- Email: [email protected]
Apparent T cell function of bone marrow cells from mice experiencing a graft-versus-host reaction
CELLULAR
IMMUNOLOGY
Apparent
21,
185-191
(1976)
T Cell Function of Bone Marrow Cells from Experiencing a Graft-Versus-Host Reaction’ ROBERT
Department
of
ELIE
Physiology, Montreal,
2 AND
McIntyre Quebec, Received
WAYNE Medical Canada
July
S. LAPP
Mice
3
Center, McGill H3C 3Gl
Unir~ersity,
30, 1975
The graft-versus-host (GVH) reaction, induced in adult F, mice by the injection of parental strain lymphoid cells (GVH mice), suppressed the ijz zitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) of spleen cells obtained from the GVH mice (GVH-SC). In vitro restoration of the PFC response of GVH-SC was carried out employing a modified Marbrook culture chamber consisting of an inner culture compartment (IC) separated from an outer culture compartment (OC) by a cell-impermeable membrane. Thymus cells (TC) and lymph node cells (LNC) but not bone marrow cells (BMC) from normal mice placed in the IC restored the PFC response of GVH-SC cultured with SRBC in the OC. The restoring ability of TC and LNC was markedly reduced following treatment with anti-theta serum plus complement. BMC taken from GVH mice 3 or more days post-GVH induction (GVHBMC) and placed in the IC restored the PFC response of GVH-SC as well as TC and LNC. Treatment of GVH-BMC with anti-theta serum plus complement did not affect their restoring ability; furthermore, the number of theta-bearing cells in the bone marrow did not increase as a consequence of the GVH reaction. Two possible explanations are proposed for the T-like function of GVH-BMC.
INTRODUCTION It is now generally agreed that antibody production to heterologous red cells in the mouse requires the cooperation of three different cell populations, thymusderived lymphocytes (T cells), bone marrow-derived lymphocytes (B cells), and macrophages (l-3). In recent years, many reports have suggested that antigenspecific (4, 5) and nonspecific (4, 6-11) T cell-derived mediators play a major role in the activation of the B cells. In this laboratory, we have used graft-versus-host (GVH) -induced immunosuppression to investigate the cellular interactions in the induction of the immune response. Experiments carried out both in Z&J (12, 13) and in zritro (14, 15) 4 on the restoration of the plaque-forming cell (PFC) response to sheep red blood cells (SRBC) h ave demonstrated that GVH-induced 1 Supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. 2 MRC of Canada Fellow. Present address : Department de Sciences Biologiques, Universite du Quebec g Mont&al, Montreal, Canada. 3 MRC of Canada Scholar. 4 Elie, R., and Lapp, W. S., “Graft-Versus-Host Induced Immunosuppression : Depressed T Cell Helper Function & vitro.” Cell. Zmmunol. in press.
Copyright
0
All
of
rights
1975 by Academic Press, Inc. reproduction in any form reserved.
186
SHORT
COMMUNICATIONS
immunosuppression was due, at least in part, to a deficiency of the T cell helper function mediated by a nonspecific factor similar to the ones described by others (496-9) * We report here a unique feature of bone marrow cells (BMC) in mice undergoing a GVH reaction (GVH-BMC). The results demonstrate that anti-theta resistant GVH-BMC can replace T cell function as defined by restoration experiments employing spleen cells from GVH immunosuppressed mice (GVH-SC) as the target cells for T cell help. MATERIALS
AND
METHODS
Animals Eight- to twelve-week-old mice of inbred strains A, C57BL/6 (B6), and the Fr hybrid B6 x A (B6AFr) were used. All animals were bred and maintained in this laboratory. Induction
of GVH Reactions
The method used to induce GVH reactions was essentially the one described previously (14-16). It consisted of injecting adult B6AFr mice intravenously with 75 x lo6 A strain spleen and lymph node cells. Single cell suspensions were prepared by gently pressing the lymph nodes and the spleens through a 50-mesh stainless-steel screen into cold Hanks’ balanced salt solution supplemented with 10% heat-inactivated calf serum (Grand Island Biological Co., Grand Island, N.Y.) . Antigen Sheep erythrocytes (SRBC) (Institut Armand Frappier, Montreal, Canada) were used as the source of antigen. SRBC obtained in a 50:50 ratio of Alsever’s solution and blood were washed twice with isotonic saline before being used. Cell Cultures Single cell suspensions were prepared by gently pressing thymuses, lymph nodes, and spleens through a sterile 50-mesh stainless-steel screen into cold CMRL 1066 medium supplemented with nonessential amino acids, pyruvate, glutamate and 15% fetal calf serum (Grand Island Biological Co., N.Y.) and 250 units/ml penicillin G (a gift from Squibb and Sons Ltd., Montreal, Quebec) ( 1066 medium). BMC cell suspensions were prepared by irrigation of tibia and femora with 1066 medium. Cells were counted by using the trypan blue dye exclusion method. A modified consisting of two concentric culture compartMarbrook culture chamber (MMCC) ments as described previously was used (14, 15). The inner compartment (IC) of the MMCC was separated from the outer compartment (OC) by a cell-impermeable membrane (0.45 pm pore size). The basic culture consisted of 10 X lo6 GVH-SC and 5 X lo6 SRBC in 1 ml of 1066 medium placed in the OC and 3 x lo6 restoring cells in 0.3 ml of 1066 medium placed in the IC. The control cultures consisted of either 10 x lo6 normal spleen cells (NSC) or 10 X lo6 GVH-SC and 5 X 10’ SRBC in 1 ml of 1066 medium placed in the OC and 1066 medium placed in the IC. The culture vessels were incubated 4 days at 37°C in a 5% CO2 humidified atmosphere. A minimum of four culture chambers was used for each experimental and control group.
SHORT
Anti-Theta
187
COMMUNICATIONS
Tveatmcnt
An anti-theta C3H serum raised in adult AKR mice as described elsewhere used (13). The treatment of the lymphoid cells with the anti-theta serum complement has been described previously (14, 16).
Detection of Antibody-Forming
was plus
Cells
The methods used for assaying the total essentially the one described by Cunningham cations (18). PFC assays were performed the MMCC.
number of direct PFC to SRBC was and Szenberg (17) with slight modifionly on cells cultured in the OC of
RESULTS
Restoration of the in Vitro PFC Response of GVH-SC Lymphoid Cells across a Cell-Imperruleable Membrane
to SRBC by Nornzal
The first series of experiments was performed to show that the PFC response to SRBC of GVH-SC could be restored by normal lymphoid cells as described previously (14-16). Table 1 shows that the PFC response of the GVH-SC was markedly suppressed; however, normal thymus cells (TC) and lymph node cells (LNC) cultured in the IC of the MMCC restored the GVH-SC cultured in the OC.
In contrast, normal BMC did not restore the GVH-SC.
Table 1 also demonstrates
that treatment with anti-theta serum plus complement virtually abrogated the ability of normal TC and LNC to restore the PFC response. These results confirm earlier TABLE RESTORATIONOFTHE LYMPHOID
1
in Vitro PFC RESPONSEOF CELLSACROSSACELL-IMPERMEABLE
Cells cultured in OCb
IC
NSC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC
TC LNC BMC TC + anti-thetad LNC + anti-thetad
GVH-SC TO SRBC BY NORMAL MEMBRANE"
Mean PFC f SE per culture chamber 588 120 422 368 131 88 173
f f f f f f f
67 17 8.5 64 35 17 21
Percentage of restorationc 100 68 53 3 12
0 Five experiments were performed. All experiments gave essentially the same results. For the sake of simplicity the results from one representative experiment are presented. b NSC and GVH-SC were cultured with SRBC in the OC. These cells served as the source of the PFC. c The percentage of restoration was calculated as follows: o restored GVH-SC) - (3 PFC of GVH-SC) x loo Percentage of restoration = (’ PFC f (1 PFC of NSC) - (1 of GVH-SC) d TC and LNC were treated with anti-theta C3H serum plus complement and washed three times before being placed in the IC. Treatment of TC and LNC with either anti-theta serum or complement alone did not affect their restoring ability.
188
SHORT
COMMUNICATIONS
TABLE PERCENTAGE OF RESTOKATION CELLS TAKEN FKOM
OF THE in Vitro PFC RESPONSE OF GVH-SC TO SRBC BY LYMPHOID B~AFI MICE AT DIFFERENT DAYS POST-GVH INDUCTION”
Day post GVH induction restoring cells were obtained -lb 0= 2 3 5 10 17 25 (2 The results of restoration of the b Restoring cells c Restoring cells
2
Percentage of restoration employing different cell types TC
LNC
6.5 66 27 79 60 68 79 57
69 72 28 30 80 70 106 63
BMC 3 0 2 5.5 39 87 110 82
three experiments were pooled and are presented in the form of percentage PFC response of GVH-SC cultured in the OC as calculated in Table 1. were obtained from normal mice. were obtained a few hours after the injection of the parental cells.
of
reports that GVH-induced immunosuppressionis due, at least in part, to a deficiency of T cell helper function ( 14-16) .4 Restoration of the in Vitro PFC Response of GVH-SC across a Cell-ImpermzeableMembrane
to SRBC by GVH-BMC
Next, experiments were performed to test the ability of lymphoid cells taken from animals undergoing a GVH reaction to restore the PFC response to SRBC of GVH-SC. Table 2 shows that TC and LNC harvested at different times post-GVH induction restored the PFC response of the GVH-SC as well at TC and LNC obtained from normal mice. Table 2 also shows that BMC taken from GVH mice on the day of parental cell injection or 2 days post-GVH induction were no better than normal BMC in restoring the PFC response of GVH-SC. The unexpected finding was that BMC taken 3 or more days post-GVH induction restored the PFC responses of GVH-SC as well as TC and LNC obtained from either normal or mice experiencing a GVH reaction (GVH mice). The ability of GVH-BMC to restore GVH-SC was not accompanied by an increased number of theta-bearing cells in the marrow (Table 3) and was not abrogated by anti-theta treatment of the GVH-BMC (Table 4). The resistance of T cell-like function of GVH-BMC to anti-theta treatment is not simply due to a loss of expression of this marker in GVH mice since this study (Table 3) and others (13) have demonstrated that GVH mice possessnear normal numbers of anti-theta sensitive cells. DISCUSSION Previous reports (12-15) 4 have demonstrated that GVH-induced immunois due, at least in part, to a deficiency of T cell helper function since the PFC responseto SRBC of GVH-SC could be restored only by anti-theta sensitive cells across a cell-impermeable membrane. The results reported in this paper demonsuppression
SHORT
TABLE PERCENTAGE
OF THETA B6AFr MICE
1x9
COMMUNICATIONS
3
C3H CELLS UNDERGOING
IN THE LYMPHOID TISSUES A GVH REACTION
Source of lymphoid tissues
Mean
percentage of theta C3H cells5
Thymus
Lymph node
98 94 95
64 42 56
Normal B6AFr Day 10 GVH-B6AFrb Day 12 GVH-B6AFrb
OF
Bone marrow 3 7 0 -
0 Mean percentage Lapp e6 al. (13). b Lymphoid tissues
of theta were
C3H
obtained
cells was on days
calculated 10 and
according
to the
12, respectively,
method
post-GVH
described
by
induction,
strate that GVH-BMC can replace T cells in restoring the in vitro PFC response of GVH-SC. At this time, we can only speculate on the type of cell in the bone marrow of GVH mice which substitutes for T cells in restoring the PFC response of GVH-SC. It could possibly be a T cell sequestered in the bone marrow, an activated T cell precursor, or some other cell capable of binding T cell products. Reports by Cohen and co-workers ( 19, 20) and by Levine and Claman (21) suggest that T cells are sequestered in the bone marrow of mice following treatment with corticosteroids. It was demonstrated that BMC from corticosteroid-treated mice induced a GVH reaction ( 19), responded to phytohemagglutinin stimulation (21)) and contained a substantial proportion of theta-bearing cells (20). It seems possible that the stress of the GVH reaction could increase the production of corticosteroid hormones and cause T cells to be sequestered in the bone marrow. However, in contrast to BMC of the hydrocortisone-treated mice (20) the GVHTABLE RESTORATION OF THE in Vi6ro PFC BONE MARROW CELLS TAKEN Cells
cultured
NSC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC
in IC
OCb
Normal TC TC + anti-thetad Normal BMC GVH-BMQ GVH-BMC” + anti-thetad Normal
4
RESPONSE OF GVH-SC TO SRBC FROM B6AFr MICE EXPERIENCING
BY ANTI-THETA TREATED A GVH REACTIONS
Mean PFC f per culture chamber 583 f 31% 28.5 f 53 f 40f 310 f 390 f
67 9 41 10 11 37 45
SE
Percentage restorationC
of
100 -
0 Three experiments were performed. All experiments gave essentially the same results. sake of simplicity the results from one representative experiment are presented. b*c*d As in Table 1. e Bone marrow cells taken from GVH mice 10 days post-GVH induction.
46 4 2 51 65 For
the
190
SHORT
COMMUNICATIONS
BMC did not exhibit an increase in anti-theta sensitive cells, which seems to exclude the possibility that restoration of GVH-SC by GVH-BMC is mediated by T cells sequestered in the bone marrow. The second possible explanation for the ability of GVH-BMC to restore the PFC response of GVH-SC is that the GVH reaction activates potential T cells that are known to be present in bone marrow (22, 23). Evidence provided by others (24, 25) tends to support the hypothesis that activated T cell precursors can perform T cell function without a concomitant expression of T cell markers which are acquired as a result of traffic through the thymus. Claman (24) has shown that BMC expressing little or no theta marker can respond to T cell mitogens. It has also been shown that cells lacking the theta marker can fulfill other T cell functions (25). On the other hand, restoration of GVH-SC by GVH-BMC could be mediated by a non-T cell capable of binding T cell products. There is evidence suggesting that excessive T cell factor is released in the early stage of the GVH reaction (Lapp, unpublished data), which probably is due to allogenic stimulation and is responsible for the allogenic effect (26). This excessive amount of T cell factor may simply bind to the surface of a cell located in the bone marrow (perhaps a monocytemacrophage) and be released when the cell is put into culture. At the present time there is no basis to favor one explanation over the other for the T cell like function of GVH-BMC. Experiments are in progress to characterize the cell responsible for the restoring ability of GVH-BMC. ACKNOWLEDGMENTS The authors thank Mrs. A. Lee Foon, Mrs. H. C. Lee, and Miss R. Siegrist careful technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
for their
,
Miller, J. F. A. P., and Mitchell, G. F., Transplant. Rev. 1, 3, 1969. Claman, M. N., and Chaperon, E. A., Transplant. Rev. 1, 92, 1969. Unanue, E. R., Advan. Immunol. 15, 95, 1972. Feldman, M., and Basten, A., J. Ex@. Med. 136, 722, 1972. Feldman, M., and Basten, A., Nature New Biol. 237, 15, 1972. Dutton, R. W., Falkoff, R., Hurst, J. A., Hoffman, M., Kappler, J. W., Kettman, J. R., Lesley, J. F., and Vann, D., Progr. Immunol. 1, 355, 1971. Schimpl, A., and Wecker, E., Nature New Biol. 237, 15, 1972. Gorczynski, R. M., Miller, R. G., and Phillips, R. A., J. Zyzmunol. lO& 547, 1972. Haskill, J. S., Byrt, P., and Marbrook, J., J. Exp. Med. 131, 57, 1970. Feldman, M., J. Exp. Med. 136, 737, 1972. Feldman, M., Transplant. Proc. 5, 43, 1973. Gruska, M., and Lapp, W. S., Transplantation 17, 157, 1974. Lapp, W. S., Wechsler, A., and Kongshavn, P. A. L., Cell. Immunol. 11, 419, 1974. Elie, R., Abrahams, R., Parthenais, E., and Lapp, W. S., In “Lymphocyte Recognition and Effector Mechanisms” (K. Lindahl-Kiessling and D. bsoba, Eds.), p. 175. Academic Press, New York, 1974. Parthenais, E., Elie, R., and Lapp, W. S., Cell. Immunol. 13, 164, 1974. Elie, R., and Lapp, W. S., In “Immune Recognition” (A. S. Rosenthal, Ed.), p. 563. Academic Press, New York, 1975. Cunningham, A. S., and Szenberg, A., Immunology 14, 599, 1968. Kongshavn, P. A. L., and Lapp, W. S., Immunology 22, 227, 1972. Cohen, J. J., Fishbach, M., and Claman, H. N., J. Zmmunol. 105, 1146, 1970. Cohen, J. J., J. Immunol. 108, 841, 1972.
SHORT
COMMUNICATIONS
‘21. Levine, M. A., and Claman, H. N., Science 167, 1515, 1970. 22. Komuro, K., and Boyse, E. A., J. Exp. Med. 138, 479, 1973. 23. Miller, H. C., Schmiege, S. K., and Rule, A., J. Zmmunol. 111, 1005, 1973. 24. Claman, M. N., J. Zmmunol. 112, 960, 1974. 25. Parkman, R., and Herler, E., Nature New Biol. 245, 14, 1973. 26. Katz, D. H., Transplant. Rev. 12, 141, 1972.
101
IMMUNOLOGY
Apparent
21,
185-191
(1976)
T Cell Function of Bone Marrow Cells from Experiencing a Graft-Versus-Host Reaction’ ROBERT
Department
of
ELIE
Physiology, Montreal,
2 AND
McIntyre Quebec, Received
WAYNE Medical Canada
July
S. LAPP
Mice
3
Center, McGill H3C 3Gl
Unir~ersity,
30, 1975
The graft-versus-host (GVH) reaction, induced in adult F, mice by the injection of parental strain lymphoid cells (GVH mice), suppressed the ijz zitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) of spleen cells obtained from the GVH mice (GVH-SC). In vitro restoration of the PFC response of GVH-SC was carried out employing a modified Marbrook culture chamber consisting of an inner culture compartment (IC) separated from an outer culture compartment (OC) by a cell-impermeable membrane. Thymus cells (TC) and lymph node cells (LNC) but not bone marrow cells (BMC) from normal mice placed in the IC restored the PFC response of GVH-SC cultured with SRBC in the OC. The restoring ability of TC and LNC was markedly reduced following treatment with anti-theta serum plus complement. BMC taken from GVH mice 3 or more days post-GVH induction (GVHBMC) and placed in the IC restored the PFC response of GVH-SC as well as TC and LNC. Treatment of GVH-BMC with anti-theta serum plus complement did not affect their restoring ability; furthermore, the number of theta-bearing cells in the bone marrow did not increase as a consequence of the GVH reaction. Two possible explanations are proposed for the T-like function of GVH-BMC.
INTRODUCTION It is now generally agreed that antibody production to heterologous red cells in the mouse requires the cooperation of three different cell populations, thymusderived lymphocytes (T cells), bone marrow-derived lymphocytes (B cells), and macrophages (l-3). In recent years, many reports have suggested that antigenspecific (4, 5) and nonspecific (4, 6-11) T cell-derived mediators play a major role in the activation of the B cells. In this laboratory, we have used graft-versus-host (GVH) -induced immunosuppression to investigate the cellular interactions in the induction of the immune response. Experiments carried out both in Z&J (12, 13) and in zritro (14, 15) 4 on the restoration of the plaque-forming cell (PFC) response to sheep red blood cells (SRBC) h ave demonstrated that GVH-induced 1 Supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada. 2 MRC of Canada Fellow. Present address : Department de Sciences Biologiques, Universite du Quebec g Mont&al, Montreal, Canada. 3 MRC of Canada Scholar. 4 Elie, R., and Lapp, W. S., “Graft-Versus-Host Induced Immunosuppression : Depressed T Cell Helper Function & vitro.” Cell. Zmmunol. in press.
Copyright
0
All
of
rights
1975 by Academic Press, Inc. reproduction in any form reserved.
186
SHORT
COMMUNICATIONS
immunosuppression was due, at least in part, to a deficiency of the T cell helper function mediated by a nonspecific factor similar to the ones described by others (496-9) * We report here a unique feature of bone marrow cells (BMC) in mice undergoing a GVH reaction (GVH-BMC). The results demonstrate that anti-theta resistant GVH-BMC can replace T cell function as defined by restoration experiments employing spleen cells from GVH immunosuppressed mice (GVH-SC) as the target cells for T cell help. MATERIALS
AND
METHODS
Animals Eight- to twelve-week-old mice of inbred strains A, C57BL/6 (B6), and the Fr hybrid B6 x A (B6AFr) were used. All animals were bred and maintained in this laboratory. Induction
of GVH Reactions
The method used to induce GVH reactions was essentially the one described previously (14-16). It consisted of injecting adult B6AFr mice intravenously with 75 x lo6 A strain spleen and lymph node cells. Single cell suspensions were prepared by gently pressing the lymph nodes and the spleens through a 50-mesh stainless-steel screen into cold Hanks’ balanced salt solution supplemented with 10% heat-inactivated calf serum (Grand Island Biological Co., Grand Island, N.Y.) . Antigen Sheep erythrocytes (SRBC) (Institut Armand Frappier, Montreal, Canada) were used as the source of antigen. SRBC obtained in a 50:50 ratio of Alsever’s solution and blood were washed twice with isotonic saline before being used. Cell Cultures Single cell suspensions were prepared by gently pressing thymuses, lymph nodes, and spleens through a sterile 50-mesh stainless-steel screen into cold CMRL 1066 medium supplemented with nonessential amino acids, pyruvate, glutamate and 15% fetal calf serum (Grand Island Biological Co., N.Y.) and 250 units/ml penicillin G (a gift from Squibb and Sons Ltd., Montreal, Quebec) ( 1066 medium). BMC cell suspensions were prepared by irrigation of tibia and femora with 1066 medium. Cells were counted by using the trypan blue dye exclusion method. A modified consisting of two concentric culture compartMarbrook culture chamber (MMCC) ments as described previously was used (14, 15). The inner compartment (IC) of the MMCC was separated from the outer compartment (OC) by a cell-impermeable membrane (0.45 pm pore size). The basic culture consisted of 10 X lo6 GVH-SC and 5 X lo6 SRBC in 1 ml of 1066 medium placed in the OC and 3 x lo6 restoring cells in 0.3 ml of 1066 medium placed in the IC. The control cultures consisted of either 10 x lo6 normal spleen cells (NSC) or 10 X lo6 GVH-SC and 5 X 10’ SRBC in 1 ml of 1066 medium placed in the OC and 1066 medium placed in the IC. The culture vessels were incubated 4 days at 37°C in a 5% CO2 humidified atmosphere. A minimum of four culture chambers was used for each experimental and control group.
SHORT
Anti-Theta
187
COMMUNICATIONS
Tveatmcnt
An anti-theta C3H serum raised in adult AKR mice as described elsewhere used (13). The treatment of the lymphoid cells with the anti-theta serum complement has been described previously (14, 16).
Detection of Antibody-Forming
was plus
Cells
The methods used for assaying the total essentially the one described by Cunningham cations (18). PFC assays were performed the MMCC.
number of direct PFC to SRBC was and Szenberg (17) with slight modifionly on cells cultured in the OC of
RESULTS
Restoration of the in Vitro PFC Response of GVH-SC Lymphoid Cells across a Cell-Imperruleable Membrane
to SRBC by Nornzal
The first series of experiments was performed to show that the PFC response to SRBC of GVH-SC could be restored by normal lymphoid cells as described previously (14-16). Table 1 shows that the PFC response of the GVH-SC was markedly suppressed; however, normal thymus cells (TC) and lymph node cells (LNC) cultured in the IC of the MMCC restored the GVH-SC cultured in the OC.
In contrast, normal BMC did not restore the GVH-SC.
Table 1 also demonstrates
that treatment with anti-theta serum plus complement virtually abrogated the ability of normal TC and LNC to restore the PFC response. These results confirm earlier TABLE RESTORATIONOFTHE LYMPHOID
1
in Vitro PFC RESPONSEOF CELLSACROSSACELL-IMPERMEABLE
Cells cultured in OCb
IC
NSC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC
TC LNC BMC TC + anti-thetad LNC + anti-thetad
GVH-SC TO SRBC BY NORMAL MEMBRANE"
Mean PFC f SE per culture chamber 588 120 422 368 131 88 173
f f f f f f f
67 17 8.5 64 35 17 21
Percentage of restorationc 100 68 53 3 12
0 Five experiments were performed. All experiments gave essentially the same results. For the sake of simplicity the results from one representative experiment are presented. b NSC and GVH-SC were cultured with SRBC in the OC. These cells served as the source of the PFC. c The percentage of restoration was calculated as follows: o restored GVH-SC) - (3 PFC of GVH-SC) x loo Percentage of restoration = (’ PFC f (1 PFC of NSC) - (1 of GVH-SC) d TC and LNC were treated with anti-theta C3H serum plus complement and washed three times before being placed in the IC. Treatment of TC and LNC with either anti-theta serum or complement alone did not affect their restoring ability.
188
SHORT
COMMUNICATIONS
TABLE PERCENTAGE OF RESTOKATION CELLS TAKEN FKOM
OF THE in Vitro PFC RESPONSE OF GVH-SC TO SRBC BY LYMPHOID B~AFI MICE AT DIFFERENT DAYS POST-GVH INDUCTION”
Day post GVH induction restoring cells were obtained -lb 0= 2 3 5 10 17 25 (2 The results of restoration of the b Restoring cells c Restoring cells
2
Percentage of restoration employing different cell types TC
LNC
6.5 66 27 79 60 68 79 57
69 72 28 30 80 70 106 63
BMC 3 0 2 5.5 39 87 110 82
three experiments were pooled and are presented in the form of percentage PFC response of GVH-SC cultured in the OC as calculated in Table 1. were obtained from normal mice. were obtained a few hours after the injection of the parental cells.
of
reports that GVH-induced immunosuppressionis due, at least in part, to a deficiency of T cell helper function ( 14-16) .4 Restoration of the in Vitro PFC Response of GVH-SC across a Cell-ImpermzeableMembrane
to SRBC by GVH-BMC
Next, experiments were performed to test the ability of lymphoid cells taken from animals undergoing a GVH reaction to restore the PFC response to SRBC of GVH-SC. Table 2 shows that TC and LNC harvested at different times post-GVH induction restored the PFC response of the GVH-SC as well at TC and LNC obtained from normal mice. Table 2 also shows that BMC taken from GVH mice on the day of parental cell injection or 2 days post-GVH induction were no better than normal BMC in restoring the PFC response of GVH-SC. The unexpected finding was that BMC taken 3 or more days post-GVH induction restored the PFC responses of GVH-SC as well as TC and LNC obtained from either normal or mice experiencing a GVH reaction (GVH mice). The ability of GVH-BMC to restore GVH-SC was not accompanied by an increased number of theta-bearing cells in the marrow (Table 3) and was not abrogated by anti-theta treatment of the GVH-BMC (Table 4). The resistance of T cell-like function of GVH-BMC to anti-theta treatment is not simply due to a loss of expression of this marker in GVH mice since this study (Table 3) and others (13) have demonstrated that GVH mice possessnear normal numbers of anti-theta sensitive cells. DISCUSSION Previous reports (12-15) 4 have demonstrated that GVH-induced immunois due, at least in part, to a deficiency of T cell helper function since the PFC responseto SRBC of GVH-SC could be restored only by anti-theta sensitive cells across a cell-impermeable membrane. The results reported in this paper demonsuppression
SHORT
TABLE PERCENTAGE
OF THETA B6AFr MICE
1x9
COMMUNICATIONS
3
C3H CELLS UNDERGOING
IN THE LYMPHOID TISSUES A GVH REACTION
Source of lymphoid tissues
Mean
percentage of theta C3H cells5
Thymus
Lymph node
98 94 95
64 42 56
Normal B6AFr Day 10 GVH-B6AFrb Day 12 GVH-B6AFrb
OF
Bone marrow 3 7 0 -
0 Mean percentage Lapp e6 al. (13). b Lymphoid tissues
of theta were
C3H
obtained
cells was on days
calculated 10 and
according
to the
12, respectively,
method
post-GVH
described
by
induction,
strate that GVH-BMC can replace T cells in restoring the in vitro PFC response of GVH-SC. At this time, we can only speculate on the type of cell in the bone marrow of GVH mice which substitutes for T cells in restoring the PFC response of GVH-SC. It could possibly be a T cell sequestered in the bone marrow, an activated T cell precursor, or some other cell capable of binding T cell products. Reports by Cohen and co-workers ( 19, 20) and by Levine and Claman (21) suggest that T cells are sequestered in the bone marrow of mice following treatment with corticosteroids. It was demonstrated that BMC from corticosteroid-treated mice induced a GVH reaction ( 19), responded to phytohemagglutinin stimulation (21)) and contained a substantial proportion of theta-bearing cells (20). It seems possible that the stress of the GVH reaction could increase the production of corticosteroid hormones and cause T cells to be sequestered in the bone marrow. However, in contrast to BMC of the hydrocortisone-treated mice (20) the GVHTABLE RESTORATION OF THE in Vi6ro PFC BONE MARROW CELLS TAKEN Cells
cultured
NSC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC GVH-SC
in IC
OCb
Normal TC TC + anti-thetad Normal BMC GVH-BMQ GVH-BMC” + anti-thetad Normal
4
RESPONSE OF GVH-SC TO SRBC FROM B6AFr MICE EXPERIENCING
BY ANTI-THETA TREATED A GVH REACTIONS
Mean PFC f per culture chamber 583 f 31% 28.5 f 53 f 40f 310 f 390 f
67 9 41 10 11 37 45
SE
Percentage restorationC
of
100 -
0 Three experiments were performed. All experiments gave essentially the same results. sake of simplicity the results from one representative experiment are presented. b*c*d As in Table 1. e Bone marrow cells taken from GVH mice 10 days post-GVH induction.
46 4 2 51 65 For
the
190
SHORT
COMMUNICATIONS
BMC did not exhibit an increase in anti-theta sensitive cells, which seems to exclude the possibility that restoration of GVH-SC by GVH-BMC is mediated by T cells sequestered in the bone marrow. The second possible explanation for the ability of GVH-BMC to restore the PFC response of GVH-SC is that the GVH reaction activates potential T cells that are known to be present in bone marrow (22, 23). Evidence provided by others (24, 25) tends to support the hypothesis that activated T cell precursors can perform T cell function without a concomitant expression of T cell markers which are acquired as a result of traffic through the thymus. Claman (24) has shown that BMC expressing little or no theta marker can respond to T cell mitogens. It has also been shown that cells lacking the theta marker can fulfill other T cell functions (25). On the other hand, restoration of GVH-SC by GVH-BMC could be mediated by a non-T cell capable of binding T cell products. There is evidence suggesting that excessive T cell factor is released in the early stage of the GVH reaction (Lapp, unpublished data), which probably is due to allogenic stimulation and is responsible for the allogenic effect (26). This excessive amount of T cell factor may simply bind to the surface of a cell located in the bone marrow (perhaps a monocytemacrophage) and be released when the cell is put into culture. At the present time there is no basis to favor one explanation over the other for the T cell like function of GVH-BMC. Experiments are in progress to characterize the cell responsible for the restoring ability of GVH-BMC. ACKNOWLEDGMENTS The authors thank Mrs. A. Lee Foon, Mrs. H. C. Lee, and Miss R. Siegrist careful technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
for their
,
Miller, J. F. A. P., and Mitchell, G. F., Transplant. Rev. 1, 3, 1969. Claman, M. N., and Chaperon, E. A., Transplant. Rev. 1, 92, 1969. Unanue, E. R., Advan. Immunol. 15, 95, 1972. Feldman, M., and Basten, A., J. Ex@. Med. 136, 722, 1972. Feldman, M., and Basten, A., Nature New Biol. 237, 15, 1972. Dutton, R. W., Falkoff, R., Hurst, J. A., Hoffman, M., Kappler, J. W., Kettman, J. R., Lesley, J. F., and Vann, D., Progr. Immunol. 1, 355, 1971. Schimpl, A., and Wecker, E., Nature New Biol. 237, 15, 1972. Gorczynski, R. M., Miller, R. G., and Phillips, R. A., J. Zyzmunol. lO& 547, 1972. Haskill, J. S., Byrt, P., and Marbrook, J., J. Exp. Med. 131, 57, 1970. Feldman, M., J. Exp. Med. 136, 737, 1972. Feldman, M., Transplant. Proc. 5, 43, 1973. Gruska, M., and Lapp, W. S., Transplantation 17, 157, 1974. Lapp, W. S., Wechsler, A., and Kongshavn, P. A. L., Cell. Immunol. 11, 419, 1974. Elie, R., Abrahams, R., Parthenais, E., and Lapp, W. S., In “Lymphocyte Recognition and Effector Mechanisms” (K. Lindahl-Kiessling and D. bsoba, Eds.), p. 175. Academic Press, New York, 1974. Parthenais, E., Elie, R., and Lapp, W. S., Cell. Immunol. 13, 164, 1974. Elie, R., and Lapp, W. S., In “Immune Recognition” (A. S. Rosenthal, Ed.), p. 563. Academic Press, New York, 1975. Cunningham, A. S., and Szenberg, A., Immunology 14, 599, 1968. Kongshavn, P. A. L., and Lapp, W. S., Immunology 22, 227, 1972. Cohen, J. J., Fishbach, M., and Claman, H. N., J. Zmmunol. 105, 1146, 1970. Cohen, J. J., J. Immunol. 108, 841, 1972.
SHORT
COMMUNICATIONS
‘21. Levine, M. A., and Claman, H. N., Science 167, 1515, 1970. 22. Komuro, K., and Boyse, E. A., J. Exp. Med. 138, 479, 1973. 23. Miller, H. C., Schmiege, S. K., and Rule, A., J. Zmmunol. 111, 1005, 1973. 24. Claman, M. N., J. Zmmunol. 112, 960, 1974. 25. Parkman, R., and Herler, E., Nature New Biol. 245, 14, 1973. 26. Katz, D. H., Transplant. Rev. 12, 141, 1972.
101