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Anti-Ig as a model for MHC-restricted help
MHC R E S T R I C T I O N IN T/B INTERACTIONS
89
ANTI-Ig AS A MODEL FOR MHC-RESTRICTED H E L P by D. C. Parker and H. P. T o n y
Department o/Molecular Genetics and Microbiology, University o/Massachusetts Medical School, Worcester, MA 01605 ( U S A ) The overall aim of our laboratory is to determine the roles of receptor Ig and helper T cells in the antibody response. Since antigen-binding B lymphocytes for any one antigen are rare, we have used anti-Ig as a model for antigen in order to increase the fraction of responding B-cell clones. Our experiments [1, 2] and similar experiments in other laboratories (reviewed in [3]), have shown that anti-Ig induces responsiveness to T-cell-derived, antigen-non-specific, MHC-unrestricted helper factors which enable the B cells to proliferate and mature to high-rate Ig secretion. In this model system as in the in vitro response to heterologous erythrocytes [4], the function of receptor Ig is to deliver an activating signal to the cell, and the role of helper T cells is limited to the secretion of stable polypeptide hormone-like growth and maturation factors. However, the response to anti-Ig or erythrocytes and helper factors may be a special case. In particular, for in vivo and in vitro secondary responses to low concentrations of protein antigens, certain helper T-cell functions cannot be replaced by such helper factors and appear to require MHC-restricted recognition by helper T cells of antigen presented on the surface of the responding B cell [5, 6]. Antigen acting alone appears to be less effective than anti-Ig in inducing early activation events in isolated, antigen-binding B lymphocytes, causing depolarization [7] and enhancing responses to mitogens [8], but failing to induce increases in total RNA or DNA synthesis [7, 8] in the absence of helper T cells. In order to study these other helper T-cell functions which require recognition of antigen presented on the B-cell surface, we have begun adding T-cell lines or hybridomas instead of T-cell-derived soluble factors to our B-cell cultures. Since we continue to use rabbit anti-Ig as a polyclonal model for antigen, we use helper T cells which are specific for rabbit immunoglobulin as a protein antigen. In our recent experiments, which will be published elsewhere, we have shown that these rabbit globulin-reactive T cells will induce good polyclonal antibody responses from small B lymphocytes under conditions of anti-Ig stimulation which are inadequate to induce blast transformation or responsiveness to stable, soluble helper factors. These conditions include concentrations of F(ab')~ anti-IgM or anti-IgD in the range of 1 to 100 ng/ml, depending on the line or clone, whereas blast transformation requires concentrations greater than 1 ~g/ml [9]. Moreover, help provided by direct addition of T cells is largely MHC-restricted at the T-cell/B-cell level, as shown by the lack of help for bystander B ceils mixed with the responding B ceils, but expressing MHC alleles not recognized by the T cells. Table I shows a typical experiment in which rabbit globulin-reactive T cells, derived from a (C3H x DBAk) F2 mouse, help (H2 k x H2 d) F1 B cells but not H2 b B cells. In a mixture of the two kinds of B cells, most of the secreting cells are F1, as shown by their sensitivity to anti-H2k plus complement. Other experiments with H-2 congenic mice have shown that this strain restriction maps to the MHC.
Small B cells as antigen-presenting cells. The MHC-restricted response of small T-depleted spleen cells to small amounts of soluble anti-Ig implies that small B cells are the relevant antigen-presenting
3e F O R U M
90 TABLE
D'IMMUNOLOGIE
I. -MI-IC-restriction of the p o l y c l o n a l a n t i b o d y response to rabbit a n t i - I g a n d rabbit globulin-specific T cells.
Additions to culture
Responding, small, T-depleted spleen cells 5 • 104 B10 (H2 b)
T-cell help
Ig-secreting cells per culture after trealmenl with
F(ab')~ anti-Ig ~g/ml
Helper factors (x) T cells2 T cells
Anti-IgM 50
5 • 104 CDF1 (H2 k X H2 d)
Helper factors T cells T cells
Anti-IgM
5 • 104 B10 plus 5 • 10a CDF1
Helper factors T cells T cells
Anti-IgM
Anti-IgM Anti-IgM Anti-IgM Anti-IgD
None
0.5
nil
C only
anti-If2k+C
6,300 10 20
7,660 -. . .
6,440
8,310
340
.
7,130 --
10 Notze 0.5
9,720
5,290
50
10 None 0.05 0.1 0.05 0.1
54,340 20 14,680 13,840 8,520 16,000
20,370
8,710
10
8,910 8,350 7,440 9,020
290 1,470 290 930
Cells were cultured for four days in 200 ~1 of RPMI-1640 containing 20 % foetal calf serum and 5 • 10-SM 2-mercaptoethanol. Cells were treated with monoclonal anti-H-2 k antibody and complement just prior to assay. Small cells were separated from anti-Thyl plus complement-treated spleen cells by centrifugal elutriation. (1) 50 % supernatant of mouse spleen cells activated with concanavalin A. (2) A continuous line (CDC-35) of rabbit globulin-reactive helper T cells derived at limiting dilution from C3H/HeJ • DBA/2 primed lymph node cells boosted in vitro with normal rabbit F(ab')2 of IgG; 3 • 104 ceils were added per culture.
cell in t h i s s y s t e m . W e can m e a s u r e a n t i g e n p r e s e n t a t i o n m o r e d i r e c t l y b y m e a s u r i n g T-cell p r o l i f e r a t i o n or IL-2 p r o d u c t i o n b y t h e T-cell h y b r i d o m a s . In c o n f i r m a t i o n of e a r l i e r e x p e r i m e n t s b y C h e s n u t a n d G r e y [10], we find t h a t a n t i - I g is p r e s e n t e d a t l e a s t t e n - t h o u s a n d - f o l d m o r e effectively t h a n n o r m a l F ( a b ' ) 2 globulin. H o w e v e r , while G r e y a n d co-workers f o u n d t h a t s m a l l B cells were ineffective in a n t i g e n p r e s e n t a t i o n unless t r a n s f o r m e d into b l a s t s b y L P S or m i t o g e n i c c o n c e n t r a t i o n s of a n t i - I g [10], we find t h a t s m a l l a n d large B cells are c o m p a r a b l y effective. T h e difference b e t w e e n t h e s m a l l a n d large cells is t h a t t h e s m a l l cells are r a d i o s e n s i t i v e a n d lose t h e i r a n t i g e n p r e s e n t a t i o n f u n c t i o n a t doses o v e r 1,000 r a d s ([11] a n d o u r u n p u b l i s h e d results), which e x p l a i n s t h e failure to s t i m u l a t e T cells in t h e e x p e r i m e n t s of G r e y a n d o t h e r s [12]. W e find no r e q u i r e m e n t for m a c r o p h a g e s or o t h e r a c c e s s o r y cells as a n t i g e n p r e s e n t i n g cells in these e x p e r i m e n t s w i t h s m a l l B cells a n d T-cell h y b r i d s or r e c e n t l y r e - s t i m u l a t e d T-cell lines. Selection of s m a l l B cells b y v e l o c i t y s e d i m e n t a t i o n u s i n g centrifugal e l u t r i a t i o n d e p l e t e s a c c e s s o r y cells for t h e T-cell r e s p o n s e to ConA as effectively as p l a s t i c a d h e r e n c e a n d t w o p a s s e s on S e p h a d e x - G 1 0 in our h a n d s . H o w e v e r , we h a v e n o t e x c l u d e d a role for r e s i d u a l a c c e s s o r y cells in s e c r e t i o n of f a c t o r s s u p p o r t i n g B- or T-cell p r o l i f e r a t i o n . R o l e s / o r the antigen receplor in M H C - r e s l r i c t e d T / B collaboration.
O v r e x p e r i m e n t s to d a t e show t h a t small B cells are e x t r e m e l y elticient a t p r e s e n t i n g s u b m i t o g e n i c c o n c e n t r a t i o n s of r a b b i t a n t i - m o u s e Ig to r a b b i t g l o b u l i n r e a c t i v e T cells. T h e M H C - r e s t r i c t e d r e c o g n i t i o n of r a b b i t a n t i - I g on t h e s m a l l B-cell surface results in a p r o l i f e r a t i v e response f r o m b o t h t h e T cells a n d t h e B cells, a n d s o m e of t h e B cells go on t o secrete Ig. T h e q u e s t i o n now is w h e t h e r r e c e p t o r Ig p l a y s m o r e t h a n a passive, a n t i g e n 4 o c u s i n g role in t h e s e responses.
MHC R E S T R I C T I O N I N T / B
INTERACTIONS
91
Although we can show unambiguously t h a t these responses are dependent on receptor Ig-mediated antigen presentation, it seems clear from the work of a number of laboratories t h a t receptor Ig need not be involved at all in MHCrestricted help if the appropriate structures for recognition by the T cell are present on the B-cell surface and the B cell or T cell is in the appropriate state of activation [13, 171. This conclusion is supported by our own unpublished experiments with rabbit globulin-specific T cells using high concentrations of normal rabbit globulin instead of low concentrations of anti-/g, and also with some apparently autoreactive T-cell lines which help B cells in the absence of nominal antigen. Although receptor Ig interactions with antigen are not necessary for MHC-restricted T cell/B cell collaboration, they m a y be particularly efficient in antigen processing and presentation and perhaps also in submitogenic signalling to enable the B cell to induce and receive T-cell help. Currently, we are investigating these possibilities b y determining requirements for cross-linking of receptor Ig and comparing the efficiency of antigen presentation via receptor Ig with t h a t via other B-cell surface molecules. I~e[erences. [1] PARr:ER, D. C., WADSWORTH, D. C. & SCHNEIDER, G. B., J. exp. Med., 1980, 152, 138. [2] PARXER, D. C., Immunol. Rev., 1980, 52, 115. [3] HOWARD, M. & PAUL, W. E., Ann. Rev. lmmunol., 1983, 1,307. [4] LErBSON, H. J., MARRACK, P. & KAPPLER, J. W., J. exp. Med., 1981, 154, 1681. [51 HiiNIG, T. & SCHIMPL, A., Europ. J. Immunol., 1979, 9, 730. [61 SINGER, A., ASANO, Y., SHmETA, M., HATHCOCK, K. D., AHMED, A., FATHMAN, C. G. & HODES, R. J., lmmunol. Rev., 1982, 64, 137. [7] MONROE, J. G. & CAMmER, J. C., J. Immunol., 1983, 131, 2641. [8] SNOW, E. C., NOELLE, R. J., UHR, J. W. & VITETTA, E. S., J. Immunol., 1983, 130, 614. [91 PHILLIPS, N. E. & PARKER, D. C., J. lmmunol., 1984, 132, 627. [10] KAKIUCHI, T., CHESNUT, R. W. & GREY, H. M., J. lmmunol., 1983, 131, 109. [11] ASHWELL, J. S., DEFRANCO, A. L., PAUL, W. E. & SCHWARTZ, R. H., J. exp. Med., 1984, in press. [12] BANDEIRA, A., PO~OR, G., PETTERSSON, S. & COUTINHO,A., J. exp. Med., 1983, 157, 312. [13] CAMMISULI,S., HENRY, C. & WOFSY, L., Europ. J. lmmunol., 1978, 8, 656. [14] AUGUSTIN, A. ~= COUTINHO, A., g. exp. Med., 1980, 151, 587. [15] TSE, H. Y., MOND, J. J. & PAUL, W. E., J. exp. Med., 1981, 153, 871. [16] JONES, ]3. & JANEWAY Jr, C. A., Nature (Lond.), 1981, 292, 547. [17] FIATCLIFFE,M. J. H. ~L JuLius, M. H., Europ. J. Immunol., 1982, 12, 634. Aided by grant-IM 327 from the American Cancer Society. Hans-Peter Tony is a fellow of the . Deutsche Forschungsgemeinschaft.
MHC R E S T R I C T I O N IN T CELL/B CELL INTERACTION: R O L E OF B-CELL SUBPOPULATIONS AND B-CELL A C T I V A T I O N STATE b y R. J. H o d e s and A. Singer Immunology Branch, Nalional Cancer Inslilule, Nalioual Inslilules o[ Heallh, Belhesda, M D 20225 ( U S A ) Early studies of T-cell-dependent (TD) B-cell activation were marked by a p p a r e n t conflicts a m o n g the findings of different investigators concerning the
89
ANTI-Ig AS A MODEL FOR MHC-RESTRICTED H E L P by D. C. Parker and H. P. T o n y
Department o/Molecular Genetics and Microbiology, University o/Massachusetts Medical School, Worcester, MA 01605 ( U S A ) The overall aim of our laboratory is to determine the roles of receptor Ig and helper T cells in the antibody response. Since antigen-binding B lymphocytes for any one antigen are rare, we have used anti-Ig as a model for antigen in order to increase the fraction of responding B-cell clones. Our experiments [1, 2] and similar experiments in other laboratories (reviewed in [3]), have shown that anti-Ig induces responsiveness to T-cell-derived, antigen-non-specific, MHC-unrestricted helper factors which enable the B cells to proliferate and mature to high-rate Ig secretion. In this model system as in the in vitro response to heterologous erythrocytes [4], the function of receptor Ig is to deliver an activating signal to the cell, and the role of helper T cells is limited to the secretion of stable polypeptide hormone-like growth and maturation factors. However, the response to anti-Ig or erythrocytes and helper factors may be a special case. In particular, for in vivo and in vitro secondary responses to low concentrations of protein antigens, certain helper T-cell functions cannot be replaced by such helper factors and appear to require MHC-restricted recognition by helper T cells of antigen presented on the surface of the responding B cell [5, 6]. Antigen acting alone appears to be less effective than anti-Ig in inducing early activation events in isolated, antigen-binding B lymphocytes, causing depolarization [7] and enhancing responses to mitogens [8], but failing to induce increases in total RNA or DNA synthesis [7, 8] in the absence of helper T cells. In order to study these other helper T-cell functions which require recognition of antigen presented on the B-cell surface, we have begun adding T-cell lines or hybridomas instead of T-cell-derived soluble factors to our B-cell cultures. Since we continue to use rabbit anti-Ig as a polyclonal model for antigen, we use helper T cells which are specific for rabbit immunoglobulin as a protein antigen. In our recent experiments, which will be published elsewhere, we have shown that these rabbit globulin-reactive T cells will induce good polyclonal antibody responses from small B lymphocytes under conditions of anti-Ig stimulation which are inadequate to induce blast transformation or responsiveness to stable, soluble helper factors. These conditions include concentrations of F(ab')~ anti-IgM or anti-IgD in the range of 1 to 100 ng/ml, depending on the line or clone, whereas blast transformation requires concentrations greater than 1 ~g/ml [9]. Moreover, help provided by direct addition of T cells is largely MHC-restricted at the T-cell/B-cell level, as shown by the lack of help for bystander B ceils mixed with the responding B ceils, but expressing MHC alleles not recognized by the T cells. Table I shows a typical experiment in which rabbit globulin-reactive T cells, derived from a (C3H x DBAk) F2 mouse, help (H2 k x H2 d) F1 B cells but not H2 b B cells. In a mixture of the two kinds of B cells, most of the secreting cells are F1, as shown by their sensitivity to anti-H2k plus complement. Other experiments with H-2 congenic mice have shown that this strain restriction maps to the MHC.
Small B cells as antigen-presenting cells. The MHC-restricted response of small T-depleted spleen cells to small amounts of soluble anti-Ig implies that small B cells are the relevant antigen-presenting
3e F O R U M
90 TABLE
D'IMMUNOLOGIE
I. -MI-IC-restriction of the p o l y c l o n a l a n t i b o d y response to rabbit a n t i - I g a n d rabbit globulin-specific T cells.
Additions to culture
Responding, small, T-depleted spleen cells 5 • 104 B10 (H2 b)
T-cell help
Ig-secreting cells per culture after trealmenl with
F(ab')~ anti-Ig ~g/ml
Helper factors (x) T cells2 T cells
Anti-IgM 50
5 • 104 CDF1 (H2 k X H2 d)
Helper factors T cells T cells
Anti-IgM
5 • 104 B10 plus 5 • 10a CDF1
Helper factors T cells T cells
Anti-IgM
Anti-IgM Anti-IgM Anti-IgM Anti-IgD
None
0.5
nil
C only
anti-If2k+C
6,300 10 20
7,660 -. . .
6,440
8,310
340
.
7,130 --
10 Notze 0.5
9,720
5,290
50
10 None 0.05 0.1 0.05 0.1
54,340 20 14,680 13,840 8,520 16,000
20,370
8,710
10
8,910 8,350 7,440 9,020
290 1,470 290 930
Cells were cultured for four days in 200 ~1 of RPMI-1640 containing 20 % foetal calf serum and 5 • 10-SM 2-mercaptoethanol. Cells were treated with monoclonal anti-H-2 k antibody and complement just prior to assay. Small cells were separated from anti-Thyl plus complement-treated spleen cells by centrifugal elutriation. (1) 50 % supernatant of mouse spleen cells activated with concanavalin A. (2) A continuous line (CDC-35) of rabbit globulin-reactive helper T cells derived at limiting dilution from C3H/HeJ • DBA/2 primed lymph node cells boosted in vitro with normal rabbit F(ab')2 of IgG; 3 • 104 ceils were added per culture.
cell in t h i s s y s t e m . W e can m e a s u r e a n t i g e n p r e s e n t a t i o n m o r e d i r e c t l y b y m e a s u r i n g T-cell p r o l i f e r a t i o n or IL-2 p r o d u c t i o n b y t h e T-cell h y b r i d o m a s . In c o n f i r m a t i o n of e a r l i e r e x p e r i m e n t s b y C h e s n u t a n d G r e y [10], we find t h a t a n t i - I g is p r e s e n t e d a t l e a s t t e n - t h o u s a n d - f o l d m o r e effectively t h a n n o r m a l F ( a b ' ) 2 globulin. H o w e v e r , while G r e y a n d co-workers f o u n d t h a t s m a l l B cells were ineffective in a n t i g e n p r e s e n t a t i o n unless t r a n s f o r m e d into b l a s t s b y L P S or m i t o g e n i c c o n c e n t r a t i o n s of a n t i - I g [10], we find t h a t s m a l l a n d large B cells are c o m p a r a b l y effective. T h e difference b e t w e e n t h e s m a l l a n d large cells is t h a t t h e s m a l l cells are r a d i o s e n s i t i v e a n d lose t h e i r a n t i g e n p r e s e n t a t i o n f u n c t i o n a t doses o v e r 1,000 r a d s ([11] a n d o u r u n p u b l i s h e d results), which e x p l a i n s t h e failure to s t i m u l a t e T cells in t h e e x p e r i m e n t s of G r e y a n d o t h e r s [12]. W e find no r e q u i r e m e n t for m a c r o p h a g e s or o t h e r a c c e s s o r y cells as a n t i g e n p r e s e n t i n g cells in these e x p e r i m e n t s w i t h s m a l l B cells a n d T-cell h y b r i d s or r e c e n t l y r e - s t i m u l a t e d T-cell lines. Selection of s m a l l B cells b y v e l o c i t y s e d i m e n t a t i o n u s i n g centrifugal e l u t r i a t i o n d e p l e t e s a c c e s s o r y cells for t h e T-cell r e s p o n s e to ConA as effectively as p l a s t i c a d h e r e n c e a n d t w o p a s s e s on S e p h a d e x - G 1 0 in our h a n d s . H o w e v e r , we h a v e n o t e x c l u d e d a role for r e s i d u a l a c c e s s o r y cells in s e c r e t i o n of f a c t o r s s u p p o r t i n g B- or T-cell p r o l i f e r a t i o n . R o l e s / o r the antigen receplor in M H C - r e s l r i c t e d T / B collaboration.
O v r e x p e r i m e n t s to d a t e show t h a t small B cells are e x t r e m e l y elticient a t p r e s e n t i n g s u b m i t o g e n i c c o n c e n t r a t i o n s of r a b b i t a n t i - m o u s e Ig to r a b b i t g l o b u l i n r e a c t i v e T cells. T h e M H C - r e s t r i c t e d r e c o g n i t i o n of r a b b i t a n t i - I g on t h e s m a l l B-cell surface results in a p r o l i f e r a t i v e response f r o m b o t h t h e T cells a n d t h e B cells, a n d s o m e of t h e B cells go on t o secrete Ig. T h e q u e s t i o n now is w h e t h e r r e c e p t o r Ig p l a y s m o r e t h a n a passive, a n t i g e n 4 o c u s i n g role in t h e s e responses.
MHC R E S T R I C T I O N I N T / B
INTERACTIONS
91
Although we can show unambiguously t h a t these responses are dependent on receptor Ig-mediated antigen presentation, it seems clear from the work of a number of laboratories t h a t receptor Ig need not be involved at all in MHCrestricted help if the appropriate structures for recognition by the T cell are present on the B-cell surface and the B cell or T cell is in the appropriate state of activation [13, 171. This conclusion is supported by our own unpublished experiments with rabbit globulin-specific T cells using high concentrations of normal rabbit globulin instead of low concentrations of anti-/g, and also with some apparently autoreactive T-cell lines which help B cells in the absence of nominal antigen. Although receptor Ig interactions with antigen are not necessary for MHC-restricted T cell/B cell collaboration, they m a y be particularly efficient in antigen processing and presentation and perhaps also in submitogenic signalling to enable the B cell to induce and receive T-cell help. Currently, we are investigating these possibilities b y determining requirements for cross-linking of receptor Ig and comparing the efficiency of antigen presentation via receptor Ig with t h a t via other B-cell surface molecules. I~e[erences. [1] PARr:ER, D. C., WADSWORTH, D. C. & SCHNEIDER, G. B., J. exp. Med., 1980, 152, 138. [2] PARXER, D. C., Immunol. Rev., 1980, 52, 115. [3] HOWARD, M. & PAUL, W. E., Ann. Rev. lmmunol., 1983, 1,307. [4] LErBSON, H. J., MARRACK, P. & KAPPLER, J. W., J. exp. Med., 1981, 154, 1681. [51 HiiNIG, T. & SCHIMPL, A., Europ. J. Immunol., 1979, 9, 730. [61 SINGER, A., ASANO, Y., SHmETA, M., HATHCOCK, K. D., AHMED, A., FATHMAN, C. G. & HODES, R. J., lmmunol. Rev., 1982, 64, 137. [7] MONROE, J. G. & CAMmER, J. C., J. Immunol., 1983, 131, 2641. [8] SNOW, E. C., NOELLE, R. J., UHR, J. W. & VITETTA, E. S., J. Immunol., 1983, 130, 614. [91 PHILLIPS, N. E. & PARKER, D. C., J. lmmunol., 1984, 132, 627. [10] KAKIUCHI, T., CHESNUT, R. W. & GREY, H. M., J. lmmunol., 1983, 131, 109. [11] ASHWELL, J. S., DEFRANCO, A. L., PAUL, W. E. & SCHWARTZ, R. H., J. exp. Med., 1984, in press. [12] BANDEIRA, A., PO~OR, G., PETTERSSON, S. & COUTINHO,A., J. exp. Med., 1983, 157, 312. [13] CAMMISULI,S., HENRY, C. & WOFSY, L., Europ. J. lmmunol., 1978, 8, 656. [14] AUGUSTIN, A. ~= COUTINHO, A., g. exp. Med., 1980, 151, 587. [15] TSE, H. Y., MOND, J. J. & PAUL, W. E., J. exp. Med., 1981, 153, 871. [16] JONES, ]3. & JANEWAY Jr, C. A., Nature (Lond.), 1981, 292, 547. [17] FIATCLIFFE,M. J. H. ~L JuLius, M. H., Europ. J. Immunol., 1982, 12, 634. Aided by grant-IM 327 from the American Cancer Society. Hans-Peter Tony is a fellow of the . Deutsche Forschungsgemeinschaft.
MHC R E S T R I C T I O N IN T CELL/B CELL INTERACTION: R O L E OF B-CELL SUBPOPULATIONS AND B-CELL A C T I V A T I O N STATE b y R. J. H o d e s and A. Singer Immunology Branch, Nalional Cancer Inslilule, Nalioual Inslilules o[ Heallh, Belhesda, M D 20225 ( U S A ) Early studies of T-cell-dependent (TD) B-cell activation were marked by a p p a r e n t conflicts a m o n g the findings of different investigators concerning the