Microbial Pathogenesis 1992 ; 12 : 27-38

Mycobacterium /eprae 65hsp antigen expressed from a retroviral vector in a macrophage cell line is presented to T cells in association with MHC class II in addition to MHC class I C . L . Silva,* A . Palacios, M . J . Colston and D . B . Lowriet Laboratory for Leprosy and Mycobacteria Research, National Institute for Medical Research, Mill Hill, London NW7 IAA, U .K. (Received July 16, 1991 ; accepted in revised form September 4, 1991)

Silva, C . L . (Laboratory for Leprosy and Mycobacteria Research, National Institute for Medical Research, Mill Hill, London NW7 1AA, U .K .), A . Palacios, M . J . Colston and D . B . Lowrie . Mycobacterium leprae 65hsp antigen expressed from a retroviral vector in a macrophage cell line is presented to T cells in association with MHC class II in addition to MHC class I . Microbial Pathogenesis 1992 ; 12 : 27-38 . Mycobacterium leprae lives free in the cytoplasm in infected macrophages . To test if an M . /eprae antigen released into the cytoplasm would associate with major histocompatibility complex (MHC) class II we introduced the gene encoding the 65 kDa heat-shock protein (ML65hsp) into a retroviral shuttle vector (pZIPNeoSV(X)) and transfected the murine macrophage cell line J774G8 . S1 nuclease mapping and Western blot analysis of the transfected cell line (CJ11) showed that specific messenger RNA and ML65hsp antigen were stably expressed . Presence of antigen at the cell surface was demonstrated by flow cytometric analysis with specific monoclonal antibodies (mAb) . Antigen-specific T lymphocytes were stimulated by CJ11 cells to proliferate and release interleukins (IL-2 and IL-3) . These responses were blocked by mAbs specific for either MHC class 11 or for the mycobacterial antigen . The endogenous antigen was also recognised by MHC class I-dependent cytotoxic T cells ; cytotoxicity was inihibited by mAbs against either MHC class I molecules or ML65hsp . Thus, production of ML65hsp within the host cytoplasm resulted in association of the antigen with both MHC class I and MHC class II antigen-presenting structures and evoked both lymphocyte proliferation and cytotoxicity towards the antigen-presenting cell . These findings may be relevant to the development of recombinant subunit vaccines against intracellular pathogens . Key words : antigen presentation ; heat-shock protein ; Mycobacterium leprae ; macrophage transfection .

Introduction The leprosy bacillus (Mycobacterium /eprae) establishes chronic infections in macrophages, where it lives free in the cytoplasm . If antigens arising from such intracellular microorganisms are preferentially presented in association with major histocompatibility complex (MHC) class I molecules, as is the case with most viral antigens which

* Permanent address : Department of Parasitology, Microbiology and Immunology, Faculty of Medicine of Ribeirao Preto-USP, 14049-Ribeirao Preto, SP, Brazil . t Author to whom correspondence should be addressed . 0882-4010/92/010027+12 $03 .00/0

© 1992 Academic Press Limited



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similarly arise within the cytoplasm, the immune response could be biased towards cytotoxicity and this may not always be appropriate . Heat shock proteins (hsp) are among the antigens which are recognized most frequently in the immune responses to intracellular pathogens, including M. /eprae,' 4 and this might reflect a particular ability of these antigens to associate with MHC class II despite their intracellular origin . Additional factors which might contribute to preferential recognition (immunodominance) include intracellular residence evoking a stress response in the microorganism with consequential enhanced synthesis and release of hsp as part of a microbial survival strategy . Immunodominance might also be assisted by homology with a host protein for which a background of immune recognition of 'self' already exists ;' the presence of T cells which respond to self epitopes on human hsps in normal individuals has been demonstrated ."' The significance of hsp immunodominance for either protective immunity or immunopathology is as yet unclear . T lymphocytes that recognize hsp can have phenotypes appropriate either for activating macrophage antimicrobial function or for lysing infected cells .' -" Interest in the immune response to microbial hsp has received a new impetus from recent evidence that immune cross reactivity between microbial and homologous host proteins might contribute to an autoimmune aetiology in rheumatoid arthritis and experimental diabetes . 12-11 To assess whether endogenously generated ML65hsp antigen has an intrinsic capacity to associate with MHC class II, we have introduced the M. leprae gene encoding the 65 kD hsp (ML65hsp) into a murine macrophage cell line (J774G8) and used immunologically specific T lymphocytes to test for association of the resultant endogenously synthesized antigen with MHC class I and class II . Results Introduction of the ML65hsp gene into eukaryotic cells When the retroviral shuttle vector containing the M. leprae gene (pZIPML65 ; Fig . 1 ) was transfected by calcium phosphate precipitation into the virus-packaging cell line psi-CRE, clones resistant to neomycin (G418 ; 0 .5 mg/ml) were obtained . When clones yielding about 1 x10' G418-resistant-colony forming units of virus per millilitre of supernatant were co-cultivated with J774G8 cells in the presence of polybrene the latter became infected, expressing resistance to G41 8 (0 .5 mg/ml) . Expression of mRNA and protein Two G418-resistant J774G8 cell lines, CJ11 and EJ, respectively infected with retrovirus carrying ML65hsp gene and with vector only, were analysed for ML65hsp specific mRNA and protein expression . Dot blot analysis of total cellular RNA extracted from CJ1 1 and EJ cell lines and probed with 32 P-labelled tubulin DNA and ML65hsp DNA showed hybridization of the M. leprae probe only to CJ11 extracts [Fig . 2(A)] . The amount of labelling with ML65hsp probe relative to labelling with tubulin probe did not decrease during 3 months in culture, although subsequent monitoring has shown that prolonged stability requires the maintenance of selective pressure with G418 . Northern blotting confirmed that the sequence specific to the ML65hsp DNA probe was absent from both EJ and parent J774G8 cells (not shown) . When CJ1 1 was cloned by limiting dilution and 16 clones were tested for the presence of the M. leprae hsp, lysates from eight of the clones were recognized by the specific mAb IIIC8 whilst extracts from the other eight were not [Fig . 3(A)] . Western blotting of lysate from CJ11 clones that were positive in dot blots showed the presence of the ML65hsp antigen with an electrophoretic mobility corresponding to a molecular mass of about

Macrophages

expressing

a M. leprae

29

gene

3’LTR

S’LTA ~neopZIPNeoSV(

X 1

3’LTR

S’LTR "00

pZIPML65

1629 I ITGA

0 207 I IATG EcoR

,

I V PROBE

3613 I EcoR I 1

Fig. 1. Construction of pZIPML65. The 3.6 kb genomic DNA fragment containing the gene for ML65hsp was excised from the EcoRl site of cloning vector pUC8 (represented by the solid bar). After end-filling, the DNA sequence was blunt-ligated into the BamHI site of the retroviral shuttle vector pZIPNeoSV(X) (closed box) to create pZIPML65. Sequences encoding the selectable marker for resistance to neomycin (Neo gene) and the retroviral promotors (LTRs) are indicated.

(Al 0

b

C

d

I

1.6-

kb

Fig. 2. Expression of ML65hsp mRNA in CJll transfectant. For dot blot analysis (A) of mRNA, total RNA was extracted from transfected cell lines CJl 1 (a and d) and EJ (b) and from parent cell hne J774G8 (c) 3 months after transfection. Equal amounts (5 fig) were blotted and separately hybridized to DNA probes for tubulin (1) and ML65hsp (2).44 Sl nuclease analysis (8) of total RNA purified from CJl 1 clones 1, 12, 3, 15 and 6, from EJ cells, J774G8 parental cell line and non-cloned CJll cells was done by hybridization to the ML65hsp probe and digestion with Sl nuclease. The protected RNA-DNA fragments were resolved by agarose gel electrophoresis alongside DNA and RNA size markers, transferred to nitrocellulose paper and hybridized with 32p-ML65hsp DNA probe. The deduced size of the protected fragment is indicated.

C L. Srlva

30 (6)

(A) I

9

12

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6

5

7

3

8

IO

II

2

13

14

16

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a

b

et al

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Fig. 3. Expression of ML65hsp in CJII cells. Protein dot blots (A) were made on nitrocellulose with lysates from 16 clones of CJll ceils and from EJ (a) and J774G8 (c) cells, which served as negative controls. Recombinant ML65hsp (40 ng) (b) sewed as a positive control. The membrane was probed with mAb Ill-C8 and bound antibody was detected with alkaline phosphatase-conjugated goat anti-mouse lg. For Western blot (B), cell lysates were subjected to electrophoresis on 15% polyacrylamide-SDS Laemmli gels, transferred to nitrocellulose membrane and probed as for the dot blot. Lanes contained: a, recombinant ML65hsp; b, CJI 1 lysate; c. EJ lysate. The position of the 65 kDa size marker is indicated.

60 kD, slightly less than the recombinant DNA-derived ML65hsp purified from transfected E. co/i [Fig. 3(B)]. No band reacting with the specific mAb was found by Western blot analysis of lysate from EJ cells. When specific mRNA from ML65hsp-positive clones was analysed by Sl exonuclease mapping, the sequence hybridizing to the ML65hsp DNA probe was found to be 1.6 kb in length [Fig. 2(b)]. No variation between clones in the abundance of this mRNA was evident. Clones that were negative for the ML65hsp antigen either failed to produce specific mRNA or produced small fragments (not shown). The expression of ML65hsp at the surface of CJI 1 cells was revealed by binding of the two ML65hsp specific mAbs (Ill-C8 and II-H9) while EJ cells bound neither (Fig. 4). CJII and EJ both bound mAbs against class I and class II MHC determinants equally.

Recognition

of endogenously

expressed ML65hsp

antigen by T lymphocytes

When CJll cells were cultured in the presence of MLGEihsp-specific T lymphocytes there was a significant T cell proliferative response [Fig. 5(A)] accompanied by production of IL-2 [Fig. 5(B)] and IL-3 [Fig. S(C)]. EJ cells that were tested in the same assays had little effect on ML65hsp-specific T cells unless they were supplied with exogenous ML65hsp. CJI 1 cells, in addition to presenting endogenous ML65hsp, were able to present an unrelated exogenous antigen, BSA, to BSA-specific T cells. mAbs against ML65hsp or against MHC class II molecules significantly inhibited the ML65hsp-specific T cell proliferation and interleukin responses to CJI 1 cells while mAb against MHC class I determinants had little or no effect (Fig. 6). CJI 1 cells were also targets for lysis by ML65hsp antigen-specific T cells. As shown in Fig. 7, they were much more susceptible than EJ cells and lysis was almost totally inhibited by mAbs to ML65hsp. Treatment with mAb to MHC class I blocked cytolysis of both ML65hsp-producing cells (CJI 1) and ML65hsp-pulsed EJ cells.



Macrophages expressing a M . leprae gene

31 EJ

CJ l l Control

UL

A~__

ai E aa

X -M9 (65hsp)

s

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Floor .sesaes intensity (log)

F ig . 4 . Surface expression of ML65hsp by transfected macrophages CJ1 1 and EJ cells were labelled with mAb to ML65hsp and MHC class I and class II determinants, stained with fluoresceinated second antibody and analysed by flow microfluorimetry (FACS) . The specificity of each mAb is indicated in parenthesis . Background staining of cells with fluoresceinated second antibody alone or with mAb or myeloma proteins of appropriate isotype and irrelevant specificity was negligible . The data are plotted as log fluorescence intensity (in arbitrary units) against cell number .

Discussion The retroviral shuttle vector used here, pZIPNeoSV(X), proved to be effective in allowing a gene from M . /eprae to be stably integrated and expressed in the murine macrophage cell line J774G8 . Since several initial attempts to transfect this cell line by calcium phosphate precipitation of the vector DNA were unsuccessful, it is likely that the natural processes of the retroviral infection facilitated the successful outcome, as has been noted in other cell systems ." ML65hsp has high amino acid sequence homology (40-50% identity) with the mitochondrial heat shock protein P1 . 16 Foreign genes for homologous proteins might be particularly predisposed to expression in heterologous systems . However, several microbial genes without such known homologies are well expressed in mammalian cells with appropriate vectors ."` 9



C . L . Silva et al.

32

APC

Antigenspecific T cells

Stimulus

CJII CJII CJII CJII -

ML65hsp ML65hsp ML65hsp ML65hsp

ML65hsp BSA -

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(A)

ML65hsp ML65hsp ML65hsp ML65hsp BSA BSA BSA BSA BSA

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ML65hsp BSA -

8

H

H

(C)

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16

3 3 H-thymidine incorporation (cDm x10 )

. Fig . 5 . Recognition by T cells of macrophages transfected with the M . leprae gene encoding ML65hsp IL-2 release (B) and IL-3 release (C) by antigen specific splenocytes (ML65hsp(A), Lymphoproliferation or BSA-specific T cells) were measured on incubation of the splenocytes with CJ1 1 or EJ cells as antigenpresenting cells (APC) in the presence or absence of added antigens (stimulant) as indicated . Bars represent mean and SD of triplicate wells .

S1 exonuclease analysis suggested that transcriptional control elements of the mycobacterial hsp may be operational here . Apparently full-length mRNA was produced which was translated into protein bearing epitopes recognised by ML65hspspecific mAbs and T lymphocytes . The ML65hsp gene has a higher GC content and consequently different frequencies of translation codon usage than the homologous gene of the host cell (GC = 60 .9% for ML65hsp, 41 .7% for human P1 protein) but



Macrophages expressing a M . leprae gene mAb blocking

Dilution

ML65hsp ML65hsp CLASSII CLASS II CLASS I CLASS I

1-2000 1-500 1-2000 1-500 1-2000 1-500

ML65hsp 1-500 CLASS II 1-500 CLASS I 1-500

33

(A)

IO 20 30

1f 40 H

=i (8) 1 3 6 9 3H-thymidine incorporation (cpm x10-3)

Fig . 6 . Blockage of lymphoproliferation and interleukin production with specific mAb . T cell proliferation (A) and IL-2 production (B) were blocked when CJ11 cells and ML65hsp-specific splenocytes were incubated in the presence of mAbs at the dilutions indicated . Anti-ML65hsp comprised an equal volume mixture of III-C8 and II-H9 . Bars indicate mean and SD of triplicate wells .

60

O ND

(A)

(8)

∎ 20

I I I I I 10 I 3 3 10 Effector : target ratio Fig . 7 . Lysis of CJ1 1 cells as targets for ML65hsp-specific splenocytes . CJ11 cells (A) or EJ cells (B) were labelled with 51Cr and used as targets in an assay of splenocyte cytotoxic activity in the presence of several dilutions of effector cells (0) . Assays were also done with added exogenous ML65hsp (20 ug/ml ; 0) or with mAb against ML65hsp (a mixture of III-C8 and II-H9 ; o) or against MHC class I (Ab9-4E9A1 ; ∎) . Cytotoxicity was not blocked by mouse IgG, myeloma protein (Sigma) (data not shown) . 0

I

such differences did not prevent operation of this heterologous system . Codon GUG at position 66 in the M. /eprae nucleotide sequence is a possible translation start site in mycobacteria and E . co/i, yielding a protein of 61 856 Da .20 However, eukaryotic cells are unlikely to initiate translation at GUG21,22 so that translation probably commenced at the first in-frame AUG, in the M . leprae sequence (position 207) and



34

C . L . Silva et al

continued to the stop codon (position 1830) to yield a protein of 56868 Da . This would be consistent with the mobility of the antigen detected on electrophoresis of CJ11 lysates [Fig . 3(B)] and the reactivity with mAbs II-H9 and III-C8 which recognize widely separated epitopes on ML65hsp, defined respectively by linear amino acid sequences at positions 11 1-122 and 531-541 (carboxy-terminal) in the protein" 13 (also P . W . Jenner, unpublished data) . The potential amino acid sequence encoded between the GUG and AUG codons does not have homology with the host P1 protein, 24 whereas putative regulatory Shine/Dalgarno, -10 and - 35 nucleotide sequences are identifiable in this region . It is evident that when the foreign ML65hsp gene was constitutively expressed within CJ1 1 the protein that was produced became available at the cell surface for immunological recognition both by T cells and antibodies . The conventional view, that endogenous antigens are presented on MHC class I and exogenous antigens are presented on MHC class II molecules 25 seems not to apply here . Endogenous ML65hsp was presented to T lymphocytes both in association with MHC class II antigen to support immune amplification reactions (lymphoproliferation and interleukin generation) and in association with MHC class I antigen to elicit cytotoxicity . Exogenous ML65hsp presented by EJ cells elicited cytotoxicity . Several other examples at variance with the conventional view are now known, 26-2a perhaps reflecting differing tendencies of different antigens to enter the endosomal compartment where association with MHC class II normally occurs . 29 Similarly, the early evidence that T cell recognition of antigen on MHC class II molecules could not be inhibited by antibody against the antigen has not been universally sustained . 30 Investigation of how the endogenously generated ML65hsp was processed, where it became associated with the different MHC structures, and its ability to stimulate T cells with a wide range of phenotype and function will be reported separately . It may suffice to say here that both endosomal processing and exocytosis seem to be involved and that specifically sensitized T cells with CD4 + CD8 - , CD4 - CD8` and CD4 - CD8 phenotypes were all cytotoxic . We may also note, in the context of the drive towards making subunit vaccines, that an endogenous single protein antigen evokes a spectrum of immune responses that are pertinent to M . leprae as an intracellular pathogen . At this stage there is no indication that the immunodominance of this particular antigen is attributable to a special affinity for the MHC class II-associated processing and presentation pathways . FACS analysis showed that the availability of MHC class I and class II for binding to specific mAbs was the same on ML65hsp-expressing cells (CJ11) as on non-expressing cells (EJ) and these cells did not differ in their ability to present exogenous antigen . Hence, there was no obvious effect on association of other antigens with the presentation apparatus, but this is not a sensitive guide to a dynamic system where only a small number of MHC molecules need to be associated with antigen to elicit maximum T cell responses ." , " A more detailed analysis and comparison with other endogenous antigens in this model may yet reveal features of ML65hsp handling by macrophages that could account for immunodominance .

Materials and methods Cell culture. NIH 3T3 cells 33 and psi-CRE cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% newborn calf serum . J774G8 34 and its transfected derivatives (CJ1 1 and EJ) were maintained in DMEM with 10% foetal calf serum . HT-2, 32D 35 and WEHI-3 (ATCC TIB68) were maintained in RPMI-1640 medium containing 100 pg/ml streptomycin, 1000 U/ml penicillin, 1 mm glutamine and 12% foetal calf serum (RPMI-C) . HT-2 cultures were supplemented with 5% supernatant from concanavalin A



Macrophages expressing a M. /eprae gene

35

stimulated rat spleen cells and 32D cultures were supplemented with 10% WEHI-3 culture supernatants . Monoclonal antibodies . The anti-ML65hsp mAbs were III-C8 and II-H9, 36 '37 specific for mycobacterial hsp and showing no cross-reaction with extracts of J774G8 in Western blots or ELISA (R . W . Stokes, unpublished data) . Anti-MHC class I and anti-MHC class 11 mAbs were obtained from Dr M . Parkhouse as Ab9-4E9A1 and Ab3-CD4C7, respectively . Vector assembly . The M . /eprae ML65hsp gene was inserted into the retroviral shuttle vector pZIPNeoSV(X) 38 using standard cloning procedures 39 to create pZIPML65 (Fig . 1) . All enzymes were obtained from Boehringer Chemicals . The gene originated from the M. /eprae genomic DNA library of Dr R . A . Young 20 and was present in a 3 .6 kb genomic DNA fragment which was excised from the EcoR I site of cloning vector pUC8 40 end-filled and blunt-ligated into the BamH I site of the shuttle vector . The ML65hsp gene spans nucleotides 207-1892 of the genomic fragment, commencing with start codon ATG, and is accompanied by 5' promotor elements from which it is apparently expressed in E. co/i. 40 The retroviral vector, besides carrying plasmid sequences necessary for propogation in E. coli, contains a retroviral transcription and integration unit derived from Moloney murine leukaemia virus and a selectable marker conferring resistance to G41 8 in mammalian cells . Infectious virus generation . pZIPML65 DNA was introduced into the retroviral packaging cell line psi-CRE 41 by calcium phosphate precipitation ." The packaging cell comprises NIH 3T3 cells containing helper retrovirus deleted of the psi sequence essential for encapsidation . Retroviral vector DNA introduced into this cell complements the deletion and vector is produced as a helper-free infectious virus . Fresh (24 h) cultures of psi-CRE cells (1 x 106 per flask) were transfected with 12 jig of either pZIPML65 or pZIPNeoSV(X) DNA then selected with 0 .5 mg G418/ml (Sigma) in DMEM . Virus production by resistant clones was assayed by titrating 24 h culture supernatants from 30-cm 2 monolayers in the presence of polybrene (4 pg/ml) for ability to confer resistance to G418 on 3T3 cells .41 Supernatant virus was concentrated by centrifugation (27000xg, 12 h) and viral RNA was probed with 32 P-ML65hsp DNA to confirm the presence of the gene . Infection of J774G8 macrophage cell line . The macrophage cell line was infected with the vector carrying the ML65hsp gene by co-cultivation with a monolayer of retrovirus-producer cells . The producer cells were grown to sub-confluence then mixed with J774G8 cells at a producer :target cell ratio of 1 :10 and cultured for 12 h with polybrene (2 pg/ml) . The nonadherent cells were replated on four consecutive days to remove residual adherent producer cells . Infected cell lines CJ11 (J774G8 infected with pZIPML65) and EJ (J774G8 infected with vector pZIPNeoSV(X) only) were then selected by culture with G418 (0 .5 mg/ml) for 10-14 days . Analysis of RNA . Total cellular RNA was extracted by the lithium chloride method 43 and RNA dot blots were done as described . 44 For S1 nuclease mapping, the ML65hsp gene excised from pUC8 with EcoR I was used as a complementary DNA probe . Twenty micrograms of extracted RNA was hybridized with 1 jig of ML65hsp DNA at 60°C for 3 h in 10 pl of 80% formamide, 40 mm PIPES (pH 6 .4), 0 .4 M NaCl, 1 mm EDTA . The mixture was then treated with 800 U nuclease S1 (Boehringer) in 0 .3 ml containing 50 mm sodium acetate (pH 7 .6), 0.28 M NaCl, 4 .5 mm ZnSO 4 , for 2 h at 37°C . The S1-resistant DNA-RNA hybrid was precipitated, denatured in glyoxal and subjected to electrophoresis on 1 .2% agarose gel in 10 mm sodium phosphate (pH 7 .0) alongside 0 .24-9 .5 kb RNA ladder (BRL) and 1 kb DNA ladder (BRL) . Nucleotide fragments were transferred to nitrocellulose paper and hybridized with the 32P-ML65hsp DNA probe which had been radiolabelled by nick-translation . 44 Detection of ML65hsp in ce/I /ysates on nitrocellulose with mAb . Cells were harvested from 2-day cultures and suspended in NP-40 lysis buffer at a concentration of 10 7 cells per ml . The suspension was kept on ice and mixed at intervals for 30 min . Cell debris was removed by centrifugation at 1000xg for 10 min . Lysates (50 pl) were directly spotted onto nitrocellulose membrane (Bio Trace, NT Gelman) using a dot blot apparatus . The membrane was allowed to air-dry and the remaining protein binding sites were blocked with 3% gelatin in Tris-buffered saline at room temperature for 1 h . The membrane was then probed with III-C8 mAb overnight at room temperature, then washed and reacted with alkaline phosphatase-conjugated goat anti-



36

C . L . Silva et al

mouse Ig (Sigma) . For Western blots, the cell lysates were subjected to electrophoresis on 15% polyacrylamide-SDS Laemmli gels, 45 transferred electrophoretically to nitrocellulose membrane and probed as described above for dot blots . F/ow cytometry. CJ11 and EJ cells (2x10 6 ) were washed with ice-cold phosphate-buffered saline (PBS) containing 0 .1% sodium azide and 0 .2% bovine serum albumin (BSA) and incubated for 1 h on ice with 100 µl of mAb diluted 1 :500 in PBS/BSA or with PBS/BSA alone . Cells were then washed twice in ice-cold PBS/BSA and incubated for 1 h on ice with fluoresceinated goat anti-mouse Ig (Sigma) . After two final washes the cells were resuspended in 0 .5 ml PBS/BSA with 1% paraformaldehyde, stored at 4°C for up to a week, and analysed using a FACScan microfluorimeter (Becton Dickinson) . Assays of T cell responses to transfected cells . Antigen-specific T lymphocytes were obtained as spleen cells from vaccinated mice as follows . Female BALB/c mice were injected subcutaneously with 100 1d of emulsion containing 5 gg of recombinant DNA-derived ML65hsp 30 or BSA in Freund's incomplete adjuvant and then injected intravenously twice at weekly intervals with 5 µg antigen in saline . On day 23, splenocytes were obtained and expanded in number through 6 days in culture with relevant antigen (25 µg/ml in RPMI-C) . Lymphoproliferation assays were done in triplicate by plating gamma-irradiated (4000 Rads) antigen-presenting cells (APC ; 5x104 /well) in 96-well round-bottomed microtitre plates (Nunclon) in the presence or absence of relevant antigen and washed antigen-specific splenocytes (1 x10 5/well) . After 60 h incubation at 37°C in a humidified incubator containing 5% CO 2 the cultures were pulsed for 18 h with 3 H-thymidine (0 .5 pCi/well ; 3 H-TdR, Amersham), collected onto glass-fibre filters and radiolabel incorporation was measured by liquid scintillation spectroscopy . For IL-2 and IL-3 assays the 60-h supernatants from lymphoproliferation assays were collected . IL-2 was assayed as an essential growth factor for HT-2 cells ." For IL-3 determination, 32D cells (1 x 10 4 /well) were incubated with 25% supernatant added to the medium for 20 h at 37 ° C then pulsed with 3 H-thymidine for 16 h, harvested and radioactivity uptake was assessed by liquid scintillation counting . Assay for cytotoxic activity was by a standard chromium release assay . In brief, target cells in 500 pl of RPMI-1 640 medium were labelled by incubation with 300 pCi "Cr-sodium chromate for 4 h . Serial dilutions of effector cells were incubated in 200 p1 RPMI-C with 2x10 4 labelled target cells in the presence or absence of relevant antigen or mAb . After 8 h incubation at 37°C, 100 p1 of supernatant was collected and assayed for released radioactivity . Specific lysis was calculated from the expression : specific lysis = (release with CTL-spontaneous release)/(release with 0 .1% SDS-spontaneous release) x100 . When mAbs were used to block cytotoxicity they were added, before mixing target and effector cells, as ascitic fluids at 200-fold dilution and were present for the duration of incubation .

C .L .S . was supported by a postdoctoral fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq, Brazil (process 201654/88-1) .

References 1. 2. 3. 4. 5.

Dubois P . Heat shock proteins and immunity . Res Immunol 1989; 140 : 653-9 . Young RA, Elliot TJ . Stress proteins, infection, and immune surveillance . Cell 1989 ; 59 : 5-8 . Kaufmann SHE . Heat shock proteins and the immune response . Immunol Today 1990 ; 11 : 129-36 . Young DB . Chaperonins and the immune response . Sem Cell Biol 1990; 1 : 27-35 . Lamb JR, Bal V, Rothbard JB, Mehlert A, Mendez-Samperio P, Young DR . The mycobacterial GroEL stress protein : a common target of T-cell recognition in infection and autoimmunity . J Autoimmun 1989; 2 : S93-1 00 . 6 . Munk ME, Schoel B, Modrow S, Karr RW, Young RA, Kaufmann SHE . T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein . J Immunol 1989; 143 : 2844-9 . 7 . Emmrich F, Thole JER, van Embden JDA, Kaufmann SHE . A recombinant 64-kilodalton protein of Mycobacterium bovis bacillus Calmette-Guerin specifically stimulates human T4 clones reactive to mycobacterial antigen . J Exp Med 1986 ; 163 : 1024-9 . 8 . Oftung F, Mustafa AS, Shinnick TM et at. Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells. J Immunol 1988; 141 : 2749-54 .



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