Immunobiol., vol. 180, pp. 272-277 (1990)

Short Communication 1 Department of Medical Microbiology and Immunology, University of Ulm, Ulm, Federal Republic of Germany, and 2 Hansen's Disease Laboratory, Division of Bacterial Diseases, Center for Disease Control, Atlanta, Georgia, U.S.A.

Epitopes of the Mycobacterial Heat Shock Protein 65 for Human T Cells Comprise Different Structures MARTIN E. MUNKl, THOMAS M. SHINNICK2 , and STEFAN H. E. KAUFMANN l Received September 6, 1989 . Accepted September 29, 1989

Abstract T cell recognition of foreign antigens is a result of a ternary complex between T cell receptor, nominal peptide and major histocompatibility complex molecule. It has been proposed that the nominal peptide, which is presented by accessory cells to T cells, has a characteristic structure which can be predicted on the basis of physicochemical criteria. To further study this aspect, we stimulated T cells from normal human blood donors with synthetic peptides (each of approximately 15 amino acids in length) from the heat shock protein 65 of Mycobacterium tuberculosis-M. bovis. We found that while the characterization of certain epitopes follows commonly used predictions, other epitopes cannot be predicted by known methods.


Mycobacterium tuberculosis is a microorganism capable of survIVlllg inside macrophages, and although humoral immune responses are demonstrable in this bacterial infection, T cells playa key role in protection (1). Evidence has been presented that a significant fraction of T cells to M. tuberculosis is directed against antigens which belong to the heat shock protein (hsp) family including the 65 kDa hsp (2-7). The hsp are a group of proteins with a molecular weight ranging from 15 to 110 kDa (8). Their synthesis increases under a variety of stress conditions, including heat, heavy metals load, low oxygen pressure and pH alteration (8). These findings support the notion that M. tuberculosis organisms residing inside phagolysosomes and under heavy stress conditions produce abundant hsp Abbreviations: hsp = heat shock protein(s); MHC = major histocompatibility complex; FBMC = peripheral blood mononuclear cells; S.!. = stimulation index.

T Cell Epitopes of Different Structure . 273

which are subsequently processed and presented by the infected phagocyte. This could then result in the stimulation of a significant percentage of T cells against hsp. T cells recognize foreign antigens in the context of molecules of the major histocompatibility complex (MHC), and functional evidence exists that T cell activation is induced via a ternary complex between the T cell receptor and foreign antigenic peptide plus MHC molecule on the antigen presenting cell (9). This interaction is preceded by intracellular events termed «antigen processing», the function of which is not only to reduce the antigen to a manageable size for interaction with the MHC molecule and the T cell receptor, but also to present it in an immunogenic form. It has been proposed that common properties of antigenic sites for recognition by T cells can be predicted on the basis of the physicochemical protein structure (10-12). Here, we provide evidence that certain peptides corresponding to amino acid sequences of hsp 65, which stimulate T lymphocytes, do not follow published predictions.

Materials and Methods Isolation and culture of human peripheral blood mononuclear cells (FBMC) PBMC from two healthy PPD positive individuals (donors I and II) and one untreated tuberculosis patient (donor III) were purified over Ficoll-density gradient (Biochrom). T cells were enriched by passage over nylon wool columns (13) and 1 x 10s/ml T cells cultured with 2 x 105 / ml irradiated (3000 R) autologous accessory cells in the presence of 15 f,lg/ml partially overlapping synthetic peptides of the hsp 65 comprising 14 to 19 amino acids. Control cultures were stimulated with purified hsp 65 from M. bovis, which is identical to hsp 65 from M. tuberculosis, (5 f,lg/ml; kindly provided by Dr. J. D. A. VAN EMBDEN, The Netherlands (14)) or with killed solubilized M. tuberculosis H37Ra organisms (5 f,lg/ml, batch 741362, Difco, U.S.A.) in 0.2 ml RPMI 1640 medium (Gibco, F.R.G.) supplemented with 10 % heatinactivated Ab+, Rh+ human serum, antibiotics and 5 X lO- s M 2-mercaptoethanol. After 6 days of culture at 37"C and 5 % COz, cells were pulsed with 1 f,lCi 3H-thymidine (Amersham) for 18 h. Proliferative responses were considered positive at stimulation indices > 5 (5.1. = cpm experiment/cpm control). Synthetic peptides Peptide synthesis has been described elsewhere (15, 16). In short, protected peptide resins were prepared by the method of simultaneous multiple peptide synthesis, and 24 peptides were cleaved at a time in a new multi-vessel apparatus (17), resulting in the generation of 50-70 mg of each peptide.

Results T cells from the three donors showed marked proliferative responses to killed M. tuberculosis organisms and the hsp 65 (Table 1). T cells from donor I responded to 5 (25 %), those from donor II to 14 (70 %) and those from donor III to 8 (40 %) of the 20 synthetic peptides tested (Table 1). T

Amino Acid Sequence


151 130 1




4 -26 15 3 23 39

1 4

30 2 15 14 25

n.d. f ) 14 95

Donor III HLA-D not determined


Stimulation index (see «Materials and Methods») MHC class II phenotype d Amino acid residue numbers of M. tuberculosis hsp 65 (23) e Amino acid sequences by single letter code f n.d. = not done

85 4 3 26 -127 48 11 197 422

N N C C A C C 1 2




83 36 74 24

125 29 3 3 -31 23

Donor II (DR7; DRw53;DQw2,3)

Lymphocyte proliferationb )



11 n.d. 1 492 1



3 3 1

Donor I (DR4,7; DRw53; DQw2,3)")


Prediction Method')

, A = T cell epitope predicted on the basis of amphiphatic peptide configuration according to (11). C = T cell epitope predicted on the basis of continuous sequence of glycine or charged residue followed by two or three hydrophobic residues and ending with a polar amino acid or glycine according to (10). N = T cell epitope not predictable according to A or C.


Peptide Residue

Table 1. Human T cell reactivity to synthetic peptides of the M. tuberculosis hsp 65



c:.." s::



;:r: rn


















:t 0






::l Z


s:: :>-


... '"

T Cell Epitopes of Different Structure .


cells from the three donors showed different reactivity patterns towards the synthetic peptides, although some of the peptides were recognized by T cells of each of the three donors tested. T cells from donor I recognized 4/7, those from donor II 3/7, and those from donor III 2/7 peptides comprising amphipatic structures according to DELISI and BERZOFSKY (11) as assessed by computer analysis performed by the programs within the UWGCG program (18). T cells from donor I recognized 3/13 peptides according to the prediction of ROTHBARD et al. (10), those from donor II 9/13 and those from donor III 5/13 of these peptides. Importantly, peptides which did not fit into either prediction nevertheless stimulated T cells of donor II (3/4) and donor III (1/4). Table 1 also lists the MHC class II phenotype of donors I and II. The two donors show partial MHC class II identity. Thus, it is possible that certain epitopes recognized by T cells from both donors were presented in the context of the same MHC-haplotype.

Discussion Our data identify a large number of epitopes for human T cells within the mycobacterial hsp 65. In earlier studies, long-term cultured lines and clones were used to define epitopes of this molecule, and it was found that the T cell clones recognized peptides, some of which had been predicted to represent T cell epitopes (4, 5). We have chosen another approach, by using freshly isolated T cells, and found that in a single individual, multiple T cells exist with specificity for distinct epitopes. Most impressively, T cells from donor II were stimulated by 14 different synthetic peptides. Although we cannot formally exclude that the peptides exerted nonspecific stimulatory activity on peripheral T cells, such mitogenic effects seem unlikely because only few peptides (amino acids 291 to 305 and 494 to 508) stimulated T cells from all three donors. We, therefore, consider it more likely that the peptides were specifically recognized by the T cells. The HLA-DR phenotype of two donors was defined and found to be partially identical. It is therefore possible that the epitopes recognized by T cells from both donors were presented in the context of the same MHC haplotype. It is also possible that some peptides possessed generic HLA-binding capacity, in contrast to motifs predicted for their specific binding by HLA-DRl. Indeed, studies by others (19-21) have provided strong evidence that different alleles of the same MHC molecule may bind different epitopes. T cell epitopes have been proposed to belong to the following structures: 1) amphipatic peptide configuration (11) or 2) a continuous sequence composed of a glycine or a charged residue followed by two or three hydrophobic residues and ending with a polar amino acid or glycine (10, 12). Our results indicate that epitopes recognized by T cells may not always conform to these predictions. For, we not only identified stimulatory peptides which had been predicted, but also peptides which had not been


predicted as T cell epitopes by either method of prediction. Furthermore, some predicted T cell epitopes failed to stimulate T cells in our study. Hsp 65 is a highly conserved molecule, and the mycobacterial and human cognate show remarkable sequence homology (22). Using synthetic peptides corresponding to shared sequences of the two hsp molecules, we found recently that human class II -restricted T cells lyse autologous target cells presenting these autoepitopes, although they were not predicted to represent T cell epitopes (23). In conclusion, these data suggest that the epitope structures which are recognized by T cells in the context of MHC molecules may follow conditions other than those deduced from currently used predictions (10-12). Acknowledgements This work received financial support to S.H.E.K. from the UNDPlWorld Bank/WHO Special Program for Research and Training in Tropical Diseases, the WHO as part of its program for Vaccine Development, the German Leprosy Relief Association, the EC-India Science and Technology Cooperation Program, the A. Krupp award for young professors, and the Deutsche Forschungsgemeinschaft (SFB 322). M.E.M. is recipient of a grant from the Conselho Nacional de Desenvolvimento Cientifico & Tecnologico (CNPq) Brazil. We thank R. MAHMOUDI for typing the manuscript, Prof. S. GOLDMANN for HLA-phenotype analysis, and Dr. J. D. A. VAN EMBDEN for hsp 65.

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Epitopes of the mycobacterial heat shock protein 65 for human T cells comprise different structures.

T cell recognition of foreign antigens is a result of a ternary complex between T cell receptor, nominal peptide and major histocompatibility complex ...
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