Minoru Shigeta, Dr. Masao Kimoto, and Dr. C. Garrison Fathman, Stanford ... C (Fig. 1). In contrast, responses to 10 -3/~g/ml TNP-Ficoll were completely ...
MAJOR
HISTOCOMPATIBILITY
COMPLEX-RESTRICTED
SELF-RECOGNITION RESPONSES
IN
TO TRINITROPHENYL-FICOLL
A N o v e l Cell I n t e r a c t i o n P a t h w a y R e q u i r i n g S e l f - R e c o g n i t i o n o f Accessory Cell H - 2 D e t e r m i n a n t s b y B o t h T Cells a n d B Cells By RICHARD J. HODES, KAREN S. HATHCOCK, AND ALFRED SINGER From the Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
Most thymus-dependent (T) lymphocyte populations are restricted in their function by a requirement for recognition of"self" major histocompatibility complex (MHC) 1 gene products. Thus, T cells that mediate help (Tn) (1-5), antigen-specific proliferation (6, 7), cell-mediated lympholysis (8, 9), delayed-type hypersensitivity (10), and immune suppression (11) require recognition of M H C products expressed by the cells with which they interact. In addition to M H C restriction of T cell recognition, the possibility that B cells as well as T cells may be capable of MHC-restricted selfrecognition has recently been evaluated (12-15). In a recent report (15), the possibility that trinitrophenyl (TNP)-Ficoll-responsive B cells are M H C restricted in their interaction with accessory cells was assessed under T-independent (TI) response conditions. In the absence of any detectable requirement for T cells, it was demonstrated that TNP-Ficoll-responsive B cells, as a consequence of their development in a fully MHC-allogeneic chimeric host environment, were unable to cooperate with accessory cells expressing syngeneic donor-type M H C determinants but could cooperate with accessory cells expressing allogeneic host-type M H C products. These findings raised a number of other questions. Although they demonstrated that B cells could express MHC-restricted self-recognition in the absence of T cells, these studies did not determine the effects that T cells or their soluble products could exert on this B cell function. Specifically, it remained undetermined whether restricted recognition by B lymphocytes of accessory cell M H C determinants could be expressed in a response that concomitantly required the function of TH cells. Moreover, since it has previously been suggested that B cells can be directly activated by antigen in the presence of T cell-derived factors (16), there was the possibility that such T cell products might be capable of bypassing or overcoming any M H C recognition requirement for the activation of B cells. In the present report, conditions were first established in which primary antibody responses to limiting concentrations of T N P 1Abbreviations used in this paper: ATS, anti-thymocyte serum; C, complement; Con A, concanavalin A; IL-2, interleukin 2; KLH, keyhole limpet hemocyanin; MHC, major histocompatibilitycomplex; MLR, mixed lymphocyte response; PFC, plaque-forming cell; RAMB, rabbit anti-mouse brain serum; SAC, spleen adherent cells; SRBC, sheep erythrocytes; TD, T dependent; (T,G)-A--L,poly-L-(Tyr,Glu)-polyD,L-Ala--poly-u-Lys;TH, T helper; TI, T independent.
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J. Exp. MEo.© The RockefellerUniversity Press • 0022-1007/83/02/0486/16 $1.00 Volume 157 February 1983 486-501
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re shown to be highly T cell d e p e n d e n t (TD). U n d e r these conditions, it was d e m o n s t r a t e d t h a t TH cell function d i d require T n cell recognition o f accessory cell M H C d e t e r m i n a n t s b u t d i d not require T n cell recognition o f B cell M H C d e t e r m i nants. However, T n cell a c t i v a t i o n o f T N P - F i c o l l - r e s p o n s i v e c h i m e r i c B l y m p h o c y t e s still r e q u i r e d B cell recognition o f s e l f - M H C d e t e r m i n a n t s expressed b y accessory ceils. Thus, these findings d e m o n s t r a t e t h e existence o f a novel cell i n t e r a c t i o n p a t h w a y in which b o t h T N P - F i c o l l - r e a c t i v e B cells a n d TH cells express r e q u i r e m e n t s for M H C - r e s t r i c t e d self-recognition o f accessory ceils. Materials and Methods Animals. C57BL/10 (B10), B10.A, B10.BR, (B10 × B10.A)F1, (B10 × B10.BR)F1, and (C57BL/6 × A/J)Fx (B6AF1) mice were obtained from The Jackson Laboratory, Bar Harbor, ME. B10.A(2R) mice were provided by Dr. David Sachs, National Institutes of Health (NIH), and [B10.A × B10.A(2R)]F1 mice were provided by Dr. Gene Shearer, NIH. Radiation Bone Marrow Chimeras. Chimeras were prepared as previously described (5). 12-15 × 106 T cell-depleted donor bone marrow cells were injected intravenously into 950-radirradiated recipients and rested for 8-24 wk before use. Chimeric spleen cell populations were routinely H-2 typed by indirect immunofluorescence and were uniformly of donor origin without detectable ( B 10 chimeric spleen cells and stimulated with TNP-FicoI1. T h e two spleen populations were identically H-2 a except that (a) the [B10.A × B10.A(2R)]FI spleen cells were heterozygous H-2D a/b, and (b) the B10.A ---> B10 chimeric spleen cells had differentiated in an/-/-2 b environment. U p o n stimulation of the co-cultures with TNP-FicolI, the activation requirements of the normal and chimeric B cells could be compared in the presence of the same T cell and accessory cell populations. Thus, any differences observed in the activation of these two B cell populations in co-culture could be
496
SELF-RECOGNITION BY T AND B LYMPHOCYTES
/ 0
2.5 5 7.5 10 12.5 (BIO X BIO.A) F~
T CELLS/CULTURE XIO-4 FIG. 7. Chimeric B cells are MHC restricted in their recognition of accessory cells. Titrated numbers of nylon-nonadherent (1310 × B10.A)FI spleen (T) cells were added to 4 X 105 anti-Thy1.2 plus C-treated B10.A ~ B10 spleen B plus accessory cells in the presence of no added SAC (m), or in the presence of 4 X 104 B10 (O) or BI0.A (A) SAC, and cultures were assayed for responses to 10-3 #g/ml TNP-FieolI. Responses in the absence of antigen were < 10 PFC/culture. TABLE I V
Hybridoma T Cell Supernatant Overcomes the Requirementfor MHC Restriction in Activation of Chimeric B Cells by TNP-Ficoll TNP-specific PFC* Group
Spleen cells cultured
IL-2
Antigen C
A B C D
BI0.A (B10.A X 2R)F~ B10.A ~ B10 + (B10.A X 2R)F1 B10.A --~ B10 + (B10.A × 2R)Fa
+
TNP-Ficoll TNP-Ficoll TNP-Ficoll TNP-Ficoll
915 800 579 1,560
Percent response Anti-D b remainplus C ing 836 19 21 633
91 2 4 41
* The indicated normal spleen cell populations or equal cell mixtures of normal and chimeric spleen cells were cultured for 4 d with the indicated antigen, with or without a 1:2 dilution of FS6 supernatant as a source of IL-2. At the end of this culture period, cells were harvested, 10v cells of each group were treated with C alone or with anti-D b antiserum plus C, and the remaining cells were assayed for anti-TNP PFC. a t t r i b u t e d d i r e c t l y to d i f f e r e n c e s in B cell a c t i v a t i o n r e q u i r e m e n t s a n d n o t solely to d i f f e r e n c e s b e t w e e n n o r m a l a n d c h i m e r i c T cells o r a c c e s s o r y cells. It w a s o b s e r v e d t h a t c o - c u l t u r e s d i d r e s p o n d to T N P - F i c o l l ( T a b l e I V , g r o u p C ) ; h o w e v e r , it c a n b e s e e n t h a t all t h e P F C g e n e r a t e d w e r e s e n s i t i v e to t r e a t m e n t w i t h a n t i - D b p l u s C a n d h e n c e w e r e e x c l u s i v e l y d e r i v e d f r o m t h e n o r m a l [ B 1 0 . A × B 1 0 . A ( 2 R ) ] F 1 B cell p o p u l a t i o n ( T a b l e I V , g r o u p C ) . I n o t h e r w o r d s , t h e c h i m e r i c B 1 0 . A ~ B 1 0 B cells h a d n o t b e e n t r i g g e r e d in c o - c u l t u r e s , w h i c h is c o n s i s t e n t w i t h t h e fact t h a t t h e coc u l t u r e s d i d n o t c o n t a i n a n y o f t h e h o s t - t y p e H - 2 b a c c e s s o r y cells r e q u i r e d for t h e a c t i v a t i o n o f t h e s e c h i m e r i c B cells. H o w e v e r , w h e n s o l u b l e s u p e r n a t a n t f r o m t h e T cell h y b r i d o m a F S 6 w a s a d d e d t o s p l e e n cell c o - c u l t u r e s r e s p o n d i n g to T N P - F i c o l l , 41% o f t h e P F C w e r e i n s e n s i t i v e to t r e a t m e n t w i t h a n t i - D b p l u s C, w h i c h d e m o n s t r a t e s t h a t t h e F S 6 s u p e r n a t a n t a c t i v a t e d b o t h t h e [ B 1 0 . A X B 1 0 . A ( 2 R ) ] F a B cells a n d t h e B 10.A ~ B 1 0 c h i m e r i c B ceils ( T a b l e I V , g r o u p D). T h u s , t h i s e x p e r i m e n t d i r e c t l y d e m o n s t r a t e d t h a t (a) T N P - F i c o l l - r e s p o n s i v e B cells t h a t h a d m a t u r e d i n a n a l l o g e n e i c
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differentiation environment are not activated in the presence of syngeneic TH cells and accessory cells even though TNP-responsive normal B cells present in the same cultures are activated, and (b) soluble T cell factors, unlike TH cells themselves, activate TNP-Ficoll-responsive chimeric B cells in the absence of chimeric host-type accessory cells. Taken together, the experiments presented in this report demonstrate that soluble T cell factors elicited from monoclonal T cell populations with either Con A or specific polypeptide antigens do activate TNP-Ficoll-responsive B cells directly without any requirement for further cell interactions, whereas activation of TNP-Ficollresponsive B cells by TNP-Ficoll-reactive T n cells requires that both B cells and T n cells recognize the M H C determinants expressed by TNP-Ficoll-presenting accessory cells. Discussion It has recently been demonstrated (12-15) that B cells, as well as TH cells, can express requirements for recognition of self-MHC determinants expressed by the accessory cells with which they interact. In particular, it has recently been shown (15) that B cells from long-term radiation bone marrow chimeras express a requirement for MHC-restricted self-recognition in T I responses to high concentrations of T N P Ficoll. These studies did not determine, however, the effects that T cells or soluble T cell products might exert on this B cell function. Thus, it remained undetermined whether interacting TH cells and B cells could each express requirements for recognition of self-MI--IC determinants on accessory cells in a T D response. Because the simultaneous expression of such recognition requirements by both B cells and TH cells would constitute a novel cell interaction pathway, the present study was carried out to assess the cell interaction requirements of TH cells and B cells in the highly T D primary antibody response to low concentrations of TNP-Ficoll. It was first shown that TH cell function under these conditions is M H C restricted, requiring T cell recognition of M H C products expressed by accessory cells but not by B cells. It was also found that the soluble products secreted by cloned T cells stimulated by Con A or by specific polypeptide antigen permitted direct B cell activation by TNP-Ficoll without M H C restriction and in fact without demonstrable requirements for either T cells or accessory cells. However, in the absence of these soluble factors and in the presence of MHC-restricted TH cells, it was demonstrated that activation of T N P Ficoll-responsive chimeric B cells still required B cell recognition of accessory cell M H C determinants. These findings thus demonstrate the existence of two distinct requirements for MHC-restricted recognition in T D responses to TNP-FicolI. TH cells are restricted in their recognition of M H C determinants expressed on accessory cells, but show no similar requirement for recognition of B cell M H C products. These results are similar to those previously described for TH cell-dependent activation of Lyb-5 + B cells in responses to the conventional T D antigens T N P - K L H and SRBC (16, 21, 25). In addition, the present report has demonstrated a requirement for MHC-restricted recognition of accessory ceils by responding chimeric B cells. A novel cell interaction model that is suggested by these findings is that direct and MHC-restricted interaction occurs between T cells and accessory cells as well as between B cells and accessory cells, but that there is no requirement for direct interaction between T and B cells in
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this activation p a t h w a y (Fig. 8A). It is not clear from the present e x p e r i m e n t s w h e t h e r M H C - r e s t r i c t e d TH cells a n d M H C - r e s t r i c t e d B cells c o o p e r a t i n g in the T N P - F i c o l l response are r e q u i r e d to recognize M H C d e t e r m i n a n t s on the same accessory cell or w h e t h e r these T a n d B cells must recognize the identical M H C d e t e r m i n a n t s expressed on these accessory cells. T h e role o f soluble T cell products as m e d i a t o r s in responses to T N P - F i c o l l is unclear. It was d e m o n s t r a t e d t h a t soluble products of m o n o c l o n a l T cells could directly activate B cells for these responses, b y p a s s i n g a n y a p p a r e n t r e q u i r e m e n t for the p a r t i c i p a t i o n of either T cells or accessory cells. T h e conclusion t h a t these s u p e r n a t a n t s acted directly on B cells, a n d not on residual T cells, was s u p p o r t e d b y the finding that, as r e p o r t e d b y H a r w e l l et al. (20), these same s u p e r n a t a n t s were not sufficient to reconstitute a n t i - S R B C responses of the same a d e q u a t e l y T - d e p l e t e d populations, whereas such a n t i - S R B C responses were reconstituted b y heterogeneous s u p e r n a t a n t s from Con A - s t i m u l a t e d spleen cells. In contrast, when help for responses to T N P - F i c o l l was p r o v i d e d b y u n p r i m e d TH cells, a r e q u i r e m e n t for M H C - r e s t r i c t e d B cell interaction with accessory cells was consistently observed. T h i s contrast implies that s t i m u l a t i o n of TH cells with T N P - F i c o l l fails to generate soluble l y m p h o k i n e s that are (qualitatively a n d quantitatively) sufficient to overcome the r e q u i r e m e n t for M H C - r e s t r i c t e d cell interaction with accessory cells in the a c t i v a t i o n of B cells. F u r t h e r m o r e , these observations predict t h a t a r e q u i r e m e n t for M H C - r e s t r i c t e d selfrecognition b y B cells will not be observed u n d e r conditions in which such l y m p h o k i n e s are i n d u c e d (Fig. 8B). It remains to be d e t e r m i n e d how this m o d e l of M H C - r e s t r i c t e d self-recognition will a p p l y to the activation of B cells p a r t i c i p a t i n g in c o n v e n t i o n a l T D responses to p o l y p e p t i d e antigens. A l t h o u g h the TH cells p a r t i c i p a t i n g in responses to T N P - F i c o l l express M H C restriction requirements similar to those o f the TH cells t h a t activate Lyb-5 + B cells for responses to p o l y p e p t i d e antigens (16, 21, 25, 26), it is likely t h a t these TH cells in fact represent distinct populations. Thus, for e x a m p l e , antigenspecific T cell proliferative responses have been generated to a wide variety o f p o l y p e p t i d e antigens, b u t not to the p o l y s a c c h a r i d e T N P - F i c o l l . In a d d i t i o n , only modest p r i m i n g effects have as yet been r e p o r t e d for a n t i b o d y responses to T N P - F i c o l l (24), a n d the a n t i g e n specificity o f TNP-Ficoll-responsive Trt cells, in contrast to t h a t o f polypeptide-specific TH cells, has not yet been established. In p a r t i c u l a r , the possibility must be considered that T N P - F i c o l l m a y differ from c o n v e n t i o n a l T D p o l y p e p t i d e antigens in its failure to efficiently s t i m u l a t e the secretion of soluble TH
]
[]
SOLUBLE
RESTRICTED~RESTRICREsMHI TED CTED~ FIG. 8. (A) The T cell-dependent activation ofTNP-Ficoll-responsive B cells can occur through a novel activation pathway in which TH ceils and B cells are each required to recognize self-MHC determinants expressed on accessory cells. (B) Soluble T cell factors can directly activate TNPFicoll-responsiveB cells by an alternative pathway that bypasses cell interaction requirements and thus overcomes any requirement for B cell recognition of self-MHC products.
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cell products capable of directly activating B cells. The ability of polypeptide antigens to stimulate such T n cell factors might well favor the direct activation of B cells in these responses (Fig. 8B), thereby obscuring alternative pathways in which B cells might express a requirement for recognition of selfoMHC products expressed on accessory cells (Fig. 8A). In conclusion, the present studies have investigated the effect of T cells and soluble T cell products on the activation requirements of TNP-Ficoll-responsive B lymphocytes. It was observed that the Tn cells required for responses to limiting concentrations of TNP-Ficoll were M H C restricted in their interactions with accessory cells. In addition, chimeric B cells responding in the presence of these Tn cells also expressed a requirement for MHC-restricted self-recognition in their interaction with accessory cells, although this requirement could be bypassed in the presence of soluble T cell factors. Thus, the present studies demonstrate that requirements for self-recognition of accessory cell M H C determinants by TNP-Ficoll-responsive B cells can only be observed under conditions in which a B cell-accessory cell interaction occurs and can be bypassed by the presence of soluble B cell activating factors derived from T cells. In addition, the present study demonstrates the existence of a novel cell interaction pathway in which TNP-Ficoll-reactive T~I cells and B cells are each required to recognize accessory cell M H C determinants, but are not required to recognize each other. Summary
In vitro primary antibody responses to limiting concentrations of trinitrophenyl (TNP)-Ficoll were shown to be T cell dependent, requiring the cooperation of T helper (TI~) cells, B cells, and accessory cells. Under these conditions, TH cells derived from long-term radiation bone marrow chimeras were major histocompatibility complex (MHC) restricted in their ability to cooperate with accessory cells expressing host-type M H C determinants. The requirement for MHC-restricted self-recognition by TNP-Ficoll-reactive B cells was assessed under these T-dependent conditions. In the presence of competent TH cells, chimeric B cells were found to be M H C restricted, cooperating only with accessory cells that expressed host-type M H C products. In contrast, the soluble products of certain monoclonal T cell lines were able to directly activate B cells in response to TNP-Ficoll, bypassing any requirement for M H C restricted self-recognition. These findings demonstrate the existence of a novel cell interaction pathway in which B cells as well as TH cells are each required to recognize self-MHC determinants on accessory cells, but are not required to recognize each other. They further demonstrate that the requirement for self-recognition by B cells may be bypassed in certain T-dependent activation pathways. The authors wish to thank Drs. J. Berzofsky, H. Dickler, R. Germain, R. Schwartz, and D. Singer for their careful readings and stimulating discussions during the preparation of this manuscript; C. Harrison and J. Williams for their expert technical assistance; and L. DeNenno, W. Hinkle, J. Israel, and F. Jones for their care of experimental animals. Receivedfor publication 3 September 1982. References 1. Katz, D. H., T. Hamaoka, and B. Benacerraf. 1973. Cell interactions between histoincompatible T and B lymphocytes. II. Failure of physiologic cooperative interactions between T
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activation. I. Macrophage presentation of TNP-FicolI: evidence for macrophage-B cell interaction. J. lmmunol. 124:989. Oi, V. T., P. P. Jones, J. w . Coding, L. A. Herzenberg, and L. A. Herzenberg. 1978. Properties of monoclonal antibodies to mouse Ig allotypes, H-2, and Ia antigens. Curr. Top. Microbiol. lmmunol. 81:115. Hodes, R. J., and A. Singer. 1977. Cellular and genetic control of antibody responses in vitro. I. Cellular requirements for the generation of genetically controlled primary IgM responses to soluble antigens. Eur. J. lmmunol. 7:892. Harwell, L., B. Skidmore, P. Marrack, and J. Kappler. 1980. Concanavalin A-inducible, interleukin-2 producing T cell hybridoma.J. Exp. Med. 152:893. Asano, Y., M. Shigeta, C. G. Fathman, A. Singer, and R. J. Hodes. 1982. Role of the major histocompatibility complex in T cell activation of B cell subpopulations. A single monoclonal T helper cell population activates different B cell subpopulations by distinct pathways.J. Exp. Med. 156:350. Hodes, R. J., M. Kimoto, K. S. Hathcock, C. G. Fathman, and A. Singer. 1981. Functional helper activity of monoclonal T cell populations: antigen-specific and H-2 restricted cloned T cells provide help for in vitro antibody responses to trinitrophenyl-poly(L-Tyr,Glu)-poly (D,I.-Ala)--poly(L-Lys).Proc. Natl. Acad. Sci. USA. 78:6431. Mond, J. J., P. K. Mongini, D. Sieekmann, and W. E. Paul. 1980. Role o f T lymphocytes in the response to TNP-AECM Ficoll. J. Immunol. 125:1066. Letvin, N. L., B. Benacerraf, and R. N. Germain. 1981. B-lymphocyte responses to trinitrophenyl-conjugated Ficoll: requirement for T lymphocytes and Ia-bearing adherent cells. Proc. Natl. Acad. ScL USA. 78"5113. Asano, Y., A. Singer, and R. J. Hodes. 1981. Role of the major histocompatibility complex in T cell activation of B cell subpopulations. Major histocompatibility complex-restricted and -unrestricted B cell responses are mediated by distinct B cell subpopulations. J. Exp. Med. 154:1100. Hodes, R. J., M. Shigeta, K. S. Hathcock, C. G. Fathman, and A. Singer. 1982. Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigenspecific and H-2-restricted monoclonal TH cells activate Lyb-5 + B cells through an antigennonspecific and H-2-unrestricted effector pathway. J. Immunol. 129:267.
