53, 4^ Correspondence^ 649

Interleukin-2-induced T-cell Response to M. leprae inLepromatous Leprosy: Reversion of a Suppressor Mechanism

or Expansion of a Small M. leprae-reactive T-cell Pool?

To THE EDITOR:

A number of recent studies indicate thata fraction of lepromatous leprosy patientsharbor circulating Mycobacterium leprae-reactive T cells, despite their well-docu-mented anergy to Al. leprae antigens ( 3 ). In-deed, a Al. /cprae-triggered, in vitro T-cellresponse has been evidenced by differentprocedures. We previously repored that invitro incubation at 37°C of peripheral bloodmononuclear cells (PBMC) of some lepro-matous patients could restore their prolif-erative response to M. leprae ('), a phenom-enon which was recently attributed to theelimination of suppressor macrophages orof macrophage-released suppressor factors(7 8 ). Experiments conducted by Mehra, etal. (9 ) indicated that depletion of suppressorOKT8+ T cells could also restore T-cellresponsiveness to M. leprae antigens in BLpatients. Finally, Haregewoin, et al. ( '. 5 ) ob-served that the addition of exogenous in-terleukin-2 (IL-2) to PBMC from lepro-matous patients restored Al. leprae-triggeredT-cell proliferation. This finding attributedto the reversion of a suppressor mechanismwas confirmed by Nath, et al. ( 8 ), but wascontradicted by Ottenhoff, et al. ( 9 ).

We have undertaken a similar study in agroup of 18 lepromatous leprosy patients ofvaried geographical origins (9 from the WestIndies, 5 from Africa, 2 from Europe, and2 from Asia), all living in Paris and followedat Saint-Louis Hospital (Dermatology De-partment, Service of Professor Cottenot). Atvariance with the above-cited report, wehave also conducted similar studies in 13tuberculoid patients and 5 healthy subjects,studying the responsiveness to PPD in par-allel to that of Al. leprae in all groups ofsubjects. We found an IL-2-induced in-crease of M. leprae responsiveness in 10 outof 18 lepromatous patients, but also in 3 outof 4 tuberculoid patients who happened tobe nonresponsive in vitro to Al. leprae, andin 2 out of 3 similarly Al. leprae-unreactivehealthy subjects. The ability of PBMC fromM. leprae nonresponders to become reac-

tive in the presence of IL-2 was closely linkedto their responsiveness to PPD.

PBMC were cultured for 6 days in RPMI1640 medium supplemented with 20% hu-man AB serum, with either M. leprae (WHObatch CD 40; 1 µg/ml), BCG-derived PPD(IP71, a gift of Dr. J. Augier, Pasteur Insti-tute, Paris; 10 µg/ml) or no antigen, in thepresence or absence of IL-2 (Lymphocult Tlectin-free, Biotest; 5%). 3 H-Thymidine in-corporation was measured after an 18-hrpulse. Results from antigen-stimulated cul-tures were expressed as Acpm = cpm withantigen — cpm without antigen. Back-ground proliferation without antigen was lowin the absence of IL-2 (around 1000 cpm inall 3 groups) but increased significantly inthe presence of IL-2, to the same extent inlepromatous patients (mean ± S.E.M. =12,078 ± 2700), tuberculoid patients(16,175 ± 3502), and healthy subjects(18,678 ± 8811). Such an effect of IL-2 onspontaneous PBMC proliferation, alreadynoted by Haregewoin, et al. (4 ) and Nath,et al. ( 8 ), might result from the proliferationof IL-2-dependent, NK-like, large granularlymphocytes and/or of autoreactive T cellsdirected at MHC class II auto-antigens.

Ten out of 18 lepromatous patients (Ta-ble 1) exhibited after IL-2 addition a sig-nificant proliferative response to M. leprae,as defined by a Acpm exceeding 5000. Theproportion of patients whose AI. leprae un-responsiveness was reversed by IL-2 in ourstudy was closely similar to that reportedby Haregewoin, et al. ( 5 ) and by Nath, et al.( 8 ) but much higher than that reported byOttenhoff, et al. (9) (2 out of 12). The geo-graphical distribution, that of BL vs LLforms, and of treatment duration was quitesimilar among the patients who did respondto M. leprae in the presence of IL-2 andamong those who did not. The level of pro-liferative response to AI. leprae observed inthe presence of I L-2 among lepromatous pa-tients, however, still remained lower thanthat seen in the absence of IL-2 among tu-berculoid patients (p < 0.05). Four lepro-matous patients, nos. 2 and 3 who did not

650^ International Journal of Leprosy^ 1985

TABLE I. Oleo q/ IL-2 addition on M. leprae and 1'1'D-stimulated proliferative responsein lepromatous patients.

Patient no. andtype of leprosy

Dura-tion oftreat-ment

M. leprae PPD

Without IL-2 With 1L-2 Without IL-2 With IL-2

1^(BL) 30 mos. -17 34 4492 (I3L) 5 mos. 36 -2,346 903 -2,2333 (LL) 4 yrs. 163 -2,774 361 5054 (LL) 6 yrs. -1,327 1,504 -1,257 3,8335 (BL) 13 yrs. -80 -3,329 3,762 3,0936 (LL) 33 yrs. -96 2,545 -199 -2,0667 (LL) 15 yrs. -135 -5,622 2,872 4,5158 (LL) 8 mos. 27 1,448 41,828 20,5729 (LL) 2 yrs. 864 8,207 1,899 21,866

10 (BL) 6 I mo. 2,040 59,272 109 30,47911^(LL) 0 -301 21,939 -1,756 13,49512 (LL) 19 yrs. 678 14,576 1,731 10,84813 (BL) 17 mos. 4,154 6,655 9,066 3,32814 (BL) 0 1,673 8,495 15,601 30,27115 (LL) 11 yrs. 220 14,020 11,848 33,25316 (BL) 11 yrs. 5 6,402 17,980 22,89317 (LL) 6 mos. 2,456 14,805 27,732 49,20318 (LL) 10 yrs. 530 7,908 92,494 142,901

Mean ± S.E.M. 679 ± 482 8,538c ± 3,461 12,455 ± 5,455 21,51 Ic 7,932

Results of 3 H-thymidine incorporation measurement arc expressed as cpm = cpm with antigen - cpmwithout antigen.

" Patient showing reversal reaction at the time of study.Significantly different from the results obtained without IL-2 addition (paired I test, p < 0.05).

respond to M. leprac in the presence of IL-2and nos. 10 and 12 who did, were re-testedto compare the inductive effect of recom-binant IL-2 (a gift of Sandoz Laboratories,Basel, Switzerland, used at concentrationsof 0.02-0.1 pg/m1) with that of the chro-matography-purified, lectin-free IL-2 rou-tinely used in our experiments. Indeed,Haregewoin, et al. ( 5 ) reported a higherfrequency of IL-2-induced reversal of M.leprac unresponsiveness when recombinantIL-2 was used instead of a crude T-cell-con-ditioned medium. In the present study, nodifference could be seen in any patient be-tween the results obtained with recombi-nant IL-2 and those obtained with Lym-phocult T lectin-free, which would indicatethat crude, unpurified T-cell-conditionedmedium is not optimal for inducing rever-sion of M. leprae unresponsiveness in le-promatous patients but that a partial puri-fication may be sufficient to improve itsefficiency.

Eleven out of 18 lepromatous patients hadno PPD-induced proliferation of PBMC (vs3 out of 13 tuberculoid patients), but 4 ofthem became PPD-responsive when IL-2

was added, and a significant increase (p <0.05) of the mean response to PPD plus IL-2was observed in this group when comparedto the mean response to PPD alone. Inter-estingly, all patients who displayed a sig-nificant proliferative response to Al. lepraeplus IL-2 also showed a significant responseto PPD, either spontaneous or IL-2 in-duced; whereas only 1 of the M. leprae-non-reactive patients did so. It is striking that90% of the patients studied by Haregewoin,et al. ( 4 ), among whom a majority showedIL-2-inducible, T-cell responsiveness to M.leprae, also presented with a significant pro-liferative response to PPD. Conversely, pa-tients investigated by Ottenhoff, et al. ( 9 ),whose T cells remained in most cases un-responsive to Al. leprac in spite of IL-2 ad-dition, exhibited insignificant or low re-sponses to PPD with or without IL-2.Moreover, 1 of the 2 patients who did re-spond to M. leprac plus IL-2 actually re-sponded to PPD plus IL-2 as well.

Among 13 tuberculoid patients (Table 2)investigated in this study, 4 (nos. 1-4, allBT) showed no proliferative response ofPBMC to M. leprae, but only 1 of them (no.

PPDlepraeNo. and type

of leprosyWithout IL-2 With IL-2 Without IL-2^With IL-2

Dura-tionof

treat-ment

BT/TT patients

Subjects

53, 4^ Correspondence^ 651

TABLE 2. Elkct of IL-2 addition on M. leprae and PPD-stimulated proliferative re-sponses in tuberculoid patients and healthy controls.

1^(13T) 2 yrs. -307' 4,220 -366 1,0402 (BT) 2 yrs. 357 5,784 3,539 10,8183^(13T) 0 298 14,857 NDb ND4^(13T) 0 -355 6,370 821 14,8475 (BT) 6 yrs. 5,004 18,471 10,503 34,8656 (BT) 0 5,403 57,202 15,534 56,9147 (BT) 0 8,268 13,104 14,418 16,6318 (BT) 4 mos. 23,698 45,997 147,744 121,2609 (BT) 0 34,542 24,739 40,436 26,552

10 (TT) 8 yrs. 38,336 26,454 114,336 57,45411^(BT) 1 mo. 54,314 90,293 158,351 137,29312 (BT) 7 yrs. 80,315 51,596 46,699 121,28013 (BT) 6 mos. 113,288 119,744 165,562 125,707

Mean ± S.E.M. 27,588 ± 10,020 36,833 ± 9,819 57,798 ± 19,240 60,388 ± 14,919Healthy subjects

1 55 -662 -17 10,8862 893 5,102 314 18,4833 43 22,511 12,107 24,4314 13,572 25,229 160,094 140,2565 39,830 22,266 179,660 128,988

Mean ± S.E.M. 10,893 ± 7,691 14,889 ± 5,377 70,432 ± 40,775 64,609 ± 28,719

a Results of 3 H-thymidine incorporation are expressed as .cpm = cpm with antigen - cpm without antigen.b ND = not done.

1) remained unresponsive to M. leprae plusIL-2. It is noteworthy that this patient didnot show any proliferative response to PPD,either with or without IL-2; whereas all oth-er patients did.

Five healthy subjects (Table 2) previouslyinvestigated for other purposes were select-ed to represent varying intensities of PBMCproliferative response to PPD. In the ab-sence of IL-2, only those subjects (nos. 4and 5) who were strongly reactive to PPDalso reacted, to a lesser extent, to AI. leprae.Among the 3 other subjects, 2 (nos. 1 and2) were also unresponsive to PPD (althoughthey had been vaccinated with BCG); whenthe proliferation assay was performed in thepresence of IL-2, both exhibited a signifi-cant proliferation to PPD but only 1 dis-played a marginal response to Al. leprae.The last Al. leprae unreactive subject (no.3), BCG vaccinated, exhibited in the ab-sence of IL-2 a significant but low prolif-erative response to PPD (increasing uponIL-2 addition), and presented with a strong,AI. leprae-triggered PBMC proliferative re-

sponse after IL-2 addition. These results aredifferent from those of Haregewoin, et al.(4 ) who failed to induce a significant re-sponse to M. leprae with IL-2 in 5 not ex-posed and not responsive healthy controls,but did not provide information about theirresponse to PPD.

In conclusion, our data indicate that amajority of M. leprae nonresponders, in-cluding all lepromatous patients, a few bor-derline tuberculoid patients, and somehealthy subjects not exposed to M. leprae,may acquire an in vitro proliferative re-sponse to Al. leprae upon IL-2 addition. Thiscapacity seems to closely correlate with theability of these individuals to develop a pro-liferative response to PPD, strongly sug-gesting that the IL-2-induced proliferativeresponse to M. leprae is directed against epi-topes shared by M. bolls and M. leprae. Assuggested by Ottenhoff, et al. (9 ), this mayexplain the differences observed in the fre-quency of IL-2-induced reversal of M. lep-rae unresponsiveness among different pop-ulations. Thus, it would be very interesting

652^ International Journal of Leprosy^ 1985

to investigate among lepromatous patientsand M. /eprae-exposed anergic contactswhether or not such an in vitro reversionwould correlate with the ability to developT-cell-mediated immunity to M. leprae af-ter BCG vaccination, or after combined im-munotherapy with I3CG and killed Al. lep-rae, as proposed by Convit, et al. ( 2 ).

Our observation on the IL-2 induction ofM. leprae responsiveness in some healthysubjects raises a significant query on themechanism of such a "reversal," both inthese subjects and in lepromatous patients.The formerly proposed hypothesis of asuppression of IL-2 production in lepro-matous patients, which could be bypassedby the addition of exogenous IL-2 (4 5, 7 ' 8),

might apply to the healthy subjects pre-sented in this study as well, since they wereunable to develop a significant T-cell re-sponse to PPD despite BCG vaccination.Alternatively, IL-2 may just amplify theproliferation of a low number of Al. leprae-reactive, precursor T cells, allowing theirproliferative response to reach the thresholdof detection. The IL-2-induced prolifera-tion to M. leprae in lepromatous patientsdid not reach the level of that attained inthe absence of IL-2 by tuberculoid patients,which may be interpreted as a partial failureof IL-2 to overcome suppression but alsocould result from a simple quantitative dif-ference of M. leprae-reactive cells betweenlepromatous and tuberculoid patients. Theevaluation of the frequency of such precur-sors by the limiting dilution techniquewould, perhaps, provide a decisive answer.

— Premlata Shankar, M.D.—Daniel Wallach, M.D.

—Marie-Ann Bach, M.D., Ph.D.Unite de Pathologic de l'ImmuniteInstitut PasteurandService de Dermatologie du Pr. Cottenot116pital St.-LouisParis, France

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2. CoNviT, J., ARAN7AZU, N., PiNARDi, M. and ULRIcii,M. Immunological changes observed in indeter-minate and lepromatous leprosy patients and Mit-suda-negative contacts after the inoculation of amixture of Mycobacterium leprae and BCG. Clin.Exp. Immunol. 36 (1979) 214-220.

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4. HAREGEWOIN, A., GoDAL, T., Musl Arn, A. S., BE-LEHU, A. and YEMANEBERHAN, T. T-cell-condi-tioned media reverse T-cell unresponsiveness in le-promatous leprosy. Nature 303 (1983) 342-344.

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6. MEIIRA, V., CONVIT, J., RUBINSTEIN, A. and Bwoni,B. Activated suppressor T-cells in leprosy. J. Im-munol. 129 (1982) 1946-1951.

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8. NATI!, 1. , SATI0sH, M., JAYARAMAN, T., BHUTANI,

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