Cell-mediated immunity in mice treated with mycobacterium leprae or with macrophages harbouring M. Leprae

Ann. Immunol. (Inst. Pasteur) 1984, 135 D, 39-50

CELL-MEDIATED WITH OR

WITH

IMMUNITY

IN

MYCOBACTERIUM

MACROPHAGES

MICE

TREATED

LEPRAE

HARBOURING

M. L E P R A E

by P.-R. Ridel 0' 3), j. S. Jhol (2) and J. L. Krahenbuhl 0)

(1) Laboratory Research Branch, National Hansen's Disease Center, Camille, Louisiana, 70721 (USA), (2) SRI International, Menlo Park, Cali/ornia 94025 (USA), and (2) Institut Pasteur de la Guyane /rancaise, 97306 Cayenne Cedex (France)

SUMMARY Following t r e a t m e n t of BALB/c or C3H/HeN mice in the hind footpads with irradiated Mycobacterium leprae, a marked enhancement of natural killer (NK) activity was observed in cells from the draining popliteal lymph node or from the spleen. NK activity was further enhanced when the treatment consisted of killed M. leprae which had been incorporated into mouse peritoneal macrophages. This effect was noted as early as 2 weeks after t r e a t m e n t and persisted for at least 9 weeks. Lymphoblastic transformation in response to suboptimal doses of the T-cell mitogen concanavalin A or to M. leprae antigen was assayed in parallel in cells from the draining popliteal lymph node and from the spleen. In contrast to NK assays, t r e a t m e n t with M. leprae alone moderately altered the response to mitogen. However, there was a prominent enhancement of the T-cell response when t r e a t m e n t consisted of M. leprae-laden macrophages. KEY-WORDS: Cell-mediated immunity, Mgcobacterium leprae, Macrophage; Natural killing, Mouse, Footpad.

INTRODUCTION Growth of the leprosy bacillus in the footpads of susceptible strains of intact mice is limited to the inoculated footpad [16]. In contrast, animals lacking T-lymphocyte function develop a disseminated infection, Manuscrit re~u le 26 octobre 1983, accept6 le 10 mai 1984. Correspondence and reprints: Pierre-Richard Ridel, Institut Pasteur de la Guyane fran~aise 97306 Cayenne Cedex (France).

40

P.-R. RIDEL, J. S. JHOL AND J. L. KRAHENBUHL

demonstrating that i m m u n i t y to Mycobacterium leprae is highly dependent upon cellular mechanisms of resistance [3, 14]. Local footpad infection of immunologically competent mice is accompanied by the development of sufficient i m m u n i t y to protect the animal from challenge infection. Protection against challenge infection can also be induced by local vaccination with killed suspensions of armadillo-derived M. leprae, and Shepard and his co-workers [18, 20, 21, 22] as well as Pattel and Lefford [11, 12] have reported the induction of delayed-type hypersensitivity to M. leprae antigens by intradermal or local subcutaneous footpad inoculation of killed M. leprae. The local nature of the cell-mediated i m m u n e (CMI) response to M. leprae is further emphasized by the persistent enlargement of the l y m p h node draining the site of inoculation [16, 17], and the report of Pattel and Lefford [ 12] demonstrated t h a t specific T-celli nteraction with M. Leprae resulted only in non-specific activation of macrophages in the footpad. Previous studies from our laboratory [6, 7] also underscored the local nature of cellular interactions which occur in response to footpad t r e a t m e n t with M. leprae: natural killer (NK) cell activity was elevated in the ipsolateral lymph node but not in the contralateral popliteal lymph node nor in cells from the spleen or peritoneal cavity. The present studies were carried out to further explore cellular interactions occurring after subcutaneous footpad t r e a t m e n t with preparations of killed M. leprae. Because of the close interaction between the leprosy bacillus and host macrophages and cells known to play important afferent and efferent roles in CMI in general, o u r continued a t t e m p t to modulate local cellular response to M. leprae included t r e a t m e n t with peritoneal macrophages harbouring intracellular M. leprae. Moreover, in addition to the NK response, a non-specific lymphoblastic transformation (LBT) test of T cells to concanavalin A (ConA) and a specific L B T response to soluble M. leprae antigen were studied.

M A T E R I A L S AND METHODS

Mice. Female BALB/c or C3H/HeN mice 6-8 weeks of age were obtained from Simonsen Laboratories, Gilroy, CA. M. leprae vaccine. A suspension of the killed 1/79 preparation of armadillo-derived M. leprae (gamma-irradiated, 2.5 megarads, 6oCo)was obtained from the IMMLEP program of the World Health Organisation. CMI ACPM FCS i.p. i.v. LBT

= = = = = =

cell-mediated immunity. m e a n of c o u n t p e r m i n u t e . foetal c a l f s e r u m . intraperitoneal(ly). intravenous(ly). l y m p h o b l a s t i c t r a n s f o r m a t i o n (test).

LHF ~ left hind footpad. LN ~ lymph node. NK = n a t u r a l killer. P E C ~ p e r i t o n e a l e x u d a t e d cell. RHF ~ right hind footpad. B P M I - T 8 0 = R P M I - k 0 . 0 2 % of T w e e n 80.

CMI TO M Y C O B A C T E R I U M L E P R A E IN MICE

41

M. leprae antigen. Soluble M. leprae antigen was prepared using purified M. leprae obtained from Dr P. Brennan, Colorado State University, Fort Collins, Co. The bacilli concentration was adjusted to 1 • 10 x~ bacilli/ml in sterile saline and sonicated 10 times wiLh ar162Branson )) sonicator (Biosonic, Inc., Cochranville, PA) for 6 rain at 4 ~ C with 80 W efficacy. The final product was centrifuged at 14,000 g for 10 rain, the supernatant decanted and saved. The precipitates were resuspended in saline and sonicated again as described above. After centrifugation, the supernatants were pooled, diluted with saline to a concentration corresponding to a theoretical bacilli count of 1 • 10S/ml and stored in 1-ml aliquots at --20o C.

Macrophages. Peritoneal exudate cells (PEC) were harvested from mice injected intraperitoneally (i. p.) 72 h previously with 0.5 ml thioglycolate medium (Difeo Labs, Detroit, Michigan). As determined by staining for non-specific esterase [24], the percentage of maerophages in suspensions of thioglycolate-stimulated PEC was

s0-s5%.

Incorporation o/M. leprae inlo macrophages. Washed suspensions of PEC were adjusted to a concentration of 4 • 107 macrophages/ml in RPMI (Irvine Scientific, Irvine, CA) containing 10% foetal calf serum (FCS), 0.02% Tween 80 and antibiotics (5 ~g/ml gentamycin; Sigma Chemical Co., St Louis, MO; in I1PMI-80), and 1-ml suspensions were placed in 5-ml snap-cap tissue culture tubes (Falcon Plastics, Los Angeles, CA). M. leprae were suspended at a concentration of 4 • 10S/ml in I:/PlVII-T80, and 1 ml was added to the macrophage suspension to give a final ratio of 10 M. leprae per macrophage. M. leprae or macrophages alone were prepared at the same concentration in the same media and placed on a rocking platform at 37 ~ C. After incubation for 1 h, the tubes were chilled at 4 ~ C for 5 min and the maerophages or M. leprae suspensions decanted. Macrophages which had adhered to the tube walls were easily removed using a cell scraper (Costar, Cambridge, MD). Staining of macrophage preparations with Fite stain confirmed the presence of M. leprae within the macrophages. Only a very small number of bacilli were found outside the maerophage cells.

Trealmenls. Mice of a given group were injected in the left and right hind footpads ( L H F and R H F , respectively) with 0.05 ml of medium containing a suspension of 107 M. leprae which had been incorporated into 106 peritoneal macrophages. Controls were treated with 0.05 ml of medium alone or medium containing 108 macrophages alone.

E[[eclor cells'. Individual popliteal lymph nodes were removed aseptically from the left and right hind legs at the appropriate time after treatment, minced finely, and a single cell suspension prepared by pressing the fragments through 100-inesh stainless steel screens. When obviously enlarged (1-2 mm), the individual nodes from a single animal were pooled. When this was not the case (controls), the lymph nodes from 3 animals were pooled.

N K cell assay. To measure N K activity in popliteal lymph node cells, a 4-h cytolytic assay was performed in which release of 51Cr-prelabelled tumour cells was quantitated as

42

P.-B. R I D E L , J. S. J H O L AND J. L. K R A H E N B U H L

percent release [7]. Briefly, 1 • 107 YAC-I mouse lymphoma target cells (syngeneic for A mice) were labelled with 100 ~Ci of 61Cro4Na2 (New England Nuclear, Boston, MA; specific activity 100-300 Ci/mM) for 45 rain at 37 ~ C. Extracellular 51Cr was removed by washing, and the cells were resuspended at a concentration of 1 • 105 cells/ml in RPMI medium containing 10~o FCS. Each well of round bottom microtitre plates (Linbro Scientific, Inc., Hamden, CT) was seeded with 0.1 ml of this cell suspension (1 • 104 target cells). Effector cells (0.1 ml) were then added in triplicate at the appropriate concentration and the plates centrifuged at 150 g for 5 rain to facilitate contact between effector and target cells. Regardless of whether the assay was performed on cells of a single mouse or a group, an E / T ratio of 20/1 was employed. After a 4-h incubation, the plates were centrifuged, 100 ~l or supernatant were removed, the a m o u n t of 51Cr released was measured in a (( Packard Auto Gamma ,) counter and the percent lysis was calculated with the following formula: test cpm -- spontaneous cpm % release ---- maximum cpm -- spontaneous cpm • 100

L B T assay. In order to perform L B T response to the mitogen ConA in parallel with the N K assay, the remaining lymph node cells were suspended at a concentration of 2• in L B T medium (RPMI-2% FCS containing 0.6% Hepes (Sigma), 10 mM sodium pyruvate (Gibco), 20 mM L-glutamine (Sigma) and non-essential amino acids, p H 7.1) and 0.1 ml was added to each well of fiat bottom microtitre plates (Linbro). A solution of ConA (Miles Laboratories, Bedford, MA) was prepared in fresh L B T medium at a concentration of 1 ~g/ml and 0.1 ml of L B T medium or ConA solution, added in quadruplicate to each group of lymph node cells. After 54 h of incubation, 0.2 ~Ci of 3H-thymidine (New England Nuclear; specific activity 6.7 Ci/mM) was added to each well for the remaining 18 h of culture. After 72 h, the cells were harvested with a (( Mash II )) (Microbiological Associates, Washington, DC) and 3H-thymidine uptake quantitated in a ((Packard Tri-Carb )~liquid scintillation counter. The level of L B T was calculated as Acpm, which is the difference in cpm 8H-thymidine uptake between cultures incubated in the presence or absence of ConA. To measure the response to M. leprae antigen, a 5-day assay was employed. M. leprae antigen was prepared at an optimal concentration (as determined in pilot studies) of 1 • 107 bacilli/ml in fresh L B T media and 0.1 ml of M. leprae antigen added in quadriplicate to each group of cells. To measure T-cell expansion after in vilro Ag stimulation, we performed cocultures, which also required 5 days. The cultures were initiated as above with M. leprae antigen in the medium, but ConA or medium was added at 54 h; the cultures were pulsed with 3H-TdR at 102 h and terminated at 120 h.

Statistics. Where the sample size was sufficient, statistical significianee was tested using the 2-sample rank test. RESULTS F i g u r e 1 shows t h e results of 6 e x p e r i m e n t s p e r f o r m e d in B A L B / c mice a t v a r i o u s t i m e s a f t e r t r e a t m e n t . N K a c t i v i t y of l y m p h n o d e cells f r o m c o n t r o l mice ( t r e a t e d w i t h R P M I or m a c r o p h a g e s alone) was c o n s i s t e n t l y less t h a n 2 % . In c o n t r a s t , f o o t p a d t r e a t m e n t w i t h killed M . leprae m a r k e d l y e n h a n c e d N K a c t i v i t y . Clearly, t r e a t m e n t w i t h M. leprae-laden

CMI TO MYCOBACTERIUM LEPRAE IN MICE

43

15

i A 10

A

A

3

4

>,. _J

5

A

o 1

2

5

6

EXPERIMENT

Fro. 1. - - N K cell response in ceils from the popliteal lymph nodes of B A L B / c mice treated in the footpads with medium (o), macropbages alone ( o), killed M. leprae alone (d~) or macrophages harbouring M. leprae (A); E f T ratio = 20/1. E x p e r i m e n t s 1 and 2 were performed on day 28 after t r e a t m e n t ; experiments 3, 4, 5 and 6 on days 14, 21, 28 and 48, respectively.

macrophages further enhanced the NK activity of lymph node cells. These effects were seen as early as 2 weeks after treatment and persisted at least 48 days. Shown in figure 2 are the LBT data performed in parallel in these 6 experiments. Regardless of treatment, the background level of 8H-TdR incorporated by the cells in the absence of ConA was neither elevated nor depressed in comparison to control values. Under the culture conditions employed in the LBT assay, the concentration of ConA utilized (0.05 or 0.1 ~g/well) were suboptimal doses for lymph node cells, as shown by the low level of LBT obtained in control mice. Local treatment with M. leprae alone did not enhance the LBT response of lymph node cells to ConA. Enhanced LBT activity was seen only in lymph node cultures of mice treated with M. leprae-laden macrophages. As in the case of NK activity, enhancement was evident within 14 days and persisted for at least 48 days. Figure 3 shows the results of experiments carried out in C3H/HeN mice, a strain known to be high NK responders. In these studies, NK activity and LBT to ConA were tested not only in popliteal lymph node cells but also in splenic lymphocytes. In addition, an LBT assay to a soluble M. leprae antigen preparation was tested, as was the response of cells to co-culture with ConA and M. leprae antigen (table I).

52

-1 I

t

44 ,Q ~" 36

r

.q

28

4

20

9

! !

'1

12

zh 2

1

5

3 4 EXPERIMENT

6

Fro. 2. - - Milogenie effect of ConA on cells obtained from the draining popliteal lymph node of mice lreated in the footpads with medium (o), macrophages alone (o), killed M. leprae (A) or maerophages harbouring M. leprae (A). E x p e r i m e n t s 1 a n d 2 were p e r f o r m e d on d a y 28 after t r e a t m e n t ; e x p e r i m e n t s 3, 4, 5 a n d 6, on d a y s 14, 21, 28 and 48, r e s p e c t i v e l y .

130 .k

35 52

30

44

25 tJ3

20

i

>,

_

15

0 o


&

A 20 12

5

L~O

/~

z~ O ~

O 4F

LN SPL LN SPL 28 D. 6 4 D.

"LN SPL LN SPL 28 O. 6 4 D.

F[6. 3. - - N K and mitogenic effect of ConA in cells from the lymph nodes and spleens o/ C 3 H / H e N mice trealed with medium (o), macrophages alone (o), killed M. leprae (A) or macrophages harbouring M. leprae (A). L y m p h node a n d spleen cells f r o m i n d i v i d u a l mice were t e s t e d on d a y s 28 a n d 64 a f t e r t r e a t m e n t .

CMI TO MYCOBACTERIUM LEPRAE IN MICE

45

TABLE I. - - Response of cells to a 5-day co-culture with ConA and (( M. leprae >~ antigen in C 3 H / H e N m i c e 28 days after treatment. Lymph node

Spleen

T r e a t m e n t of mice

Baseline

ConA(a)

Ag(2)

Ag+CgnA

Baseline

ConA

Medium alone

197 4-19

1,045 -k697

261 :L58

1,017 :t-ll

127 •

1,560 •

•

253 •

1,038 •

3,859 :t_193

469 •

157 •

1,136 •

•

166 •

4,993 •

22,585 •

39,688 •

191 -t-22

1,332 •

M. leprae alone M. leprae + M

Ag

Ag+ConA

157

979 •

923

838 553

3,072 •

6,407 :k23

(1) C~nA, 0.05 ~g/well, added 72 h prior to harvesting. (2) M. leprae antigen, 10:/well, added at the beginning of the cultures. M = macrophages.

As in the case of BALB/c mice, the local lymph node NK response was enhanced by treatment with killed M. leprae alone, but treatment with macrophages harbouring M. leprae was clearly more effective. As also seen in figure 3, and in contrast to results with lymph node cells, the baseline level of splenic NK activity from control mice was considerable, in the range of around 20% lysis at 28 days after treatment (E/T = 100/1). Nevertheless, splenic NK activity was also markedly enhanced by local footpad treatment with killed M. leprae and was enhanced even further when M. leprae-laden macrophages were employed. There appeared to be little difference in the NK activity of lymph node cells from either group of M. leprae-treated mice at 64 days, and the enhancement of splenic NK activity induced by treatment with M. leprae with or without macrophages also appeared to be diminished at 64 days. The LBT response in treated C3H/HeN mice differed from that of BALB/c mice, because treatment with killed M. leprae alone was sufficient to significantly enhance the ConA response of lymph node cells. A further marked enhancement was observed in mice treated with M. leprae-laden macrophages. Enhanced LBT to ConA in M. leprae-treated mice was more evident in lymph node cells than in spleen cells 28 days after treatment (fig. 3). As shown by the results depicted in table I, 28 days after treatment, there was no apparent LBT response to soluble M. leprae antigen observed in lymph node or spleen cells from control mice. In animals treated with M. leprae, lymph node and splenic lymphocytes clearly responded to M. leprae antigen alone but not to M. leprae ~- ConA. Local treatment with leprosy bacilli harboured inside macrophages was markedly more effective at enhancing LBT to ConA or M. leprae antigen and was the only treatment able to enhance the LBT response to M. leprae + ConA.

46

P.-R. RIDEL, J. S. JHOL AND J. L. KRAHENBUHL

DISCUSSION The present studies were concerned with enhancement of local CMI response following t r e a t m e n t of the mouse footpad with M. leprae antigen. Markedly enhanced NK-cell activity was observed in cells of the popliteal l y m p h nodes draining the site of inoculation of killed leprosy bacilli. A greater enhancement of NK activity was observed when the t r e a t m e n t consisted of M. leprae antigen which had been phagocytized by mouse peritoneal macrophages prior to administration. Moreover, the local administration of killed M. leprae-laden maerophages induced a conspiciously elevated response of popliteal lymph node cells to the T-cell mitogen ConA. Previous studies from our laboratory emphasized the local nature of the enhanced NK activity induced in response to footpad t r e a t m e n t with M. leprae and other mycobacterial preparations. In the present report, regardless of whether the leprosy bacilli were administered alone or after phagocytosis by macrophages, t r e a t m e n t induced a significant increase in size in the draining popliteal l y m p h nodes; this is a characteristic feature of localized administration of M. leprae [11, 21]. In contrast with C3H mice, the extent of lymph node enlargement did not correlate with enhancement of local CMI in BALB/c mice. Killed M. leprae alone clearly induced the greatest lymph node enlargement (unpublished observations), but did not enhance the response to mitogen; only t r e a t m e n t with M. Ieprae-laden macrophages resulted in a prominent increase in T-cell response to ConA. C3H mice were better NK responders t h a n BALB/c mice, and the ConA L B T of their lymph node cells was enhanced regardless of whether the M. leprae were administered alone or in macrophages. Other than being an additional quantitative measure of t h e local cellular response to M. leprae, the relevance of enhanced local NK activity and elevated mitogen-induced L B T remains to be determined. The biological function of NK cells could lie in immunosurveillance of t u m o u r s [1, 5], the regulation of normal cell proliferation and differentiation [2] or in the early host response to infection [4, 15]. However, in our own work, the enhancement of local l y m p h node NK activity was unrelated to the efficacy of footpad vaccination with killed M. leprae (Krahenbuhl el al., 1981, unpublished observations). Similarly, it is doubtful t h a t local enhancement of mitogen-induced L B T plays a role in resistance to M. leprae. Enhanced NK and ConA LBT most likely represent epiphenomena which are influenced by the interweaving interactions between M. leprae and elements of the host CMI system. Shepard and his co-workers [17, 19, 20, 21, 22] have clearly shown the development of a local cellular response to M. leprae in CFW mice. Similarly, Patel and Lefford [11] showed that sub-cutaneous footpad injection

CMI TO MYCOBACTERIUM LEPRAE IN MICE

47

of killed (irradiated) M. leprae in B6/D2 mice induced delayed-type hypersensitivity to soluble protein antigen of M. leprae. These same workers also reported t h a t the footpad inoculation of killed M. leprae resulted in local but not systemic activation of macrophages to non-specifically kill Lisleria monocglogenes[ 12] and BCG [13]. These latter workers showed t h a t in mice bearing footpad infection with BCG, lymphocyte transformation was readily demonstrable locally in the lymph node lymphocytes, but not systemically in those from the spleen, and it is obvious that, in this animal model, there is a problem of circulation of the Ag-sensitized T cells and/or an inability of T cells to recruit and activate macrophages. In our experiments, M. leprae was introduced within syngeneic macrophages in the mouse footpad, and it is conceivable that this Ag presentation allows good dissemination by the lymphatic system, with t h e Ag being (~ sheltered >)from the cellular environment by the macrophage. It is also conceivable t h a t this presentation modified the local cellular response, inducing the development of a circulation memory T-cell population and/or a better recruitment and activation of the macrophage. Alternatively, because the macrophages used were thioglycolate-elicited, this treatment could act as a primary signal for macrophage activation, resulting in an <( amplification >~ of the local response. However, we did not observe a significant difference when non-elicited macrophages were used. Because it is difficult and mouse-consuming to obtain a sufficient number of macrophages from non-elicited mice, we chose the thioglycolate solution. Whatever the modifications induced by the treatment, it is impossible without additional studies - - to understand the mechanisms involved. However, results obtained with the co-culture system favour t h e hypothesis of modifications at the level of cellular cooperation (only the treatment with leprosy bacilli harboured inside t h e macrophages is able to enhance the ConA response when the co-culture system is used, t h o u g h the type(s) of cell(s) involved remains to be determined. The main interest of such experiments is to demonstrate t h a t it is possible to induce, in the mouse leprosy model, a modification of cellular response by a c~ biological >~manipulation Of antigen presentation. These observations could be of great interest for the development of a strategy vaccine because, as in the case of an intracellular bacterial agent, resistance to M. leprae infection in mice and h u m a n beings is probably effected by the microbiocidal capacity of activated macrophages resulting from the interaction of antigen with specifically sensitized T cells which subsequently elaborate t h e appropriate lymphokines [9, 10, 23]. As discussed above, the studies of Patel and Lefford [11, 12] revealed t h a t footpad injection of killed M. leprae activated t h e macrophages, but only locally; systemic activation of macrophages only followed the i. v. inoculation of M. leprae antigen into mice sensitized by local treatment. Footpad vaccination did not always protect in Shepard's work nor in ours. There is no correlation with elevated NK, but it is conceivable that the enhancement of L B T response to mitogen and M. leprae antigen induced by t r e a t m e n t with M. leprae-burdened macrophages is relevant to the enhanced protec-

-

Ann. lrnrnunol. (Inst. Pasteur), 135 D , n o I , 1 9 8 4 .

4

48

P.-R. RIDEL, J. S. JHOL AND J. L. KRAHENBUHL

t i o n afforded by M. leprae vaccines. On-going experiments should: shed light on this question. In conclusion, from this and our previous reports [6, 7], it appears t h a t elevated local N K and L B T activity represent additional q u a n t i t a t i v e m e a s u r e m e n t s of host cellular response to t h e leprosy bacillus in t h e mouse footpad and t h a t this local response could be modified by a biological t r e a t m e n t . These findings m a y have direct bearing on the fact t h a t local r a t h e r t h a n systemic CMI events are p a r a m o u n t in resistance to experim e n t a l M. Ieprae infection in mice and perhaps in man as well.

RESUME IMMUNIT]~ ~_ MI~DIATION CELLULAIRE CHEZ LES SOURIS TRAITI~ES ,~ L ' A I D E DE (( MYCOBACTERIUM LEPRAE )) OU DE (( M . LEP1RAE )) INCORPORI~ DANS DES MACROPHAGES

Apr~s t r a i t e m e n t de souris B A L B / c ou C3H/HeN dans le coussinet plantaire fi l'aide de Mycobacterium leprae tu~ par irradiation, on observe une augmentation de l'activit6 (( natural killer ), (NK) dans les cellules p r o v e n a n t du ganglion poplit6 drainant ou de la rate. Cette activit~ N K est encore plus augment6e si le t r a i t e m e n t est effectu~ avec M. leprae incorpor~ pr6alablement dans des macrophages p~riton6aux de souris. Cet effet est not6 h partir de la seconde semaine et dure au moins 9 semaines. Le test de transformation lymphoblastique avec une dose sub-optimale de concanavaline A ou d'antig~ne de M. leprae, a ~t6 6tudi~ en parall~le sur des cellules du ganglion poplit~ drainant ou de la rate. La comparaison des r6sultats de l'activit~ N K m o n t r e que le t r a i t e m e n t par M. leprae seul ne modifie que tr~s 16g~rement la r~ponse au mitog~ne. Cependant, la r6ponse des cellules T e s t tr~s augment~e quand les souris ont ~t~ trait~es par M. leprae incorpor~ dans des macrophages. MOTS-CLI~S : Immunit~ cellulaire, Mgcobaclerium leprae, Macrophage; Souris, Coussinet plantaire, Activit~ NK. ACKNOWLEDGMENTS

These studies were supported by grant A1 19069 from the National Institute for Allergy and Infectious Diseases. Dr Ridel is a recipient of a fellowship from the Victor Heiser Foundation for Research in Leprosy. The authors wish to thank Renee A. Painter, Myran Booker and Marie France Gamblin for excellent secretarial assistance.

REFERENCES [1] BLOOM, B., MINATO, N., NEIGtIBOUR, A., REID, L. & MARCUS,L., Interferon and NK cells in resistance to persistently virus-infected cells and tumors,

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