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Immunology Today, vol. 3, No. 4, 1982

Immortalizing h u m a n T-cell function In the past few years the appHcation of hybridoma technology to murine T lymphocytes has resulted in the development of T-hybridoma cell lines with several different immunological activities: cytotoxic T-cell hybridomas and T-hybridomas secreting IgE class-specific suppressor factor, antigen-specific suppressor factor, T-cell replacing factor (TRF), T-cell growth factor ( T C G F or interleukin (IL)-2), as well as antigen-specific helper factors. Homogeneous populations of such hybridomas have made it easier to isolate and chemically characterize some T-cellderived immunoregulatory molecules. Recent work in a number of laboratories has shown that it is also possible to isolate human T-cell hybridomas with specific T-cell functions. A first report' described the fusion of a bromodeoxyuridine-resistant (TK~) derivative of a human T cell line (KE 37) (derived from a patient with acute lymphocytic leukemia (ALL)) with peripheral T lymphocytes from a patient with agammaglobulinemia and high frequencies of la-positive T cells having the putative suppressor/cytotoxic phenotype ( O K T 5 + , O K T 8 + cells). O n e hybrid line isolated in H A T medium constitutively produced a factor which suppressed pokeweed mitogen (PWM)-induced B-cell differentiation. A second report- described the successful fusion of both unstimulated and lectin or alloantigen stimulated peripheral T lymphocytes with an azaguanineresistant ( H G P R T ~ ) derivative of another human T ALL line ( C C R F - C E M ) from which clones of hybrids were obtained. One of these could be induced to produce IL-2 by Con A treatment while another was found to be able to produce IL-2 only in the presence of human adherent cells and phytohemagglutinin (PHA). It is hypothesized that the latter hybridoma required macrophage interaction (IL-1?) to drive it into an IL-2 producer state, as recently described in mice'. One last clone was shown to produce a supernatant devoid of IL-2 activity but enabling the induction of cytotoxic T lymphocytes against a lymphoblastoid cell line treated with U.V. light. In a third reporf, T-cell hybrids were produced by fusion of normal PHA-activated T cells and a 6-thioguanineresistant line derived from the human T-cell line C E M . Some of the growing hybrids, initially selected for expression of T3 antigen, were shown to provide helper activity in the PWM-driven generation of antibody-forming cells. We have been using another human ALL-derived line, an HGPRT-derivative of Molt 4, for fusions with human purified tuberculin (PPD)-stimulated T-cell blasts. As in other experiments of this type only a low number of hybrids were obtained. One hybridoma produced large amounts of IL-2 in response to PHA stimulation while others produced immune interferon or provided helper activity. © Elsevier Biomedical Press I9H2 0167-49 iy/H2/0()()(l-IUIIIll/S2,7.1

However, with T-cell hybridomas, the interpretation of the findings is often complicated by the possible contribution of the transformed fusion partner to the effect observed (see Ref. 4). In addition, preservation of functionally stable hybrids requires early and repeated cloning. H u m a n T lymphomas'"" and non-transformed, TCGF-driven clones of human T lymphocytes may offer alternative approaches for obtaining functional homogeneous populations of T cells capable of indefinite growth in culture. For instance, specific proliferative '"' or cytotoxic'" human T-cell cloned lines were recently developed and maintained in T C G F for 2 months and 16 months, respectively. The development of such cloned lines of antigen-specific T cells will surely be paralleled by the construction of antigen-specific and HLA-restricted T-celf hybridomas. At a recent workshop on T-cell hybridomas held at the Basel Institute for Immunology, Elaine de Freitas reported" the first successful attempts to generate human T-cell hybridomas with antigen-inducible helper activity. An H G P R T - derivative of the human T lymphoma line Jurkat, a cell line previously shown to produce IL-2 after mitogen stimulation', was fused with tetanus-toxoid specific human T cells able to deliver antigen-induced helper activity to autologous B cells. The hybrids obtained produced IL-2 in response to tetanus toxoid and in the presence of autologous monocytes. In addition one clone had a significant helper activity for in-vitrn anti-tetanus antibody production by autologous B cells. If able to provide help for B-cell responses to unrelated antigens through a 'bystander effect'"', such T - T hybrids may constitute a means of p r o d u c i n g in vitro specifically stimulated human B-cell populations, a step which would be expected to improve the chances of obtaining B-cell hybrids. It is likely that there will be much activity in this area during the years to come, allowing the characterization of the role of surface structures in particular immune functions, the biochemical definition of antigen-recognition structures and the isolation of various lymphokines, enabling exploration of their clinical potential. BERNARD MALISSF.N JESPER ZEUTHEN

Cfnirr il 'Immiimilooie, IXSERM-CNRS de Mnrseille-Lumm\\ Case 906 imS Maneille mlex 9, Frame: hslitiUe of Ilumm denelus, I'mversity iif Aarhus. DK-HOOO Aarhus C, Denmark.

References 1 Grillot-Courvalin, C , Brouet, J. C , Berger, R. and Bernheim, A. (1981) .Mature (Lanrhn) 292, 844-845 2 Okada, M., Yoshimura, N., Kaieda, T., Yanamura, Y. and Kishimoto, T. (W%\) Pme. .\atl Aeaii. .Sti. U.S.A. 78, 7717-7721

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3 C;illis. S. a n d M i z e l . S. B. (1 9 8 1 ) / ' ™ , ,\«//,l,«rf. .V,7, VS.A.l^. 1133-1137 4 Irimnf'n. C)., Uizzolo. P. \ ., I h o m a s , \ . Ro^oziiiski, I., a n d Chess. I.. ( 1 9 8 1 ) 7 . /-.'v/,. Med. 134, 1827-1837 5 Cillis, S. a n d W a t s o n . J. (1980) J . /•.'V/J. Mrd. 152. 1709-1719 6 ( k i o t c n b c r g , ]. T).. R u s c e l l i . I". VV.. M i c r . J. VV.. Ciazdar. .\a n d O a l l o . R.'c.. (1981) / . /•\/,, Mrd. 154, 140.3-1418 7 Pawelec. G.. R e b h c i m . A., .Mlcr. B. J. a n d VVernet. P. (1980) InumiHooftu'tic^ 1 1. 3 2 7 - 5 3 2 8 M a l i s s e n . B., C h a r m o t , 1). a n d M a w a s , C. (1981) Hmmm

Immuntd. 2. 1-13 9 Srcdni, B., V o l k m a n , 1)., S c h w a r t z , R. H. a n d F a u c i . .\. .S. (1981) I'm,. .\all .Acad. Sn. I '.,V..4. 78, 1858-1852 10 Malissen, B., Kristensen, P.. Cioridis, C , M a d s e n , M . a n d M a w a s , C. (1981) .S;«nr/. J . Inmninol. 14. 2 1 3 - 2 2 4 1 1 I)c Freitas. F, C , Vclla. .S., F i n n c n b a c h , .X., C-roce. C \ F a n d K n p r o w s k i . H, (1982) in T-Cidl Hyhndimuii: Snmre-. nf ^penfic nu'dialny, in the inunimi' system .Sprint^cr-Verlag (in press) 12 Schreier. M . H. a n d T e e s , R. (1980) Ird. Anhs. .Allergy A/ipl. hinmmil.(A, 221-2V

Acquisition of diverse T-cell isotypes The earliest recognizable T-cell function in ontogeny and phylogenv is the ability to recognize and respond to allo-antigen. By day 14 cells from fetal organ cultures of embryonic mouse thymocytes can f)c sensitized to allo-antigcns - indeed a functional mixed lymphocyte reaction can be achieved after 7 days implying that cells express functional antigen receptors as soon as, or prior to, the expression of the earliest T-cell marker (Thy 1 at day 13 of gestation)'. Similarly, hagfish, the most primitive of vertebrates, have only a primitive IgM analogue, but have a temperature-sensitive afiility to reject allografts-. PreT cells arising in ' n u d e ' (athymic) mice can be induced with T-cell growth factor (TCGF) to become functional cytotoxic T cells'. These lines of evidence suggest that the receptor for allo-antigens might be considered the most primitive of the T-cell antigen receptors. Evolutionarily, one would expect that receptor to be ecjuated most closely with a T-eell analogue. The (juestion of interest is whether all functional T cells use the same 'constant region' and a different spectra of varial)le regions to recognize antigens or whether isotypes exist for T-ccll receptors. Recent studies bv Binz and Wigzeil' illustrate that an identical molecule can be isolated from both Lyt I- and Lyt 2-bearing allo-rece[)tive T cells in the moused Using a rabbit anti-receptor serum, they observed that a major polypeptide of 70,000 mol. wt could be cleaved with plasmin into characteristic fragmerits, one of which bound antigen. This suggests a domain organization of the molecule which may or may not be characteristic, since a ubiquitous arsonate binding protein can be similarly cleavecT. Two trace additional bands at 75.000 and 88.000 mol. wt are sus[)eet candidates for cross-reactive additional receptors; although these appeared in both Lyt 1- and Lvt 2bearing cell populations. One might speculate that a rabbit anti-mouse serum might have enough antiframework determinants to cross-react weaklv with other, more differentiated receptors which could be functionally distinct, but perhaps originate from a common |)rimordial gene. In the immunoglobulin family of molecules, anti-n chain reagents show less cross-reactivity with Ig(i2b than with anti-IgGl reagents because of the differences or similarities in

sequence between the three genes". If the frequency of an allo-receptor bearing cell population is ten times that of a second isotype bearing cell, little material may be purified for a second isotype, especially if the starting cell preparation is composed of M L C reactive T cells, already enriched biologically for the desired receptor'. Since the frequencies of T cells in mice which are committed to allo-recognition^ is higher than those which recognize a single antigen, then the total number of T-eell receptors for alloantigens may be expected to be disproportionately high. :\ major reason for speculation that T-cell receptor isotypes might be diverse is that T-cell subpopulations, induced with the same antigen, exist and are functionally discrete". The simplistic explanation is that there are characteristic effector functions for each constant region and that the presence of a particular isotype determines the functional capability of the T cell. Because subpopulations of cells have been shown to parallel lyt I and Lyt 2 subpopulation markers, one might have predicted that the T-cell isotype family would also parallel Lyt type. It is known that the majority of thymocytes express Lyt 1 before Lyt 2''. If the ability to function in allo-recognition ontologically precedes Lyt 1 and Lyt 2 diversihcation, it is possible that the surface isotype(s) developed prior to Lyt restrictions and that both Lyt I and Lyt 2 bearing alloreaetive 'T cells would express the same spectrum of antigen receptors. In contrast, other functional subtypes (suppressor) which developed after Lyt divergence might appear to have an isotype which would parallel Lyt phenotype. If there is a spectrum of T-cell isotypes in mice, would one necessarily expect to see characteristic isotypes for each functional T-cell type? Perhaps the 'Tcell receptor family parallels immunogloliulin isotype diversification. It is possible that the 'T-cell isotype expression is a feature of functional maturation and not potential functional capability. Early 'T cells might express a n analogue (allo-rcactive receptors being the most likely candidate) and as the immune response is triggered (by self- a n d / o r allo-environmental antigens), the maturing T cells might acquire additional isoty|)es. One would expect to see a spectrum of cells with an increased diversity of isotypes after the