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Transfer of Cell-Mediated Immunity in Leprosy by Tn;Jnsfer of Lymph

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Transfer of Cell-Mediated Immunity in Leprosy by Tn;Jnsfer of Lymph I NTERNATION AL JOU RNAL OF L EPR OSY Volume 42. Number I PrintN/ ill Ihl' US.A . Transfer of Cell-Mediated Immunity in Leprosy by 1 J Tn;Jnsfer of Lymph Node Cells N. H. Antia and S. R. Khanolkar 2 Landsteiner and Chase (7. H) demonstrated Fasal (2) who showed that the addition of the transfer of cell-mediated immunity for PHA impaired the rate of DNA synthesis in tuberculosis by lymphocytes in inbred guinea leukocytes cultured from leprosy patients. pigs. The experiment could not be repeated Wong et al (1 6) similarly observed a depres­ in nonsy ngeneic animals. They presumed sion in immunological response in leprosy. that living cells were necessary for transfer In the present study an attempt has been of cell-mediated immunity (CM I). made to transfer cell-mediated immunity Lawrence (9) demonstrated the transfer of from lepromin positive (tuberculoid) to lep­ CM I in tuberculosis in non-inbred guinea romin negative (lepromatous) patients. This pigs. He and his associates further showed study reports the findings of lepromin test­ (1 2) that transfer of immunity could be ing and of lymphocyte transformation in sev­ achieved by using cellular extracts, and even en lepromatous recipients before and after by a dializable factor obtained from sensi­ the transfer of lymph node cells from a tu­ tized leukocytes. This factor is heat stable berculoid donor. at 56° C for 30 minutes. It is a dializable moiety of <10,000 molecular weight which is MA TERIALS AND METHODS not immunogenic nor immunoglobulin-like. Selection of patients. Lymphocytes were Lawrence (9) demonstrated the practical use obtained from the venous blood of the fol­ of the transfer factor (TF) in human tuber­ lowing three groups: I) normal controls; 2) culosis by injecting subcutaneously 85 x 106 lepromatous leprosy; 3) tuberculoid leprosy leukocytes from tuberculin positive to tuber­ at the Tata Department of Plastic. Surgery, culin negative persons. The degree of trans­ 1.1. Hospital in Bombay. Brief history of the ferred sensitivity was measured in vivo by patients is summarized in Table I. the tuberculin test and in vitro by lympho­ Preparation of lymphocyte cultures. Ten cyte transformation. The negative recipients ' milliliters of venous blood were collected became sensitized within 48 hours and sensi­ in 4 ml of 6% Dextran citrate solution and tivity remained up to two years ('0). allowed to sediment at room temperature Lawrence (II) postulated the use of trans­ (1-2 hrs), the supernatant fluid of leukocyte­ fer factor in leprosy. De B'onaparte et al (3. 5) rich plasma was withdrawn and an equal demonstrated such transfer in leprosy in 5 volume of Hank's BSS was added to the su­ of 13 cases as measured by lepromin skin pernatant. Relatively rich, 73-75% lympho­ testing. cyte culture was obtained. The cell suspen­ Dierks and Shepard (4) and Paradisi et al sion was centrifuged at 2,000 rpm for ten (14) have reported that lymphocytes from lep­ minutes and the resulting pellet was diluted romatous leprosy patients do not undergo in TC-199 (Difco) with 15% AB group hu­ transformation as freely as those from nor­ man serum to a concentration of I x 106 mal individuals when phytohemagglutinin cells/ ml; penicillin 100 units/ ml, and strep­ (PHA) and the mycobacterial antigens are tomycin 100 mg/ ml were added. Two tubes added. The response to PHA was moder­ from each patient containing 5 ml of the ately depressed in tuberculoid leprosy. Sim­ above cell suspension was incubated at 37° C ilar results were obtained by Bullock and in 3% CO2 . PHA-P (Difco) I Ill / ml and lepromin (R. J. W. Rees. London) containing I Received for publication 6 October 1972. 1.1 x 10 7 M . leprae bacilli in 1.4 x 10 2 ml were 2N. H. Antia, M.B. , F.R.C.S., Professor of Plastic Sur­ added per ml of suspension and incubated gery, Tata Department of Plastic Surgery, J.J . Group for three days following which a morpholog­ of Hospitals, Byculla, Bombay-S, India; S. R. Khanolkar, M.Sc. , Jr. Research Officer, Tata Department of Plastic ical count was made of the cells to ascertain Surgery, J.J. Group of Hospitals, Byculla, Bombay-S, the percentage of lymphoblast transforma­ India. tion. The cells were washed in hypotonic 28 29 International Journal of Leprosy 1974 TABLE I. Brief history ol the patients. Leprosy Duration of leprosy Lepromin test No. type a (in years) Treatment b (3 week) I LL 6 ± 2 LL 20 ± 3 LL 22 + 4 LL 22 + 5 LL 17 + 6 LL 14 + 7 LL 22 + 8 TT 5 + 6 mm 9 TT 25 + II mm 10 TT 12 + 5 mm aLL = lepromatous leprosy; TT = tuberculoid leprosy. b + = regular; ± = irregular or doubtful. TABLE 2. Lrmphocrte trans/ormation in normal controls. No. PHA-P% Lepromin % Lepromin sk in tes t (3 week) I 70. 1 3.1 6 mm 2 72.6 4. 1 8 mm 3 80.0 7. 1 10 mm 4 72.2 2.6 II mm 5 61.4 1. 8 II mm 6 60.0 1.1 5 mm 7 62.4 3.4 4 mm A\ crage 68.4 3.3 7.8 mm saline solution, fixed in ethanol and glacial ' containing 120 x 10 6 cells (80% viable by the acetic acid (3 : I v! v) with final suspension in erythrocin B exclusion test) was injected 0.2 ml fixative and a smear of the suspension subcutaneously in the deltoid region in each was stained with Leishman's stain. The slides of the seven lepromin negative recipients. were mounted and 1,000 to 1,500 cells were After 48 hours and 21 days, lepromin was scored from each culture. The number of injected intradermally over the site of the spontaneously transformed cells in the con­ previous deltoid injection as well as into the trol culture was subtracted from the number opposite forearm. The lepromin test was of cells transformed by the antigens in each read at 48 hours and 21 days. Blood was col­ case. We have generally followed the method lected for the study of lymphoblast transfor­ described by Dierks and Shepherd (4) . Lym­ mation with PHA and lepromin after 48 phocyte transformation was studied before, hours and 21 days. and 48 hours and 21 days after injecting the lymphoid donor cells into the lepromatous RESULTS patients. Controls. The response of lymphocytes Inguinal lymph nodes surgically removed collected from seven normal controls was from the I I mm lepromin positive donor N.o. similar to that seen in other studies (4. 15). The 9 were collected and teased in 5 ml of normal average response to PHA was 68.4% and to saline. The cell suspension was allowed to lepromin was 3.3%. The results are summa­ stand for 15 minutes to settle the debris and rized in Table 2. All normal volunteers were the supernatant was pi petted off and washed lepromin positive on skin testing, the average twice with normal saline. The cells were being a 7.8 mm response, Which was read counted and one milliliter of cell suspension after three weeks. 42, I Antia & Khanolkar: Transfer of Cel/~Mediated Immunity in Leprosy 30 TABLE 3. Percem Irmphoc.l'te trans/ormation in lepromatous leprosy patients hefore and a/ier tuberculoid cell transFer. PH A-P (o/c transformation) Lepromin (% tra nsforma ti on) Before Before Lepromin No. cell tra nsfer 48 ho urs 2 1 days cell tra nsfer 48 ho urs 2 1 days test I 53.2 65 40 3.2 6.6 3.1 - 2 64 67 47.4 4.2 12.8 7.8 - 3 6 1.3 49.7 36 3.2 1.3 2.6 - 4 38.5 33.8 15.4 4.7 1.6 2.8 - 5 2 1.2 63.7 38. 1 4.2 30.7 4.0 - 6 28.2 50.2 24.0 1. 1 1. 2 7.4 - 7 38.0 40.0 Noa 1. 7 2.0 Noa - Average . 43.5 52.7 33.5 3.3 8.1 4.6 it N D = not done. TABLE 4. Lymphocyte transformation in tuberculoid leprosy. PHA-P Lepromin Lepromin skin test No. % tra nsformation % tra nsforma ti on (3 week) I 45.7 10.5 6 mm 2 53 .2 7.0 II mm 3 38.7 3.7 5 mm Average 45.9 7. 1 7.3 mm Lepromatous leprosy. This group, consist­ DISCUSSION ing of seven lepromin negative lepromatous De Bonaparte and associates (3) stated leprosy patients (Table 3), showed a signifi­ that it is possible that preparation of transfer cant increase in lymphoblast transformation factor from lepromin sensitive donors would after cell transfer. The transformation was also function as a prompt therapeutic immu­ greater for the specific antigen (lepromin) nizing agent and convert the progressive lep­ than for nonspecific antigen (PHA) and in romatous type of leprosy to the more benign both cases was greater at 48 hours after cell tuberculoid type. transfer than after 21 days by which period Immunity in mycobacterial disease is me­ the PHA transformation was less than be­ diated by lymphocytes which can be studied fore the cell transfer, while that of the re­ by observing the transformation of circulat­ sponse to lepromin remained· higher. Despite ing lymphocytes into blast cells by PHA the rise in rate of lymphoblast transforma­ (6. 13). Mitosis and DNA synthesis have been tion following cell transfer the lepromin skin studied by using tritiated thymidine (I. 2. 16) test at 48 hours and 21 days remained nega­ and the authors observed depression in the tive.
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