An insight into the T-cell recognition systemusing antigen-specific functional T-cell lines

Ann. Immunol. (Inst. Pasteur) 1983, 134 D, 123-131

AN

INSIGHT

INTO

THE

USING

T-CELL

RECOGNITION

SYSTEM

ANTIGEN-SPECIFIC

FUNCTIONAL

T-CELL

LINES

by Edna M o z e s

Department o/Chemical Immunology, The Weizmann Institute o[ Science, Rehovot 76100 (Israel)

SUMMARY For a better understanding of the molecular nature of the antigenspecific T-cell recognition system, continuous T-cell lines specific to the synthetic polypeptide (T, G)-A- -L, have been established from murine and human activated T cells. These lines are constitutive secretors of antigen-specific T-cell replacing helper factors. Lines prepared from (T,G)A - - L activated C3H.SW (high-responder) T cells were cloned and characterized. The cells and their secreted products were identical in their fine antigenic specificity, as tested using synthetic polypeptides closely related to (T,G)-A--L. The secreted T-cell helper factor was shown to possess MHC determinants as well as V-region determinants or, more specifically, idiotypic determinants. Using the fluorescence-activated cell sorter (FACSII), we demonstrated the expression of cross-reactive idiotypic markers on the monoclonal helper T cells. Gel analysis of the twice affinity-purified eluate of a (T,G)-A--L column revealed the existence of iodinated bands with a molecular weight of 15 kd and 17 kd, in addition to a diffuse band of high molecular weight. The specific helper activity of the factors was associated with a 65-75 % a m m o n i u m sulfate precipitate. Gel electrophoresis experiments indicated t h a t both a high (less than 67 kd) and a low (15-17 kd) molecular weight fraction contained the biological activity of the factor. The two fractions were also shown to be synthesized by the T-cell lines, as indicated by internal labelling experiments using 35S-methionine. A (T,G)-A--L-specific long-term T-cell line of human origin which constitutively secreted a helper factor, was established from antigen-activated T cells of a donor previously determined to be a responder to (T, G)A- -L. In addition to the antigenic specificity of the factor, it was found to possess V. determinants in addition to the relevant HLA-DR determinants. M a n u s c r i t requ le 15 f6vrier 1983.

124

E. MOZES

Tile latter finding was confirmed by the demonstration t h a t the factor reacted with monoclonal anti-Ia antibodies. Thus, the molecular characterization of the active products of the antigen-specific T-cell lines of murine and h u m a n origin will hopefully lead to elucidation of the T-cell receptor. KEY-WORDS: T lymphocyte, Recognition, Polypeptide; (T, G)-A- -L, Antigen specificity, Histoeompatibility.

INTRODUCTION.

In order to understand immune response phenomena, it is important to elucidate the antigen-specific receptor of the cells participating in the i m m u n e reaction. In the past, profound advances have been made in the understanding of the B-cell receptor; however, up to the present time, no definitive answer has been given to the question of the nature of the antigen-specific T-cell recognition system. This is due in large part to the difficulty in obtaining antigen-specific materials from a heterogenous population of T cells in an amount sufficient for biochemical analyses. To overcome this obstacle, several groups have a t t e m p t e d the establishment of either antigen-specific functional T-cell hybrid lines, or T-cell growth factor (TCGF)-dependent continuous T-cell lines and clones which provide well-defined, homogenous material for molecular analyses of the T-cell receptor. To approach the T-cell receptor problem, we have attempted to generate antigen-specific factor-producing helper T-cell lines specific to t h e synthetic polypeptide (T, G)-A- -L, the i m m u n e response to which is genetically regulated and has been thoroughly studied [13, 10]. Since antigen-specific T-cell replacing factors are considered as the soluble form of the T-cell recognition unit, t h e y provide a useful tool for approaching the T-cell receptor issue. This report describes the establishment of TCGFdependent continuous helper T-cell lines and clones of murine high-responder (C3W.SW, H-2 b, Igh-D) origin, which are also constitutive secretors of (T,G)-A--L-specific T-cell replacing helper factors [3]. The T-cell lines and their derived factors were characterized, and attempts were made to purify the active moiety of the factor. In addition, we developed a (T,G)A--L-specific human T-cell helper line and at the same time analyzed the nature of the constitutively secreted h u m a n T-cell replacing factor.

DTH FACS HGG MHC NIP

~ ~ ~ ~ ~

delayed-type hypersensitivity. fluorescence-activated cell sorter. human gamma globulin. major histocompatibility complex. 3-iodo-4-hydroxy-5-nitrophenylacetic acide.

NIP-OVA PFC PBL TCGF

= = ~ ~

NIP-ovalbumin. p l a q u e - f o r m i n g cell. peripheral blood leukocyte. T-cell growth factor.

ANTIGEN-SPECIFIC T-CELL RECOGNITION SYSTEM

125

DATA AND DISCUSSION

Establishment and characlerization of a (T,G)-A--L-specific helper line.

continuous

Functional lines with (T, G)-A--L-specific helper activity were derived from in vitro (T,G)-A--L-activated T cells [6] of C3H.SW mice. The activated cells were further enriched for helper activity by an additional 24-h pulse on antigen-bearing splenic adherent cells [1], and subsequently propagated in TCGF-containing medium without additional antigenic stimulation [3]. One of the lines obtained (E-9M(§ was cloned by the limiting dilution technique, and the derived clones were similarly maintained in TCGF-containing medium. The lines and their derived clones secreted T-cell replacing factors constitutively into the culture medium [3]. Cells and supernatants of the lines possessed helper activity, as determined by their ability to cooperate in vitro with spleen cells primed to the conjugate nitroiodo-phenyl-ovalbumin (NIP-OVA) for the production of NIPspecific plaque-forming cells ( P F C ) w h e n challenged with NIP-(T,G)A - - L [3]. The fine antigenic specificity of the E-9M(§ T-cell line and its secreted factors was studied utilizing synthetic polypeptide antigens which are closely related to (T,G)-A- -L [9]. Two experimental approaches were used: (a) NIP-OVA-primed B cells were cultured with T cells of the line and with the secreted factor, and were challenged with NIP conjugated to the different polypeptide antigens; (b) Supernatants of the E-9M(+) line were passed through immunoadsorbents made up of the different synthetic polypeptide antigens, and the (T, G)-A--L-specific helper activity was tested in the effluents and eluates recovered from the columns. The fine antigenic specificity of the T cells and their products was shown to be identical, but differed somewhat from that of antibodies. Table I, which summarizes the properties of the T-cell line and its products, represents the fine antigenic specificity of the line. As shown in that table, the (T,G)-A--L-specific E-9M(+) ceils or their derived factors completely cross-reacted with (Phe, G)-A--L and G-A--L and only partially cross-reacted with (H,G)-A--L, whereas no cross-reaction was observed with (T,G)-Pro--L. In contrast, anti-(T,G)A - - L antibodies bound well to all the above-mentioned synthetic polypeptides. Using ordered synthetic polypeptides, it was shown that the T cells and factors were specific mainly to the major antigenic determinant of the randomly polymerized (T, G)-A- -L, namely T-T-G-G, but not to T-G-T-G as shown for specific antibodies [9]. Cells of the line and clones were stained in immunofluorescence studies with antibodies directed against the variable region of the Ig heavy chain (V.) [4] considered as general reagents with the capacity to react with the T-cell receptor. Similarly, factors derived from the E-9M(+) line could be absorbed on and eluted from affinity columns built from anti-V, antibodies (table I and [3]). More specific probes of the V-region gene products were antiidiotypic

126

E. MOZES TABLE I. - - Characteristics of the (T, G)-A- -L-specific murine T-cell lines and clones and their products (1).

Antigens and reagents

Cross-reaction with cells and their derived factors (2)

(T, G)-A- -L (Phe, G)-A- -L (H, G)-A- -L G-A- -L (T, G)-Pro- -L (T-T-G-G)-A- -L (T-G-T-G)-A- -L

+ + • -r

Anti.VH (3) Anti-Id (4) Anti-mouse IgG

-b +

Anti-H-2 b Anti-H-2 k

+

~-

(i) (T, G)-A- -L specific T-cell continuous lines were of C3H.SW (H-2 b, IgH-1i) origin. (2) Reactivity was tested as indicated in the text. (3) R a b b i t antibodies against the variable region of the heavy chain of mouse immunoglobulin [4]. (4) Antiidiotypic antibodies produced in C57BL/6 mice against C3H.SW antibodies specific to (T, G)-A--L [8].

antibodies shown to be involved in the recognition system of antigenspecific effector T cells [5]. We had previously shown the presence of idiotypic determinants on a (T, G)-A--L-specific helper factor obtained from antigen-(( educated ,, T cells [12] and from (T, G)-A--L-specific helper and suppressor T-cell hybridomas [2, 11]. We further showed that (T, G)-A- -Lspecific proliferating T cells [8] as well as delayed-type hypersensitivity (DTH)-mediating T cells [14] possessed idiotypic determinants crossreactive with (T, G)-A- -L-specific antibodies. Thus, specific T cells of different functions shared idiotypic determinants. The presence of idiotypic determinants in the antigen-specific helper factor derived from the E-gM(&) continuous line has been demonstrated by the removal of its activity following affinity chromatography on an anti-idiotype Sepharose column [3]. Antiidiotypic sera produced in individual C57BL/6 mice against (T,G)A--L-specific antibodies (Id) of C3H.SW mice [8] were used for indirect immunofluorescence staining of the T cells themselves using the fluorescenceactivated cell sorter (FACSI1). The anti-idiotypes significantly stained cells of the E-gM(+) line, but did not stain a helper T-cell continuous line of C3H.SW origin specific for human y globulin (HGG), nor did they stain a (T,G)-A--L-specific helper T-cell line of CWB (H-2 b, Igh-1 b) origin. Similarly, when clones derived from the E-9M(+) line were tested, a correlation

ANTIGEN-SPECIFIC T-CELL RECOGNITION SYSTEM

127

was demonstrated between the antigen-specific helper activity and the expression of cell surface idiotypic determinants (R. Lifshitz, R. N. Apte and E. Mozes). Thus, cross-reactive idiotypes linked to heavy chain allotypes were demonstrated on antibodies and on cloned T-helper cells. In addition to V-region gene products, cells and factors of the E-gM(-[-) line were shown to contain markers of the mouse major histocompatibility complex (MHC). Thus, the activity of the factor was removed by an antiH-2b-Sepharose column, but not by an immunoadsorbent built from the nonrelevant anti-H-2 k antibodies (table I and [3]). Although the results suggest t h a t V-region genes and MHC products are linked together in the active moiety of the factor, its exact molecular organization remains to be resolved.

Molecular characterization of the E-9M( + ) derived factor. The antigen-specific factors produced by the E-9M(§ continuous T-cell line were further characterized. Gel analysis of the twice-affinity-purified eluate of a (T, G)-A--L Sepharose column revealed iodinated bands with molecular weights of 17 and 15 kd in addition to a diffuse band of high molecular weight. The factor of the T-cell line was fractionated using increasing concentrations (10-85 %) of ammonium sulfate. The (T, G)-A- -L-specific helper activity was associated with a 65-75 % ammonium sulfate precipitate. In order to determine the molecular weight of the antigen-specific biologically active entities in the T-cell helper factor, either the active eluate of a (T,G)-A--L-Sepharose column or the 65-75 % ammonium sulphate precipitate were subjected to SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Gels were then cut into 9-10 strips of 1 cm each and the fractions were eluted by electrophoresis. The results obtained with both preparations were similar, and indicated t h a t both a high (less than 67 kd) and a low (15-17 kd) molecular weight fraction contained the biological activity of the factor. Culture supernatants of the ~S-methionine internally labelled E-9M(+) line were subjected to a combined purification of sequential ammonium sulphate precipitation followed by affinity chromatography. Polyacrylamide gel patterns of the eluates of the different fractions demonstrated that the 65-75 % ammonium sulphate precipitate contained two s~S-labelled bands with apparent molecular weights of 60 and 15 kd similar to the activity pattern (R. Lifshitz, R. N. Apte and E. Mozes). It is likely t h a t the low molecular weight (15-17 kd) fraction, which fully preserved the antigenspecific helper activity of the T cell line, was a subunit of the high molecular weight ( < 67 kd) fraction. However, further purification and sequence analyses are needed to answer this question, and are now in progress.

Establishment and analyses of a (T, G)-A--L-specific human helper T-cell continuous line. Information on the function and specificity of T cells in man is very limited due to the ethical constraints of immunizing humans with antigens, and also because of the complexity of the antigenic systems related to

128

E. MOZES

disease occurrence. An antigen-specific functional factor produced by (T, G)-A- -L activated T cells appeared to be a desirable tool for approaching the mode of action as well as the specific recognition system of human helper T cells. We had previously reported the successful production of (T, G)-A--Lspecific human T-cell helper factors by human peripheral blood leukocytes (PBL) of normal donors which were activated in vitro to the antigen [7]. The supernatants collected were tested for their ability to replace (T, G)A- -L specific helper T cells in a hapten-carrier in vitro antibody production system. Since (T, G)-A- -L-helper factors appeared to activate B cells across the MHC and the species barrier [7], the helper activity of the factors was tested using NIP-OVA-primed murine B cells. About 50 % of the donors from a random population responded by the production of an active factor, suggesting differences in the immune response potential of the individuals tested | 7]. TABLE II. - - Activity and characteristics of a (T, G)-A- -L-specific human helper T-cell factor secreted by a continuous T-cell line. NIP-OVA-primed spleen cells cultured with:

NIP-specific PFC/108 viable cells (*)

Factor diluted 1/10 q- NIP-(T, G)-A--L Factor diluted 1/10 -[- NIP-HGG

620 4- 11 290 5= 28

Factor diluted 1/10,000 + NIP-(T, G)-A--L Factor diluted 1/10,000 -[- NIP-HGG

395 5= 100 139 5= 28

Effluent from anti-VH-Sepharose Eluate from anti-Va-Sepharose

196 5= 54 517 5= 44

Effluent from anti-IgG-Sepharose Eluate from anti-IgG-Sepharose

576 5= 120 128 5= 33

Effluent from anti-DR5-Sepharose Eluate from anti-DR5-Sepharose

183 5= 67 775 • 38

Effluent from anti-DR1-Sep//arose Eluate from anti-DR1-Sepharose

150 5= 27 608 5= 121

Effluent from anti-DR3-Sepharose Eluate from anti-DR3-Sepharose

700 5= 112 175 5= 61

Effluent from monoelonal anti-human Ia Sepharose Eluate from monoclonal anti-human Ia-Sepharose

133 5= 58 705 5= 128

(*) Indirect PFC were determined using NIP-SRBC indicator cells. Results are expressed as the mean of quadruplicate tests • S. D. Rabbit antibodies against the variable region of the heavy chain (VH) of human immunoglobulin were prepared by Y. Ben Neriah, A. Cohen, N. Eshhar, D. Givol and Z. Bentwich. Supernatants of the (T, G)-A- -L-specific T-cell continuous line were passed through Sepharose columns. Elutions were performed with 0.1 M NH4OH. Anti-DR5 (BRA 677) was from the laboratory of Dr Brautbar. Anti-DR1 (KZ 261827) and anti-DR3 (MO 54784) were kindly provided by Dr J. van Rood. Monoclonal antibody (Ll.l.6) against human Ia was kindly provided by Dr J. Kalil.

ANTIGEN-SPECIFIC T-CELL RECOGNITION SYSTEM

129

The minute amounts of T-cell factors available from the (T, G)-A--Lactivated T cells were sufficient only for functional studies and partial serological analysis. For molecular characterization of the factors, it was desirable to establish a continuous T-cell line secreting a biologically active homogenous factor. Using our earlier experience with murine T-cell lines, P B L of a donor previously shown to be a high responder to (T, G)-A--L were educated to (T, G)-A--L and further enriched by an additional 24-h incubation on (T, G)-A--L-pulsed fresh adherent cells [1]. The cells were then transferred to and maintained in m e d i u m supplemented with 30-50 % TCGF of human origin. Supernatants of the cultures were found to be very potent in their biological activity and could be diluted to 1/10,000 before their antigen-specific helper activity was reduced (table II). The line-derived factors possessed V. determinants, since their activity was removed by immunoadsorbents made up of rabbit antibodies against h u m a n V., as demonstrated in table II. The factor did not react with antibodies against h u m a n IgG (table II). The factor was also passed through columns of sepharose coupled to antisera against DR determinants. Since the T-cell line was established from a donor typed to possess HLA-DR 1,5, columns made up of the relevant anti-DR antibodies were used, as was a control column of anti-DR3. As shown in table II, the activity of the factor was removed by columns of Sepharose coupled to anti-DR1 and anti-DR5, and could be eluted by 0.1 M ammonia, whereas no effect on the activity of the factor was observed when the irrelevant anti-DR3 column was used. The table also shows t h a t Sepharose coupled to a monoclonal antibody which recognizes a monomorphic determinant on HLA-DR antigens removed the activity of the factor, confirming the presence of Ia determinants as well as V. deLerminants in the active moiety of the (T. G)-A--L-specific h u m a n helper T-cell product (D. Katz, H. Brautbar, Z. Bentwich and E. Mozes). The (T, G)-A- -L specific helper T-cell line and its active constitutively-secreted product will hopefully provide the means for future studies on the mode of action as well as on the nature of antigen-specific receptors of human T cells. RI~SUME UTILISATION

D E LIGNI~ES

FONCTIONNELLES POUR

L'ANALYSE

DU

DE

SPI~CIFIQUES

CELLULES

T

D'ANTIGI~NES

SYSTt~ME D E R E C O N N A I S S A N C E

DES LYMPHOCYTES

T

Dans le but de mieux comprendre la nature mol6culaire du syst6me de reconnaissance sp6cifique des antigbnes exprim6s par les cellules T, des lign6es continues de eellules T sp@ifiques du polypeptide synth6tique (T, G)-A--L ont 6t~ 6tablies ~ partir de cellules T murines et humaines activ6es. Ces lign6es s6cr~tent de fa~on constitutive des facteurs faeilitants sp6cifiques de l'antig6ne et capables de remplacer les cellules. Des lign6es, obtenues & partir de cellules T de souris C3H.SW (hautes r6pondeuses) Ann. lmmunol. (Inst. Pasteur), 134 D, n ~ 1, 1983.

9

130

E. MOZES

activ6es in vitro par le (T, G)-A- -L, ont 6t6 clon~es et caract~ris~es. L'utilisation de polypeptides synth~tiques tr~s proches du (T, G)-A--L a permis de m o n t r e r que la sp~cificit~ antig~nique fine des cellules et des produits qu'elles s~cr~tent est identique. Le facteur facilitant s6cr6t6 par les cellules T poss~dent des d~terminants du complexe majeur d'bistocompatibilit~ des d6terminants de la r6gion V e t , plus sp6cifiquement, des d6terminants idiotypiques. Des experiences realis6es avec un trieur de cellules ont d~montr~ l'expression, sur les cellules T facilitantes monoclonales, de marqueurs idiotypiques voisins. L'analyse en gel du mat6riel purifi6 deux fois par chromatographie d'affinit~ sur un i m m u n o a d s o r b a n t de (T,G)-A--L r6v~le l'existence de bandes de poids mol@ulaire 15 et 17 kd, et d'une bande diffuse de poids mol6culaire 61ev6. L'activit6 facilitante sp6cifique des facteurs peut-~tre pr~cipit6e par une concentration de sulfate d ' a m m o n i u m comprise entre 65 et 75 %. L'activit6 biologique du facteur est associ6e avec des fractions de poids mol6culaire 61ev6 (moins de 67 kd) et faible (15-17 kd). Des exp6riences de marquage isotopique interne par la 35S-m6tbionine d~montrent 6galement que ces deux fractions sont synth6tis6es par les lign~es cellulaires T. Une lign6e continue de cellules T humaines sp6eifiques du (T, G)-A--L et qui s6cr~te de fa~on constitutive un facteur facilitant, a 6t6 6tablie ~ partir de cellules T activ~es in vitro par l'antig~ne. Ces derni~res provenaient d'un donneur capable de r6pondre au (T, G)-A--L. Le facteur obtenu est sp~cifique de l'antig~ne et poss~de des d~terminants V. ainsi que des d~terminants t I L A - D R identiques A ceux exprim6s par le donneur. Ce dernier point a 6t6 confirm~ par la fixation du facteur sur des anticorps monoclonaux anti-Ia. La caract6risation mol~culaire des produits synth6tis6s par les lign6es de cellules T murines et humaines sp@ifiques d'antig~nes, conduiront 61ucider la nature d u r6cepteur des cellules T. MOTS-CLI~S : L y m p h o c y t e T, Reconnaissance immunologique, Polyp e p t i d e ; (T, G)-A--L, Sp~cificit6 d'antig~ne, Histocompatibilit6.

REFERENCES

[1] APTE, R. N., DAYAN, M. & MOZES, E., Modulation of the helper activity of educated T cells to the synthetic polypeptide poly(Tyr, Glu)-poly(DLAla)-poly(Lys) by adherent cell-bound antigen. Cell. Immunol., 1981, 61, 104-113. [2] APTE, R. N., ESHttAR, Z., LSwY, I., ZINGER, H. • MOZES,E., Characteristics of a poly(LTyr, LGlu)-poly(DLAla)-poly(LLys)-specific helper factor derived from a T-cell hybridoma. Europ. J. Immunol., 1981, 11, 931-936. [3] APTE, R. N., LSwY, I., DE BAETSELIER, P. & MOZES,E., Establishment and characterization of continuous helper T-cell lines specific to poly(LTyr, LGlu)-poly(DLAla)-poly(LLys). J. Immunol., 1981, 127, 25-30.

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[4] BEN-NERIAtl, Y., WULIMART, C., LONAI, P. & GIVOL, D., Preparation and characterization of anti-framework antibodies to the heavy-chain variable region (V.) of mouse immunoglobulin. Europ. J. Immunol., 1978, 8, 791-801. [51 EICHMANN,K., Expression and function of idiotypes on lymphocytes. Advanc. Immunol., 1978, 26, 195-254. [6] ESHHAR, Z., STRASSMANN, G., WAKS, T. & MOZES, E., In vitro and in vivo induction of effector T cells mediating DTH responses to a protein and a synthetic polypeptide antigen. Cell. Immunol., 1979, 47, 378-389. [7] KATZ, D., BENTWICH, Z., ESHHAR, N., Lbwv, I. & MOZES, E., Immune response potential to poly(Tyr, Glu)-poly(DLAla)-poly(Lys) of human T cells of different donors. Proc. nat. Acad. Sci. (Wash.), 1981, 78, 4505-4509. [8] LwsmTz, R., PARHAMI, B. & MOZES, E., Enhancing effect of murine antiidiotypic serum on the proliferative response specific for poly(LTyr, LGlu)-poly(DLAla)-poly(LLys) [(T, G)-A-L]. Europ. J. Immunol., 1981, 11, 27-31. [9] Lbwv, I., APTE, R. N. & MozEs, E., Analysis of the fine antigenic specificity of the (T, G)-A-L specific helper T-cell continuous line E-9M(+). J. Immunol., 1982, 128, 190-194. [10] MOZES,E., Expression of immune response (It) genes in T and B cells, lmmunogenel., 1975, 2, 397-410. [11] MozEs, E., ESHHAR, Z. & APTE, R. N., Soluble products of functional T-cell continuous lines and hybridomas specific to the synthetic polypeptide (T, G)-A--L, in (( Lymphokines )) (M. Feldmann & M. H. Schreier), 5 (p. 223-252), Academic Press, London, New York, 1982. [12] MozEs, E. & HAIMOVmH, J., Antigen-specific T-cell helper factor cross-reacts idiotypically with antibodies of the same specificity. Nature (Lond.), 1979, 278, 56-57. [13] SELA, M., FUCHS, S. & ARNON, R., Studies on the chemical basis of the antigenicity of proteins. - - 5. Synthesis, characterization and immunogenicity of some multichain and linear polypeptides containing tyrosine. Biochem. J., 1962, 85, 223-235. [14] STRASSMANN, G., LIFSHITZ, R. & MOZES, E., Elicitation of delayed-type hypersensitivity responses to poly(LTyr, LGlu)-poly(DLAla)-poly(LLys) by antiidiotypic antibodies, d. exp. Med., 1980, 152, 1448-1452.