITY1software), where the band intensity of the product was compared with the 500 bp (50 ng/5µl) band (reference band) of the 100 bp ladder.

2.8 Statistical analysis

All the statistics were performed using Graph Pad Prism soft ware. Analysis of Variance was (one way ANOVA) was performed followed by Tukey’s HSD Test to compare between the groups. A p-value of p < 0.05 was considered statistically significant.


3 Results

3.1 TCR beta variable gene expressions for EN

As expected, all the cDNA samples from PBMCs of EN showed the amplification of the house keeping gene β-Actin (202 bp), when amplified with gene specific primers. Table 3 TCRVβ 1–24 genes in EN (ANOVA for significant difference between stimulants for

each gene)

Stimulants Genes TCRVβ gene primers (1–24) Control PHA PPD BmA F Value P Value

TCRBV1 34.26a 37.82a 97.35b 70.48ab 10.0519 0.006**

TCRBV2 27.29a 35.24ab 82.83c 62.47bc 12.1143 0.002**

TCRBV3 30.28a 30.52a 68.79b 63.34b 8.0145 0.001**

TCRBV4 29.99a 42.42ab 82.48bc 87.25c 8.2544 0.001**

TCRBV5.1 33.84a 37.23a 92.86b 74.02ab 5.4545 0.008** TCRBV5.2 28.57a 51.30ab 107.6c 93.46bc 9.429 0.000** TCRBV6 44.48a 45.65a 82.29ab 96.96b 4.5021 0.017* TCRBV7 44.54a 48.50a 82.36a 89.26a 4.0779 0.025* TCRBV8 35.30a 45.49ab 74.95ab 84.62b 4.3936 0.019* TCRBV9 31.39a 40.40ab 78.72bc 88.32c 7.4705 0.002** TCRBV10 31.96a 46.77ab 85.01b 80.89b 5.7432 0.007** TCRBV11 27.79a 46.13ab 90.90b 81.45b 5.4368 0.009** TCRBV12 32.14 42.75 74.39 59.18 2.9799 0.626 TCRBV13.1 37.85 45.13 74.69 78.79 2.5812 0.896 TCRBV13.2 38.67 39.34 64.49 79.32 4.1888 0.229 TCRBV14 27.26a 31.51ab 80.09c 63.13bc 8.3902 0.001** TCRBV15 23.35a 43.95ab 74.06b 65.65b 8.9061 0.001** TCRBV16 21.47a 24.01a 71.92b 38.42ab 4.7675 0.014* TCRBV17 25.61 27.91 69.28 51.85 2.59565 0.088 TCRBV18 19.24a 26.16a 67.46b 35.61ab 5.0872 0.011* TCRBV19 26.16 32.02 69.63 45.47 2.2503 0.121 TCRBV20 25.05a 24.42a 68.77b 41.88ab 3.8864 0.029*

TCRBV21 22.57a 27.99a 79.70b 33.03a 6.1479 0.005**

TCRBV22 30.54a 31.79ab 88.14b 53.69ab 3.6308 0.035*

TCRBV23 30.08a 34.43a 100.74b 48.49ab 5.2141 0.010*

TCRBV24 25.93a 39.84ab 102.16b 49.74ab 4.6247 0.016*

Notes: *Denotes significance at 5% level Based on data from five patients **Denotes significance at 1% level Different alphabets between genes denotes significance at 5% level based on Tukey HSD Test and the gradation of % of expression from low to high is shown in the alphabetical form, from a to z.


Table 4 TCRVβ 1–24 genes in patients with CP (ANOVA for significant difference between stimulants for each gene)

Stimulant Genes TCRVβ gene primers (1–24) Control PHA PPD BmA F Value P Value TCRBV1 56.77ab 26.81ab 104.14bc 153.77c 11.6856 0.000** TCRBV2 42.85ab 21.22a 104.39bc 140.56c 12.8002 0.000** TCRBV3 37.38a 22.02a 80.15b 124.77c 23.5207 0.000** TCRBV4 46.00a 30.10a 100.97b 118.42b 9.849 0.001** TCRBV5.1 39.07a 21.37a 83.49b 120.57b 18.2087 0.000** TCRBV5.2 49.82ab 30.25a 101.56bc 133.24c 11.0426 0.000** TCRBV6 46.27a 28.11a 101.33b 132.74b 13.5293 0.000** TCRBV7 46.67ab 33.20a 106.12bc 157.53c 12.839 0.000** TCRBV8 40.41ab 24.59a 88.23bc 140.64c 11.3485 0.000** TCRBV9 53.38a 34.51a 90.57ab 132.57b 9.3225 0.001** TCRBV10 41.75a 30.01a 87.68b 116.18b 13.7369 0.000** TCRBV11 42.96a 29.20a 99.24b 104.35b 11.4964 0.000** TCRBV12 45.01ab 27.20a 87.13bc 128.53c 13.5473 0.000** TCRBV13.1 45.57a 25.14a 108.40b 129.79b 11.1708 0.000** TCRBV13.2 54.09a 35.58a 98.75ab 133.08b 7.7007 0.002** TCRBV14 41.12a 21.66a 89.92b 106.73b 14.9498 0.000** TCRBV15 44.10a 35.98a 92.89b 112.63b 9.6336 0.001** TCRBV16 21.78a 12.47a 87.68b 105.68b 52.856 0.000** TCRBV17 18.50a 12.33a 95.09b 85.89b 21.4812 0.000** TCRBV18 22.63a 12.96a 76.11b 80.47b 23.0227 0.000** TCRBV19 20.62a 14.49a 86.12b 81.25b 26.2871 0.000** TCRBV20 22.56a 13.27a 80.13b 92.01b 22.9431 0.000** TCRBV21 27.26ab 15.08a 86.55c 63.69bc 13.1472 0.000** TCRBV22 31.48a 19.90a 88.48b 87.88b 17.0938 0.000** TCRBV23 29.11a 16.86a 100.28b 78.86b 11.8596 0.000** TCRBV24 28.57a 17.05a 113.95b 99.95b 12.818 0.000**

Notes: *Denotes significance at 5% Level Based on data from five patients **Denotes significance at 1% Level Different alphabets between genes denotes significance at 5% level based on Tukey HSD Test and the gradation of % of expression from low to high is shown in the alphabetical form, from a to z.

Differential expression levels were observed for all the TCR genes assessed, under different stimulated conditions using PHA or PPD or BmA as well as in the unstimulated conditions (Table 3). BmA induced a significant increase in the expression of TCRVβ 4, TCRVβ 6, TCRVβ 7, TCRVβ 8, TCRVβ 9 and TCRVβ 10 genes. With respect to PPD stimulation, elevated expression was observed in genes like, TCRVβ 1, TCRVβ 2, TCRVβ 6, TCRVβ 5.1, TCRVβ 5.2, TCRVβ 8, TCRVβ 9, TCRVβ 11, TCRVβ 14, TCRVβ 16, TCRVβ 18, TCRVβ 20, TCRVβ 21, TCRVβ 22, TCRVβ 23 and TCRVβ 24. Among these,


TCRVβ 3, TCRVβ 6, TCRVβ 8, TCRVβ 11 and TCRVβ 15 genes exhibited similar expression levels upon both BmA and PPD stimulation. Besides this, PHA stimulation induced similar expression of all the above TCRVβ genes.

3.2 TCR beta variable gene expressions for CP

The house keeping gene β-actin expression was predominant in all the cDNA samples. In patients with CP, increased expression of all the TCRVβ genes was observed under PHA or PPD or BmA stimulated as well as unstimulated conditions. BmA induced the expression of all the TCRVβ genes in CP, except for TCRVβ 21. In the case of genes like, TCRVβ17, TCRVβ19, TCRVβ21, TCRVβ 22, TCRVβ 23 and TCRVβ 24, PPD induced higher levels of expression compared to BmA. PHA induced cultures also exhibited the expression of all the TCR genes (Table 4).

Thus, the TCRVβ gene expression in PHA stimulated PBMC cultures from EN and CP individuals was in the order EN > CP while in BmA or PPD stimulated conditions or in the unstimulated conditions, it was CP>EN. Further the PBMCs from CP showed elevated levels of TCRVβ gene expression both in the unstimulated and stimulated conditions.

4 Discussion

The presence of a biased TCR repertoire in peripheral blood provides supporting evidence that antigen selection process occurs and the magnitude of TCR bias is an important indicator for the role of a small number of discrete antigens that might lead to disease manifestations resulting in clinical groups. Till date, the presence of a TCR repertoire bias has not been examined in the peripheral blood of patients with human lymphatic filariasis particularly in Indian population. To address this, the repertoire of TCRVβ gene segments in peripheral blood was studied in patients with CP, endemic normals and in the microfilaraemics harboring W.bancrofti infections.

There was an overrepresentation of TCRVβ 6, TCRVβ 5.2, TCRVβ 9, TCRVβ 8, TCRVβ 11, TCRVβ 13.2, TCRVβ 13.1, TCRVβ 23 and TCRVβ 16 in response to BmA in the EN individuals. This indicated a preferential expansion of T-cells with certain TCR types to filarial antigens. Most of the TCRVβ families that were expressed were shared by all five different individuals. Similar observations were made by Sartono et al. (1997) in Indonesian population in EN upon BmA stimulation A number of TCRVβ gene families were also over-expressed in PPD stimulated cultures of EN when compared to PHA-stimulated and control conditions. The PPD used in this study served as a non-parasite-specific antigen control and is also a potent inducer of immune response in filarial patients. In this TCRVβ 1, TCRVβ 2, TCRVβ 5.1, TCRVβ 5.2, TCRVβ 8, TCRVβ 14, TCRVβ 16, TCRVβ 18, TCRVβ 20, TCRVβ 21, TCRVβ 22, TCRVβ 23 and TCRVβ 24 were expressed upon PPD stimulation. Although some of the expressed TCRVβ gene families like TCRVβ 3, TCRVβ 6, TCRVβ 8, TCRVβ 11 and TCRVβ 15 were shared in PPD and BmA stimulated cells, it is possible that these antigens could drive the expansion of distinct TCRVβ families in the same individuals. Moreover, the over expression of the same TCRVβ genes in all these five different individuals, in response to


both PPD and BmA, might involve different clonal expansions that could be predicted only after sequencing of these expressed receptor families. In a previous study, TCRVβ 8 and TCRVβ 23 were reported to share a common overrepresentation by both BmA and PPD stimulants (Sartono et al., 1997). Studies carried out by Shanmugalakshmi et al. (2003) in patients with Pulmonary Tuberculosis from south Indian populations showed that there was an overrepresentation of TCRVβ1, TCRVβ5, TCRVβ 6, TCRVβ 9, TCRVβ 10, TCRVβ 11, TCRVβ 13, TCRVβ14 and TCRVβ18 in the presence of PPD antigen.

It should be pointed out that TCRVβ gene profiles should have been done in PBMC cultures stimulated with PHA for 48 hours and not at 96 hours as cells may die at the later time period. This time period of 96 hours was chosen for comparative analysis with antigen stimulated cultures and further PHA stimulated cultures will serve as a good positive control. This could be one reason for the low levels of TCRVβ gene expression profiles observed in PHA stimulated cultures in EN and CP and further it closely resembles those of unstimulated cultures. We have found that proliferative capability of PHA stimulated PBMC cultures were appreciable in EN and CP even at 96 hours (Unpublished observations).

In Patients with CP, TCRVβ 4, TCRVβ 5.1, TCRVβ 6, TCRVβ10, TCRVβ 11, TCRVβ 13.1, TCRVβ 14, TCRVβ 15, TCRVβ 16, TCRVβ 17, TCRVβ 18, TCRVβ 19, TCRVβ 20, TCRVβ 22, TCRVβ 23 and TCRVβ 24 were over-expressed in BmA stimulated PBMC cultures. Besides this PPD induced expression of TCRVβ genes exhibited a similar profile. Unstimulated cultures however showed basal level expression of all the TCRVβ genes. Our findings on patients from Indian population support the previous studies carried out on patients of Indonesian origin (Sartono et al., 1997). TCRVβ gene expression in response to PPD could be due to the constant exposure of the filarial patients to typical and atypical mycobacteria in the TB endemic areas and the memory response that is generated as result of this exposure (Bothamley et al., 1992; Fonseca et al., 1992; Pitchappan et al., 1991). Further this may also attribute to the spectrum of immune reactivity observed in ENs as well. In this study, out of the 200 cases, screened for the presence of microfilaria, only two were positive and showed significant microfilaria count during night blood smear. Nevertheless, these two patients failed to show any parasite induced TCRVβ gene expressions, in spite of the fact that non-parasite antigen, PPD resulted in over-representation of TCRVβ genes (unpublished observations).

Thus, the enhanced TCRVβ gene expression upon BmA stimulation in CP compared to EN may be a contributing factor that leads to the inflammatory responses and pathology associated with the disease.

Acknowledgements

This work received funding from the University Grants Commission, New Delhi. MSS was a recipient of Lady Tata Fellowship. We thank Dr B. Sasisekar and Dr N.S.A. Krushna for their scientific suggestions and Ms K. Haripriya and Ms C.S. Kirthika in the preparation of the manuscript.


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