Human thymocytes expressing gamma/delta T-cell receptors

Int. J . Cancer: Supplement 4, 39-42 (1989) 0 1989 Alan R. Liss, Inc.

Publication of the International Union Against Cancer Publication de I‘Union lnternationale Contre le Cancer

HUMAN THYMOCYTES EXPRESSING GAMMNDELTA T-CELL RECEPTORS M.C. MINGARI’,P. VARESE,C. BOTTINO, G. TAMBUSSI and L. MORETTA’.~ Istituto Nazionale per la Ricerca sul Cancro, Genoa; and ’Istituto di Oncologia Clinica e Sperimentale and Centro Interuniversitarioper la Ricerca sul Cancro (C.I.R.C.),Genoa, Italy. Expression of T-cell receptors of gammddelta type characterizes a small subset of peripheral T lymphocytes which is homogeneously composed of cytolytic cells and, in most instances, lack CD4 and CD8 differentiation antigens. By the use of anti-TCR gammddelta MAbs it is possible t o identify two distinct subsets of TCR gammddelta+ cells that are characterized by a Cy l or Cy2-encoded forms of gamma-chain, respectively. While the 863 MAb-reactive (Cy l encoded) cell subset is prevalent in peripheral blood (PB), these cells represent < 10% in TCR gammddelta+ thymocyte populations. In thymus, the majority of cells was found t o react with delta-TCS I (or A13) MAbs. Culture of CD4-8- thymocytes (highly enriched in TCR gammddelta+ cells) in IL-2 resulted in the de novo expression of CD8 surface antigen and of non MHC-restricted cytolytic activity. Cloning of CD4-8thymocytes resulted, for the most part in CD3’TCR gammddelta+ cells. Moreover, the majority of clones expressed the unusual delta-TCS I+CD8+’phenotype and lysed the NK-sensitive K562 target cells. Analysis of the immunoprecipitated TCR molecules showed the existence of the (rare) heavy (55 kDa) form of gamma-chain. A redirected killing assay using murine P8I5 target cells and appropriate “stimulatory” antibodies was further employed for functional analysis of thymus-derived TCR gammddelta+ clones. While anti-CD3 MAbs efficiently triggered the cytolytic activity of all clones irrespective of their phenotype, MAbs directed t o TCR gammddelta induced efficient lysis only of BB3+ or delta-TCSI+CD8- clones, but not of delta-TCSI+CD8+ clones. Therefore, it appears that a receptor type which appears t o be relatively inefficient in mediating activation signals is predominant in the thymus and rare in the periphery.

A small subset of CD3+ lymphocytes expresses TCR molecules different from the typical alphdbeta heterodimer which are composed of the molecular products of TCR gamma and delta genes. A remarkable phenotypic feature of most TCR gammddelta+ cells present in the peripheral blood is the simultaneous lack of CD4 and CD8 surface-differentiation antigens which define the 2 major subsets of TCR alphdbeta+ cells (Brenner et al., 1986; Lanier and Weiss, 1986; Lanier et al., 1987). This phenotypic feature has been originally used as a criterion for enrichment, analysis and cloning of TCR gammddelta+ cells (Moretta et al., 1987). The recent availability of anti-TCR gammddelta MAbs has allowed the direct identification of TCR gammddelta+ cells in different lymphoid tissues and a more precise analysis of the various forms of TCR gammddelta molecules and of the mechanisms of cell activation (Jitsukawa et al., 1987; Ciccone et al., 1988a; Ferrini et al., 1989; Bottino et al., 1988; Borst et a l . , 1988; Band et al., 1988). In addition to MAbs which identify the total pool of TCR gammddelta, MAbs have been selected which recognize 2 different molecular forms of TCR. Thus, BB3 MAb reacts with approximately 2/3 of CD3 +4-8-WT31- peripheral blood cells. These cells express a Cyl-encoded, disulphide-linked form of TCR gammddelta (Ciccone et al., 1988). Virtually all of the remaining TCR gammddelta+ peripheral blood cells react with delta-TCS1 or A13 MAbs. These PB-derived cells express a CyZencoded, non-disulphide-linked form of TCR (Ferrini et a l . , 1989). Two molecular forms of non-disulphide-linked TCR gammddelta have been identified. The major difference between these 2 forms is the size of the gamma chain. Thus, while most of the non-disulphide-linked forms of TCR gammddelta isolated from PB express a 44-kDa gamma chain, a very small percentage of these cells is characterized by a 55-kDa chain (Moretta et a l . , 1988). Moreover, an association has been observed between expression of this form of TCR gammddelta and of CD8 surface molcules (Moretta et al., 1988). Analysis of the TCR gamma/delta+ cells in thymus indicated that these cells represented a small fraction of the total thymocyte population and that (as in PB) they were mostly confined to the CD4-8- subset. Therefore, this minor thymocyte population has been utilized to enrich for TCR gammddelta+ cells and for the analysis of phenotypic and functional characteristics of these cells (Bank et al., 1986). De novo expression of CD8 antigen and cytolytic activity by CD4-8- thymocytes cultured in IL-2 CD4-8 - thymocyte populations obtained by treatment with anti-CD4 and antLCD8 MAbs and complement contained variable proportions (15-50%) of CD3+ (CD4-CD8-) cells. Most of these cells were found to react with delta-TCS1 (or A13) MAb while very few were BB3+ (Mingari et al., 19886). CD4-8- thymocytes cultured in recombinant interleukin-2 (IL-2) were found to progressively acquire cytolytic activity against the NK-sensitive K562 target cell line. Moreover, phenotypic analysis showed that increasing proportions of cells expressed surface CD8 antigens, while neither CD4+ nor WT31+ cells (TCR alphdbeta+) could be detected. Analysis of the reactivity with anti-TCR gammddelta MAbs indicated that TCR gammddelta+ cells underwent proliferation and accounted for 80-90% cells after 2-3 weeks of culture. These experiments indicated that most of the CD4-8- thymocytes undergoing proliferation in response to IL-2 are represented by TCR gammddelta+ cells. Figure 1 shows that virtually all cells cultured for 3 weeks are CD3+; moreover, the majority of CD3+ cells express a delta-TCSl-reactive TCR gammddelta; finally, most delta-TCS 1 cells expressed CD8 surface antigen. The finding that increasing proportions of cells expressed surface antigens and displayed cytolytic activity suggested that these cells originated from CD4 - 8 - non-cytolytic precursors. However, an alternative explanation could be the preferential expansion, under the culture conditions utilized, of very few CD8 cells contaminating the starting population and progressively outnumbering the CD4-8 - thymocytes during culture (Mingari et al., 1988a,b). +

+

2To whom correspondence should be addressed at: Istituto Nazionale per la Ricerca sul Cancro Viale Benedetto XV, 10, 16132, Genoa, Italy.

6-TCS-1 FlTC FIGURE 1 - Two-colour FACS analysis of thymocyte copulations depleted of CD4+ and CD8+ cells and cultured for 3 weeks in rIL-2. Essentially all cells are CD3+. The majority of the CD3 cells were reactive with delta-TCS1 MAb (left panel). Expression of CD8 surface antigen was (for the most part) associated with delta-TCSl+ cells. Clonal analysis of TCR gammaldelta thymocytes In order to distinguish between the two possibilities above, we performed cloning experiments, in which purified CD4-8thymocytes were cultured under limiting dilution conditions. We applied a microculture system that had been shown to allow the clonal expansion of virtually all PB-derived T lymphocytes. The cloning efficiencies obtained with CD4 - 8 - thymocytes were relatively high (up to % cells). Most of the clones analyzed (over 100) expressed the CD3+WT31-delta-TCSl+ cell surface phenotype and approximately half of them expressed the CD8 antigen. Analysis of cytolytic activity showed that all clones lysed the NK-sensitive K562 target cells and, in some instances, the NK-resistant fresh melanoma cells. Therefore, our data indicate that the expression of both CD8 surface antigen and of cytolytic activity reflects an in vitro evolution of CD4-8- thymocyte precursors. In addition, under the culture conditions used, no WT31+ or CD4+ cells (TCR alpha/beta+) could be obtained starting from CD4-8 - thymocytes. All together, data at the population or the clonal level indicate that the prevalent types of TCR gammddelta+ cells present in thymus or in peripheral blood are rather different. In particular, a large fraction of TCR gammddelta+ cells derived from thymus express a surface phenotype unusual in peripheral blood (delta-TCS1 +CD8+). The CD3-associated molecules immunoprecipitated from TCR gammddelta+ thymic clones have been analyzed by 2-D gel electrophoresis (Bottino et al., 1988). In these experiments, immunoprecipitated molecules were first separated according to their PI by the NEPHGE, followed, in the second dimension, by SDS-PAGE. Figure 2 shows the surface TCR molecules immunoprecipitated from 2 representative delta-TCSl-reactive clones. The molecules of the right panel were derived from a CD8- clone, whereas those in the left panel were immunoprecipitated from a CD8+ one. It appears that the two TCR molecules differ not only in molecular size but also in charge mobility (the 55-kDa gamma chain expressed in the CD8' clone is slightly more acidic than that immunoprecipitated from the CD8- one). +

Anti-CD3lTCR MAb-mediated activation of TCR gammaldelta+ clones derived from thymus MAbs directed to CD3 or TCR complex (either alphdbeta or gammddelta) have been shown to induce T-lymphocyte activation, leading to lymphokine release or triggering of the cytolytic machinery. We studied the effect of anti-CD3 or anti-TCR gammddelta (delta-TCS1) MAb on the triggering of the cytolytic activity of representative TCR gammddelta+ clones derived from thymus. To this end, clones were tested for their ability to kill appropriate target cells in a redirected assay. Target cells were represented by the Fcy-receptor-bearing P815 murine mastocytoma cell line. As shown in Table I, the addition of soluble anti-CD3 MAb efficiently triggered delta-TCS 1+CD8 + as well as delta-TCS 1 +CD8- clones to lyse target cells. On the other hand, delta-TCS1 MAb efficiently activated CD8-delta-TCSlf clones. Similar data were obtained with other MAbs, including A13 and the anti-TCR gammddelta-1, a MAb which reacts with all forms of TCR gammddelta (Mingaxi et al., 1989). A similar activation pattern was observed for the (rare) delta-TCS1 +CD8+ clones derived from PB (Moretta et al., 1988). These results provide experimental evidence that distinct forms of TCR gammddelta may differ in their ability to transduce signals to the cell upon binding with appropriate MAbs. Our data suggest that the relative inefficiency of anti-TCR gammddelta MAbs to induce activation of CD8+delta-TCS1+ clones reflects a partial inability of this TCR type to transduce activation signals. On the other hand, since optimal activation could be induced by anti-CD3 MAbs, the defect appears to be inherent to the TCR itself or to a poorly efficient association with CD3 molecules. Interestingly, the receptor type which was found to be relatively inefficient in mediating activation signals is predominant in the thymus and rare in the periphery. Therefore, it is possible to speculate that thedifferences in the distribution of TCR gammddelta+ subsets may possibly reflect a change in the TCR gammddelta repertoire during the course of evolution from TCR gammddelta+ thymocytes to TCR gammddelta PB lymphocytes. This change may be consequent to preferential elimination of this particular type of TCR gammddelta+ cells within the thymus (due to selfrecognition?). The possibility also exists that the low percentages in PB may be consequent to the particular homing properties

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GAMMAIDELTA T-CELL RECEPTORS TABLE I - ANTI-CD3 OR ANTI-TCR MAb-MEDIATED RE-DIRECTED KILLING BY TCR GAMMNDELTA+CD8+ OR CD8

CLONES DERIVED FROM HUMAN THYMUS

~

MAb added to the cytolytic test1 Clone

Surface phenotype

21.5 21.6 G29 G34 G177 21.57 G14 21.53

CD8+6-TCS-l+ CD8+S-TCS-lf CD8+6-TCS-l+ CD8 6-TCS-1 CD8 6-TCS- 1 CD8 - S-TCS-l+ CD8-6-TCS-l+ CD8 BB3+ +

+

~

+

+

None

172 8 1 2 15 3 14 13

Anti-CD3

8-TCS-1

BB3

100 91 31 86 87 97 57 81

26 28 15 38 32 93 54 9

26 22 7 3 9 10 16 68

‘The various MAbs were added at the onset of the 4 hr 51Cr-releaseassay containing the FcyR-receptor-positiveP815 cells -*Data are expressed as percentage specific 5’Cr-release at an approximate effector:target ratio of 1:l.

FIGURE 2 - 2D-PAGE analysis of the CD3/TCR molecules immunoprecipitated from 2 representative TCR gammddelta+ clones derived from human thymus. These clones both reacted with delta-TCS1 MAb. However, one comprised CD8+ (left panel), and the other CD8- cells labelled with lZ5I and lysed in digitonin-containing buffer. Cell lysates were then immunoprecipitated using the anti-Leu4 (anti-CD3) MAb. All the samples were analyzed by NEPHGE and the second dimension analysis was performed using 11% acrylamide gels for SDS- PAGE analysis under reducing conditions.

of these cells (Grossi et al., 1989). Moreover, on the basis of the poor ability of these cells to be triggered via their TCR, it is possible to speculate that they have a limited capability to undergo clonal expansion in response to antigen stimulation in the periphery. ACKNOWLEDGEMENTS

This work was supported in part by grants from the Consiglio Nazionale delle Ricerche (CNR) “Piano Finalizzato Oncologia” to M.C.M. and L.M., and by the Associazione Italiana per la Ricerca sul Cancro (AIRC). REFERENCES F., MCLEAN,J., HATA,S., KRANGEL, M. BAND,H., HOCHSTENBACH, and BRENNER, M.B., Immunochemical proof that a novel rearranging gene encodes the T-cell receptor delta-subunit. Science, 238, 682 (1988). M.B., CASSIMERIS, J., ALT,F.W. BANK,I., DE PINHO,R.A., BRENNER, and CHESS,L.A., A functional T3 molecule associated with a novel heterodimer on the surface of immature human thymocytes. Nature (Lond.), 322, 179-182 (1986). BORST,J., VAN DONGE,J.M.M., BOLHUIS,R.L.H., PETERS,P.J., R.J.,Distinct molecHAFLER,D.A., DE VRIES,E. and VANDE GRIEND, ular forms of human T-cell receptor gammddelta detected or viable T cells by a monoclonal antibody. J. exp. Med., 167, 1625-1644 (1988). G., FERRINI, S., CICCONE,E., VARESE,P., BOTTINO, C., TAMBUSSI, MINGARI, M.C., MORETTA, L. and MORETTA, A,, Two subsets of human T lymphocytes expressing gammddelta antigen receptor are identifiable by monoclonal antibodies directed to two distinct molecular forms of the receptor. J. exp. Med., 168, 491-505 (1988). BRENNER, M.B., MCLEAN,J., DIALYNAS, D.B.P., STOMINGER, J.L.,

SMITH,J. A., OWEN,F.L., SEIDMAN, J.G., IP, S., ROSEN,F. and KRANGEL,M.S., Identification of a putative second T-cell receptor. Nature (Lond.), 322, 145-149 (1986). CICCONE, E., FERRINI, S., BOTTINO,C., VIALE,O., PIUGIONE, I., PANG., TAMBUSSI, G., MORETTA, A. and MORETTA, L., A monocloTALEO, nal antibody specific for a common determinant of the human T-cell receptor gammddelta directly activates CD3 WT3 1- lymphocytes to express their functional program(s). J. exp. Med., 168, 1-11 (1988). FERRINI,S., PRIGIONE,I., BOTTINO,C., CICCONE, E., TAMBUSSI, G., MAMMOLITI, S., MORETTA, L. and MORETTA, A , , Monoclonal antibodies which react with the T-cell receptor gammddelta recognize different subsets of CD3+WT31- T lymphocytes. Europ. J. Zmmunol., 19, 57-61 (1989). GROSSI,C.E., CICCONE, E., MIGONE,N., BOTTINO,C., ZARCONE, D., G., VIALE,O., CASORATI, G., MINGARI, M.C., FERRINI,S., TAMBUSSI, MORETTA, L. and MORETTA, A,, Human T cells expressing the gamma/ delta T-cell receptor (TCR-1). Cyl and Cy2-encoded forms of the receptor correlate with distinctive morphology, cytoskeletal organization and +

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growth characteristics. Proc. nut. Acad. Scr. (Wash.), 86, 1619-1623 (1989). JITSUKAWA, S., FAURE,F., LIPINSKI,M., TRIEBEL,F. and HERCEND, T., A novel subset of human lymphocytes with a T-cell receptor-gamma complex. J. exp. Med., 166, 1192-1204 (1987). N. A,, RUITENBERG, J. J . , PHILLIPS, J.H., ALLANIER,L.L., FEDERSPIEL, LISON,J.P., LITTMAN,D. and WEISS,A,, The T-cell antigen receptor complex expressed on normal peripheral blood CD4-, CD8- T lymphocytes. A CD3-associated disulphide linked gamma-chain heterodimer. J. exp. Med., 165, 1076-1094 (1987). LANIER,L.L. and WEISS,A,, Presence of Ti (WT31)-negative T lymphocytes in peripheral blood and thymus. Nature (Lond.), 324, 268-271 (1986). MINGARI, M.C., TAMBUSSI, G., BOTTINO, C., VARESE,P. and MORETTA, A,, Partial inefficiency of T-cell receptors gammddelta composed of a heavy (55-kD) gamma chain to mediate cell activation upon binding to specific monoclonal antibodies. Res. Clin Lab., 19, 3 9 4 4 (1989). A. MINGARI, M.C., VARESE,P., BOTTINO,C., MELIOLI,G . , MORETTA,

and MORETTA,L., Clonal analysis of CD4-CD8- human thymocytes expressing a T-cell receptor gammddelta chain. Direct evidence for the de novo expression of CD8 surface antigen and of cytolytic activity against tumor targets. Europ. J. Zmmunol., 18, 1831-1834 (1988~). MINGARI,M.C., VARESE, P., BOTTINO,C., MERLI,A. and MORETTA, L., Interleukin-2-induced proliferation of CD4 -CD8 - human thymocytes. Zn vitro expression of CD3 and CD8 antigens and cytolytic activity. Res. Clin. Lab., 18, 67-93 (1988b). MORETTA,A,, BOTTINO,C., CICCONE,E., TAMBUSSI,G., MINGARI, M.C., FERRINI, S., CASORATI, G., VARESE,P., VIALE,O., MIGONE,N. and MORETTA,L., Human peripheral blood lymphocytes bearing T-cell receptor gammddelta. Expression of CD8 differentiation antigen correlates with the expression of the 55-kD C 2-encoded gamma-chain. J. exp. Med., 168, 234%2354 (1988). MORETTA,L., PENDE,D., BOTTINO,C., MIGONE,N., CICCONE,E., FERRINI,S . , MINGARI,M.C. and MORETTA,A . , Human CD3 + 4 - 8 -WT3 1 - T-lymphocyte populations expressing the putative T-cell receptor gamma-gene product. A limiting dilution and clonal analysis. Europ. J. Immunol., 17, 1229-1234 (1987).