Cell-mediated immunity in experimental cutaneous leishmaniasis

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Cell-Mediated Immunity in Experimental Cutaneous Leishmaniasis F,Y. Liew Department of Experimental Immunobiology The Wellcome Research Laboratories Beckenham Kent BR3 3BS,UK

Forms of cutaneous leishmaniasis are caused by Leishmania major, L. tropica, L. mexicana, L. amazonensis and L. panamensis. Like all leishmanial species, these are obligate intracellular parasites of the mononuclear phagocyte system, with a restricted range of vertebrate hosts including humans, dogs, rodents and arboreal animals. The disease evolves chronically, usually with slow healing, but can sometimes become nonhealing, diffuse disseminating or relapsing. The parasite exists within the macrophages of the vertebrate host in the amastigote form. These transform into extracellular flagellated promastigotes in the gut of the sandfly vectors. The promastigotes can then be injected into new vertebrate hosts as the insects feed. Promastigotes, and to a lesser extent amastigotes, can now be grown in tissue culture. This, together with the use of inbred mouse strains that are susceptible to most of the Leishmania species which are pathogenic for man, has facilitated great advances in our understanding of the immunological control of leishmaniasis. However, as Eddy Liew points out, there are still many unanswered questions. Anti-leishmanial antibodies have been shown in vitro to lyse promastigotes in the presence of complement1,z, to promote phagocytosis3, and to induce surface patching and capping on promastigotes and amastigotes4. However, there is little evidence of a corresponding in vivo role for

Box 1. A Primer on Immune Responses

There are two broad types of antigen-specific immune response: humoral in which antibodies are produced by B-lymphocytes; and cell-mediated in which T-lymphocytes and other ceils combat pathogens in several ways, including lysis of foreign cells and digestion of protein antigens. All lymphocytes are derived from bone marrow stem cells and develop in the lymphoid organs (such as the lymph nodes and spleen). T-cells undergo a further stage of development in the thymus (a ductless, gland-like organ situated in the chest cavity). Removal of the thymus prevents maturation of T-cells. In mice, thymectomy followed by total body irradiation (to kill any peripheral lymphocytes) and replacement of bone marrow (i.e. stem cells) from a genetically identical individual results in the animal having B-cells but no T-cells. Conversely, treatment of mice from birth with anti-IgM (= anti-0) antibody renders the animals profoundly deficient in B-cells with minimal direct effect on T-cells. One manifestation of cell-mediated immunity is delayed-type hypersensitivity - the reaction of a sensitized subject to antigen. The lesions, in which lymphocytes and macrophages are usually prominent, appear 24 h after the contact with antigen. During this lag time the T-cells which reacted with the antigen are proliferating and producing chemicals, or factors, called lymphokines which attract other lymphocytes and macrophages to the site. Lymphokines are also secreted by T-cells stimulated experimentally with plant lectins such as concanavalin-A, or with cells from a genetically different individual. There are many lymphokines with diverse effects on their target cells. For example, under the influence of lymphokines the target ceils may stop moving or move more rapidly, they may be killed, or be stimulated or inhibited in some activity. The characteristics and activities of the lymphokines mentioned in this article are listed in Table 1. Several subsets of T-cells can be serologically distinguished from each other by their surface markers (see Table 2). The Lyt 1÷ 2,3 + cells mentioned in the text are the precursors of the more stable Lyt 1- 2,3 + (cytotoxic/ suppressor) peripheral T-cells. ©1986, ~lsevier Science Publishers B V,, Amsterdam 0169 4758/86150200

antibody in determining the outcome of leishmanial infection. Although some monoclonal antibodies5 or their Fab fragment 6 reduce the infectivity of promastigotes when mixed in large quantities with the parasites before infection, they had no effect on the disease development if injected separately from the promastigotes. In fact, the available evidence argues strongly against a protective role for antibody in controlling leishmaniasis. The disease outcome in different inbred mouse strains is not correlated with the titre or isotype of the antibody response 7, nor is the relatively late appearance of a low level of antibody during healing likely to be effective against the wholly intracellular parasite. Mice genetically selected for low antibody response (Biozzi AB/L, selection 1) are highly resistant to L. major infection and develop small self-healing lesions despite the low level of antibody produced 8. Furthermore, prolonged administration of large amounts of hyperimmune serum or antibody fractions from donor mice protectively immunized against L. major fails to influence the incidence or outcome of infection in susceptible BALB/c mice9. Collectively, these results are clearly incompatible with an important l~roL:~dve role for antibody. Cell-Mediated Immunity (CMI) In contrast to humoral immunity (see Box 1), the case for a causal role of cellmediated immunity (CMI) in acquired resistance to leishmaniasis is based on a

Parasitology Today, voL 2, no. I0, 1986

range of impressive clinical and experimental evidence. Resistant strains of mice rendered relatively T-cell deficient by thymectomy followed by irradiation and reconstitution with syngeneic bone marrow cells (see Box 1) are less able to control L. major infection and have delayed healing 1°. Athymic mutants of the highly resistant CBA and C57BL/6 mice are totally unable to control L. major infection which progresses and visceralizes. Normal resistance can be fully restored by reconstituting these Tcell deficient mutants with syngeneic Tcells u. Acquired protective immunity against L. major 12 and L. donovanP3 (as a result of recovery from infection or prophylactic immunization 9) in resistant or susceptible mice can be adoptively transferred by T-cells but not B-cellsl4,15. Treatment of resistant C3H mice from birth with anti-la antibody rendered them defective in antibody response and also susceptible to L. major infection. However, lesion progression in these treated mice can be arrested and the disease outcome reversed by adoptive transfer of T-cells alone from normal C3H donors 16 without any restoration of humoral antibody formation. These results underline the importance of cell-mediated immunity in resistance to leishmaniasis. CMI in Resistance

CMI can be defined as immunological responses that can be adoptively transferred by cells, where the effector stage involves direct cellular interaction. Several characterized immunological phenomena come under this heading including cytotoxic Tcell reactivity, delayed-type hypersensitivity (DTH, see Box 1), T-cell mediated macrophage activation and natural killer (NK) cell function. Cytotoxic T (Tc)-Cells may have a role in acquired immunity against leishmanial infection 17, although no convincing evidence for a mechanism involving them has so far emerged15, is. This is perhaps not surprising since the efficacy of To-cells against infectious agents is based primarily on the destruction of infected host cells harbouring replicating pathogens which, when released prematurely, fail to reinfect other host cells. This generally works well against viruses but not against protozoa such as Leishmania which is even more infectious in the intracellular amastigote stage. N K cells can play a role in immunity against visceral L. donovani infection in C57BL/6 bg/bg (beige) mice but not in resistance against cutaneous L. major infection 19.

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Table I. Characteristics and activities of some lymphokines Interferon gamma

IFN- T

Macrophage activating factor

MAF

can stimulate increased killing of intracellular parasites by mononuclear phagocytes

Interleukin 2

IL-2

acts on other lymphocytes to amplify immune responses

Interleukin 3

IL-3

stimulates mast cell proliferation

T-cell growth factor

TCGF

B-cell stimulating factor

BSF

G ranulocyte and macrophage colony stimulating factor

G M-CSF

self-explanatory: consists of IL-2 and other as yet unidentified factors essential for B-cell proliferation and maturation self-explanatory

The role of D T H in cutaneous leishmaniasis is controversial. Earlier studies indicated a good correlation between D T H reactivity and resistance to clinical 2o and experimentall2,Zl, 22 cutaneous leishmaniasis, but more recent evidence suggests otherwise. Genetically susceptible BALB/c mice immunized intravenously (i.v.) with lethally irradiated or formalin-fixed promastigotes develop substantial resistance to L. major infection. These protected mice not only fail to express 15 D T H to leishmanial antigen, they also develop significantly lower levels of D T H than unimmunized controls when subsequently challenged intradermally (i.d.) with killed parasites 23. This antigen-specific suppressive effect is adoptively transferable with splenic T-cells which express the Lyt 1+ 2 +, L3T4 + phenotype 23 (see Box 1). In contrast to i.v. immunization, mice injected subcutaneously (s.c.) or i.d. with killed promastigotes produce strong specific DTH. However, these percutaneously immunized mice not only fail to develop evidence of protection, they inevitably show exacerbated disease upon a challenge infection 24. BALB/c mice injected intraperitoneally (i.p.) with cyclophosphamide or pertussigen (a purified protein from Bordetella penussis) shortly before i.d. immunization with killed pmmastigotes, produced greatly enhanced D T H compared to mice injected with parasites

There is good evidence that cell-mediated immunity is important in acquired resistance to leishmaniasis: T-cell deficient mice are less able to control L, major infection and take longer to recover than normal mice, while the progression of lesions in immunodeficient mice can be halted by adoptive transfer of normal T-celts.

Table 2. Cell-surface differentiation markers for T-cells Mouse marker name Human marker name Alternative human name Cell type thymus cortex thymus medulla helper T (Th) suppressor T (Ts) cytotoxic T (To)

Lyt I TI Leu I

-T3 Leu 4

L3T4 T4 Leu 3

Lyt 2,3 T5/8 Leu 2

+ + + +

+ + -t+

+ + + -

+(80%) + -+ +

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alone. Interestingly, mice with higher levels of D T H also develop earlier lesions and accelerated mortality when infected with L. major (J.S. Dhaliwal and F.Y. Liew, unpublished). Even more directly, T-cell lines derived from mice injected s.c. with killed L. major promastigotes, can passively transfer D T H yet cause exacerbation of L. major disease in BALB/c mice25. Collectively these findings provide compelling evidence that D T H is not involved in protective immunity, and that it can actually facilitate the development of cutaneous leishmaniasis. Box 2. Cutaneous D T H Reactions

Cutaneous D T H is a gross measurement of reactivity of interacting T-cells and phagocytes. It comprises the Jones-Mote reaction and the classical tuberculin reaction, described in 1890 by Robert Koch. Subcutaneous injection of tuberculin into normal people gives no reaction, but in people with tuberculosis the same injection causes a red, indurated, painful swelling which peaks 24--48 h after the injection. This reaction can be used to test for past or current infection with Mycobacterium tuberculosis. DTH induced by intradermal infection of killed Leishmania promastigates is a Jones-Mote type of reaction. The Jones-Mote reaction is an early occurring form of delayed-type hypersensitivity described in 1934 by T.D. Jones and J.R. Mote. It is also known as basophilic cutaneous hypersensitivity and is characterized by massive infdtration of basophils (up to 50%) at the site of antigen injection, presumably attracted by an as yet unidentified lymphokine. It is milder than the classical delayed-type hypersensitivity, peaks at 12-15 h and disappears after 48 h.

parasitesare found inside host macrophages, so the killing mechanisms of these cells are important antileishmanial devices. Interactions of leishmanial antigens with immune T-cells stimulates the Tcells to produce factors which increase the leishmanicidal activity of macrophages. Leishmania

to effective cytocidal activity towards both L. donovani and L. olr/eR/26,27. Although

this was fLrSt demonstrated with lymphokines released from T-cells by exposure to concanavalin-A or allogeneic cells, similar leishmanicidal activity can be induced by interaction of specific leishmanial antigens with immune T-cells18,28,29. The intracellular killing of Leishmania in vitro by macrophages involves either the generation of unstable oxygen metabolites or a non-oxidative process30,31. Interferon-7 (IFN-7) 32, as well as a smaller (25kDa) non-IFN-7 molecule33, can activate human peripheral blood monocytes to destroy intracellular L. donovani. IFN-7 and macrophage activating factor (MAF) are also effective in stimulating destruction of intracellular L. major in parasitized murine peritoneal macrophages34. (There is now direct evidence that IFN-7 and MAF are separate molecules3L) BALB/c mice recovered from L. major infection following prior sublethal irradiation are highly resistant' against reinfection. Splenic T-cells from these mice can transfer protective immunity, secreting MAF and activating parasitized macrophages for the destruction ofintracellular L. major. In contrast, T-cells from s.c. immunized or normal BALB/c mice are devoid of all such activities (Dhaliwal and Liew, unpublished). Evidence available so far favours the notion that the major protective immunity against cutaneous leishmaniasis is mediated by T-cells which produce IFN-? and MAF leading to the activation of macrophages and the destruction of intracellular leishmanial parasites.

Cutaneous D T H is a gross measurement of cellular reactivity involving a series of interacting T-cells and phagocytes (Box 2). It comprises the Jones-Mote reaction (which peaks 12-15 h after contact with antigen) involving basophils and polymorphs, and the classical tuberculin reaction (which peaks 24-28 h after contact with antigen) with mainly mononuclear cell infiltration. It is likely that different lymphokines (see Box Characteristics of Protective T-Cells There now appears to be a broad consen1) mediate the early and late DTH, but this is not yet clear. D T H induced by i.d. injec- sus that T-cells conferring protective tion of killed promastigotes is clearly a immunity belong to the subset bearing the Jones-Mote type of reaction with minimal Lyt 1+2- phenotype. The main evidence is presence of mononuclear cells at the peak of as follows. response. It may be that this early D T H (1) As indicated above, when normally resistant recruits cells which serve as targets for CBA and C57BL/6 mice are homozygousfor the infecting parasites without the ability to nu gene (i.e. they do not have a thymus) and are limit their subsequent replication, and so it thereby genetically deficient in T-cell function, accelerates disease progression. In contrast, they become totally unable to control L. major the tuberculin form of D T H which involves infection which progresses and disseminates. the activation of mononuclear phagocytes Normal resistance can be fully restored by may decelerate disease progression. How- reconstituting them with as few as 106splenic Tever, no direct evidence exists for a protec- cells of the Lyt 1+2- but not the Lyt 1-2 + phenotypell from uninfected mice. tive role of classical D T H in leishmaniasis. (2) CBA mice recovered from L. majorinfection Due to the predominant intracellular are highly resistant to reinfection. Such acquired location of the parasite, it is reasonable to immunity can be adaptively transferred with assume that the major leishmanicidal effec- splenic T-cells but not B-cells. The T-cells tar mechanism in viva is the mononuclear involved have been identified by cell depletion phagocyte. Prolonged exposure of mouse experiments in vitro as belonging to the Lyt macrophages to lymphokines in vitro leads 1+2- subsetl4.

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(3) Susceptible BALB/c mice given repeated i.v. injections of killed promastigotes are resistant to L. major challenge infection. The protective immunity so induced is passively transferable with splenic and lymph node T-cells. The protective T-cells again express the Lyt 1+2_ phenotype. (4) BALB/c mice develop resistance to infection with virulent clones of L. braziliensis after vaccination with mutagenized temperatureselected avirulent clones of parasites. Immunity can again be adoptively transferred with Lyt 1+2 - but not Lyt 1-2 + T-cells 36. These complementary adoptive transfer and replacement studies establish the essential role of the Lyt 1+2- T-cell subset in immune control of murine cutaneous leishmaniasis. Understanding of the precise nature of its curative role however, may have to await the availability of monoclonal T-cells with comparable function in vivo. Impairment of Protective Immtmity The development in highly susceptible BALB/c mice of fatal disseminating cutaneous leishmaniasis is accompanied by a parallel induction of specific suppression of CMI. Mice with progressive disease express much lower levels of D T H to leishmanial antigens compared with controls, despite normal reactivity early in the disease. Antigen-specific suppression can be adoptively transferred by splenic T-cells 21. The same spleen cell population also produces significantly reduced levels of interleukin-2 (IL-2) in response to concanavalin-A compared with that from normal mice 37,38. Even more strikingly, culture supernatants of T-cells from mice with progressive L. major infection fail to activate leishmanicidal activity in parasitized macrophages and do not contain detectable levels of M A F (Dhaliwal and Liew, unpublished). In spite of this lack of response, available evidence argues against any intrinsic defect in the ability of BALB/c mice to mount curative immunity against cutaneous leishmaniasis. There is no impairment in the initial induction or expression of CMI in the form of D T H , IL-2 production or M A F secretion for the first three weeks after infection21, 3s. Moreover BALB/c mice recovered from L. major infection following sublethal irradiation, express strong CMI and are refractory to subsequent L. major

infection 39. The active suppression of CMI in mice with progressive fatal infection has been associated with the generation of suppressor ceUs39. This is based on the following observations. (1) BALB/c mice exposed to sublethal (550 rad)

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y-irradiation prior to infection are able to control the disease39 and retain elevated levels of IL-2 (Ref. 38) and MAF production (Dhaliwal and Liew, unpublished). This outcome can be entirely reversed and normal disease progression restored if the irradiated mice are injected with as few as 106 T-cells isolated from the CMIsuppressed donors that have progressive disease. Normal T-cells can also effect this reversal, following a transient period of disease arrest, whereas T-cells from cured donors transfer protective immunity39. Similar results have been obtained with L. donovani infection in genetically susceptible B10.D2 mice4°. The diseasepromoting cells in both systems bear the Lyt 1+2- phenotype14,4°. (2) BALB/c nu/nu mice injected with small doses (106) of normal syngeneic lymphoid cells become resistant to L. major infection, but remain highly susceptible when injected with 100 times greater cell dose. However, T-cells from mice with progressive disease not only fail to protect BALB/c nu/nu mice at any cell dose, but can prevent the protective effect of low numbers of syngeneic normal mouse spleen cells when transferred together. The suppressive or disease-promoting function is again mediated by Lyt 1+2- T-cells H. (3) BALB/c mice rendered completely antibody deficient by anti-IgM antibody treatment from birth can control L. major infection4L This effect can be reversed by T-cells from BALB/c mice with progressive lesions without a concomitant restoration of the antibody response. 15

I

Fig. I. Effect of subcutaneous injection of irradiated L. major promastigotes after intravenous immunization on the subsequent disease developments. Groups of 5 BALB/c mice were given I or 4 x weekly Lv. immunization followed by I or4xweeklys.c. injections with irradiated promastigotes. Together with uninjected controls (0), they were infected with 2 x 105 L. major promastigotes and the subsequent lesion development followed. (If_I) Ixs.c. alone; (~7), I x Lv. alone; ( I ) , 4 x i.v. alone; ( A ), I x i.v. + 4 x s.c.;(O)4xi.v. + 4x~c.; (O)4xi.v. + I xs.c.

I

A

E n.ILl

I--

LU

a

z LU

5

0

2O

60 DAYS

100

268

Intravenous or intraperitoneal immunization with killed Leishmania promastlgotes allows mice to develop resistance to L. major, L. mexicana and L. braziliensis. Conversely, the same anogen given by any percutaneous route can inhibit induction of prophylactic immunity.

Both resistance and susceptibility to cutaneous leishmaniasis are conferred byLyt I + 2 3 - T-cells. Either this cell population is heterogeneous or the quantity of cells in the individual determines their effect. At present the true reason is unknown.

Parasitology Today, voL 2, no. I0, 1986

These findings therefore argue forcibly for the existence of a disease-promoting Lyt 1+2 - subpopulation of T-cells which are operationally called suppressor cells. A similar population of disease-promoting counter-protective T-cells can also be found following s.c. immunization with killed or fractionated promastigotes. T-cell lines derived from mice injected s.c. with freeze-thawed L. major promastigotes can also enhance the development of the disease when transferred to normal recipients 25. The cell lines are L. major-specific and again express the L3T4 +, Lyt 1+2- phenotype. As mentioned earlier, BALB/c mice develop significant resistance to L. major infection following repeated i.v. or i.p. injections of killed promastigotes. The same antigens given intradermally, subcutaneously or intramuscularly are not only ineffective, they markedly exacerbate disease development following s.c. infection with L. major. Furthermore, these percutaneous routes of injection can inhibit the induction and, less strongly, the expression of prophylactic immunity (see Fig. 1). The inhibitory effect can be adoptively transferred by Lyt 1+2 - cells bearing L3T4 + marker 42. This observation has now been extended to resistant mouse strains and to L. mexicana and L. braziliensis43.

A Consensus of Opinion The following consensus on the immune regulation of cutaneous leishmaniasis has emerged from the discussion above. • Protective immunity in cutaneous leishmaniasis is mediated by Lyt 1+2 - Tcells and not by antibody. • BALB/c mice (and by inference susceptible individuals) are intrinsically capable of generating protective immunity. Macrophages from genetically susceptible mice can be activated to give a curative leishmanicidal response. Recovery from otherwise fatal disease can be achieved by prior adult thymectomy, irradiation and bone marrow reconstitution, sublethal dose irradiation, anti-I~ or anti-L3T4 + antibody treatment, or by prophylactic i.v. and i.p. immuniTation. • Abrogation of protective immunity and exacerbation of normal disease progression can be demonstrated with: Lyt 1+2T-cells from mice suffering progressive disease, Lyt 1+2- L3T4 + cells from mice after s.c. immunization, or in vitro propagated, s.c. immunization derived Lyt 1+2% L3T4 + lymph node cells. • D T H is not only a cellular phenomenon

dissociable from protective immunity, also its earlier phase and subsequent nonspecific manifestation is detrimental to the host in facilitating disease progression.

Unresolved Questions Despite this impressive consensus, there is still controversy about the mechanism of immune regulation in cutaneous leishmaniasis. Since resistance and susceptibility are both conferred by Lyt 1+2 - T-cells, what are the mechanisms leading to the difference in disease outcome? It has been argued that protection or counterprotection merely reflect a quantitative difference in the requiremem for L3T4 +, Lyt 1+2 - Tcells25,44. In other words, too many protective T-cells are detrimental, reminiscent of the 'too much help leads to suppression' concept. Alternatively, the Lyt 1+2 phenotype cell population is functionally heterogeneous, and may be subdivided qualitatively into subsets mediating protection and those promoting disease progression45,46. Supporting the former interpretation are the findings that transfer of 106 Lyt 1+2 - cells to nu/nu BALB/c mice led to protection wherease l0 s of the same cells would exacerbate the disease11. Less direct evidence is the observation that infected susceptible BALB/c mice contain a higher ratio of L3T4+/Lyt 1+2 - cells than similarly infected resistant CBA mice44. Furthermore, treatment of mice with anti-L3T4 + antibody in vivo led to containment of disease in BALB/c mice47. However, evidence supporting the latter interpretation appears to be equally persuasive. Firstly, unlike normal T-cells, Lyt 1+2 - cells from mice with progressive infection are not protective at any cell dose in nu/nu BALB/c micell. This argues for a qualitative difference between activated, disease-promoting cells and unactivated T-cells which contain normal ratios of precursor cells capable of differentiating into protective or counterprotective phenotypes. The larger cell doses required to exacerbate disease in nu/nu mice merely indicate that the threshold of activation of these cells is different from that of protective cells. Secondly, in a series of titration experiments, it was found that freshly isolated T-cells from mice with progressive disease 14 o r i m m u n i z e d s.c. with killed promastigotes42 either inhibit protective immunization ( > 1 0 7 cells) o r have no effect (<10 7 cells). No protection was observed at any dose. Conversely, freshly isolated T-cells from mice recovered from infection39 or given prophylactic i.v.

Parasitology Today, voL Z no. I0, 1986

immunization 15 are either protective (> 107 cells) or ineffective (< 107 cells). No exacerbation of disease process was observed at any dose. Furthermore, the protective Tcells from i.v. immunized mice are Lyt 1+2-, L3T4-, whereas the counterprotecfive T-cells following s.c. injection of killed parasites are Lyt 1+2-, L3T4 + (Liew et al., unpublished). Functional heterogeneity of T-cells of the helper (Lyt 1+2 -) phenotype is also supported by recent evidence from other experimental systems. Antigen-specific Lyt 1+2-, L3T4 + murine T-cell clones can be divided into those producing IL-2, IFN-y, GM-CSF (granulocyte-macrophage colony stimulating factor) and IL-3, or those producing IL-3, BSF-1 (B-cell stimulating factor 1), a non-IL-3 mast cell growth factor and a non-IL-2 TCGF (T-cell growth factor)4s. The inducer/helper Tcells in the rat also comprise two distinct functional subsets: one that synthesizes high level of IL-2 and another that helps Bcells into antibody synthesis49. It is clear that stringent numeration and careful testing/n vivo of well characterized T-cell subsets are necessary to resolve this current uncertainty. What is the mechanism underlying the preferential induction of host protection and counterprotection? The same parasite infecting susceptible or resistant individuals can lead to completely different disease outcome. Macrophages from susceptible mice are less capableS0,51 of limiting the replication ofintracellular L. major by virtue of Scl (Ref. 52) and H-1I b (Ref. 53) genes expression in the mononuclear phagocyte lineage54. It is thus reasonable to believe that antigen presenting cells (APC) would be the prime determining factor in preferential Tcell activation. The crucial role of APC is also evident from the finding that the same parasite antigen injected i.v. or s.c. lead to either protection or counterprotection respectively. An interesting hypothesis has been put forward by Mitchell and Handman 46 who argue that a lipid-containing glycoconjugate (L-GC), and the delipidated water soluble glycoconjugate (DL-GC) derived from L. major promastigotes by enzyme cleavage play a central role in murine cutaneous leishmaniasis. They proposed that L-GC when anchored by lipid and oriented in relation to class II major histocompatibility complex (MHC) molecules on infected macrophages is both an inducer and a target for certain class II MHC-restricted T-cells. These are macrophage-activating protective Lyt 1÷ 2 T-cells and are presumed to be present

269

in high frequency in normal lymphoid organs capable of transferring protection to nu/nu micelk Their activation and effectiveness, however, would be greatly diminished in susceptible mice whose macrophages are chronically infected and have decreased MHC class II expression. On the other hand, DL-GC bound to macrophages via specific receptors and unassociated with MHC is thought to activate the diseasepromoting T-cells which are also Lyt 1+ 2-. Supporting this hypothesis is the finding that L-GC when injected intraperitoneally conferred protection against L. major infection in susceptible BALB/c mice6. The specificity and comparative counterprotective effect of L-GC and DL-GC administered by various routes are as yet unknown. The differences in expression and orientation of MHC class II molecules on normal or infected macrophages from resistant or susceptible mice are also conjectural at present. In the past, the H-2 (mouse MHC) gene complex has been shown to play a relatively minor role in genetically determined susceptibility to L. major infection55. Until more direct evidence emerges, the question of preferential induction must be regarded as still conjectural. Is there a strict correlation between leishmanicidal activity in vitro and disease resistance in vivo? As argued earlier, by virtue of the intra-macrophage location of the amastigotes, the most likely final effector mechanism in the elimination of leishmartial parasites would be the macrophages activated by lymphokines secreted by specifically sensitized T-cells. Indeed, there is a good correlation between the capacity of a T-cell population to produce MAF activate normal macrophages for leishmanicidal activity, and its ability to transfer protection (Dhaliwal and Liew, unpublished). Thus upon stimulation in vitro with leishmanial antigens, T-cells from mice recovered from infection elaborate MAF as measured by macrophage tumoricidal as well as leishmanial microbicidal activities. In contrast, T-cells from mice with progressive disease or after s.c. immunization are non-responsive. However, a completely different set of results was obtained 25from a similar line of investigation. Draining lymph node cells from mice injected s.c. with freeze-thawed L. major in Freund's complete adjuvant were restimulated, cultured, enriched for T-cells and repeatedly propagated in vitro with interleukin 2. These T-cell lines or clones which are L. major specific, L3T4 ÷ and Lyt 1+2- enhanced the development of the dis-

There is good correlation between the amount of macro'phage octivoting factor produced by Leishmania-stimulated T ceils, i e. their ability to activate normal macrophages to leishmanicidal activity, and the protection tran/erred by such T-ceils. T-cells from mice with progressive disease, or after subcutaneous ~mmun~zation,do not transfer protection.

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ff vaccines against human cutaneous leishmaniasisare to be developed, the routine phenomenon of inhibition of immuniL7 by percutaneous immunization must be examined in man.

Parasitology Today, vot 2, no. 10, 1986

ease when transferred to either genetically susceptible (BALB/c) or resistant (CBA) mice. This suppressive function was observed even though the cell line exhibited helper activity for antibody production, produced MAF upon stimulation with the parasite antigen, and transferred DTH to BALB/c recipients. A major variation between these two sets of experiment is that the latter used long term T-cell propagation in vitro. It has always been suspected but never proven that continuous antigenic activation and growth-factor stimulation may deregulate unipotential T-cells to those capable of multiple biological activities. What is the prospect for vaccination against clinical leishmaniasis? The i.d., s.c. and i.m. routes of immunization with killed leishmanial antigens not only fail to protect but block the prophylactic i.v. immunization against cutaneous leishmaniasis in mice. Full understanding of this phenomenon seems mandatory, as does determination of whether this critical dependency on route of immunization extends to man and to biochemicaUy defined antigens. If the latter associations are found, then new approaches to induction of protective immunity will be required. References

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