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Demonstration of active suppressor cells in spleens of young NZB mice
(I.1 I I I ,\K Ih~MI’voI
OGY 51, 419-423
Demonstration
(1980)
of Active Suppressor Cells in Spleens Young NZB Mice’
of
D. A. LERMAN .&ND C. E. CALKINS
NZB mice. a strain prone to the development ofautoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells \uppres\ the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth i5 similar to that of spleen cells from 4-day-old C57BL/6 mice. a strain which does not strains of mice, suppressor cell spontaneously develop autoimmune disease. As in “normal“ activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent In the suppressor cells detected in the spleens of newhorn and young NZB mice and suggest that the development of autoimmune responses doe\ not result from a lack of suppressor cells in the young animals.
INTRODUCTION Suppressor cells have been demonstrated in states of tolerance to a number of non-self-antigens and may play a role in the induction and/or maintenance of tolerance (1). The possibility that suppressor cells may also be important in selftolerance has led to a consideration of suppressor cell activity in autoimmune responses, Several suppressor cell deficiencies have been demonstrated in adult NZB mice (3). a strain of mice in which antibodies to autologous thymocytes INTAY have been demonstrated as early as I month of age (3) and to autologous erythrocytes somewhat later (2). There is considerable evidence that immunoregulatory deficiencies occur early in life in these mice. At birth NZB mice, unlike other strains of mice. respond to SRBC i/l \si\.o (4) and their lymphocytes produce high levels of IgM i/l I‘iftw (5). In addition, weanling NZB mice cannot sustain experimentally induced tolerance for the long periods of time characteristic of tolerance induced in mice of other strains (6). If a defect in suppressor cell activity is responsible for the abnormal responsiveness of young NZB mice and for the development of autoimmune responses, then this defect should be detected in NZB mice earlier than 1 month of age. the time of onset of the first detectable autoimmune response. Other strains of i Supported In part by Grant IM-139 from the American Cancer National Institutes of Health, and by a Grant from the Pew Pennsylvania Chapter of the Arthriti\ Foundation. ” .4bhreviation\ used: NT. not tested: NTA. naturally occurring plaque-forming cell\: SRBC. sheep red blood cell\.
Society. by Grant AI-l.5789 from Foundation through the Eastern thymocytotoxlc
antihody:
PFC,
4 I9 0008.8749/80/060419-05$02.00/O Copyright All rights
(. 19X0 by Academic of reproduction in
Press.
any form
Inc. rexwed.
420
SHORT COMMUNICATIONS
NEWBORN
CELLS
1x706)
FIG. 1. Ability of young spleen cells to suppress adult anti-SRBC antibody responses in rzitro. 0.5 x lo6 to 5.0 x lo6 spleen cells from 7-day-old NZB (0 -.- 0) or 4-day-old C57BLl6 (0 0) mice were added with SRBC to cultures of 1 x lo7 syngeneic adult spleen cells. PFC responses of cultures of adult cells which received syngeneic young cells were compared with those of control cultures which received no young cells. Mean control responses were 311 5 69 and 4850 t 538 PFC/culture for NZB and C57BL/6 cell cultures, respectively. Brackets represent the range of values in two to four experiments.
mice have active suppressor cells present in their spleens in the first few weeks of life which act in vitro to prevent the cells of these mice from responding to Tdependent antigens (7). Since these suppressor cells may function to suppress antiself-responses in young mice, it is important to compare suppressor cell activity in the spleen cell populations of young NZB mice and of a strain which is not prone to autoimmune disease. MATERIALS
AND METHODS
Animals. Adult NZB and C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Additional NZB mice were generously donated by Dr. J. Bruce Smith (Institute for Cancer Research, Phila., Pa.). Newborn NZB and C57BL/6J mice were bred in the animal colony at Thomas Jefferson University. In each experiment newborn and adult mice were matched according to age, sex, and source of origin. Adult mice were used at 8- 16 weeks of age and young mice from 4 to 21 * 1 days of age. Cell culture and assay. Adult spleen cells were cultured according to the method of Mishell and Dutton (8). Varying numbers of newborn spleen cells were added to duplicate adult spleen cell cultures containing lo7 adult cells and 3 x lo6 sheep erythrocytes (SRBC, obtained from a single sheep by Colorado Serum Co.,
421
SHORT COMMUNICATIONS
Denver, Cola.). Direct IgM plaque-forming cells (PFC) to SRBC were enumerated 4 and 5 days after the initiation of culture using a slide modification of the Jerne plaque assay (8). The peak response which occurred on Day 4 or on Day 5 is reported. NZB mice are low responders to SRBC in \litr’o (9). When the peak response was less than 150 PFCiculture (in approximately 20%, of the experiments), the number of plaques counted in unsuppressed control cultures were too low to accurately evaluate the degree of suppression in cultures to which suppressor cells had been added. Therefore, only those experiments where control responses were greater than 150 PFCiculture are included in the present communication. The data are expressed as percentage of control, with the control response being the number of PFC per culture in cultures of 1 x IO’ syngeneic adult spleen cells + SRBC. RESULTS To determine the presence and activity of suppressor cells in spleens of young NZB and C57BLi6 mice, varying numbers of spleen cells from mice of each strain aged 4-21 days were cultured with 1 x 10’ adult spleen cells of the same strain in the presence of SRBC. Suppressor cells were detectable in the spleens of both strains in the first week after birth (Fig. I). The degree of suppression of the adult response was dependent upon the number of young spleen cells added. As few as 0.5 x 10” NZB spleen cells obtained from 7-day-old mice reduced the adult PFC response to 60% of the control response; 5.0 x 10” young spleen cells reduced the adult response to 15% of control. At 4 days of age, C57BLi6 mice had suppressor cells in their spleens capable of suppressing the homologous adult response to 19% of control when 5.0 x IO” young cells were added to the adult cultures. The addition of fewer young C57BLi6 cells produced no apparent suppression. By 7 days of age, the suppressive capacity of the C57BLi6 spleen cells was less strong than either that of younger C57BLi6 cells or that of 7-day-old NZB cells (Table I). TABLE
1
Suppression of Antibody Responses in Cultures of Adult NZB or C57BL;6 Spleen Cells by Addition of Spleen Cells from Syngeneic Mice of Varying Ages” Percentage of control Age of suppressor cell donors Days 4 7 I4 21 Adult
PFC response (range)”
NZB ( x IO”’ NT” I6 (O-29) 64 (44- 103) 108 (98- 118) I48 (77-242)
C57BL;6 I x IO” NT 54 (21-74) 37 (266.51) I21 (91-151) NT
5 x IO”
I x 10”
19 (3-36) 61 (21-86) 76 (63-88) NT ! 16 (X3- 153)
86 (74-98) 86 (52- 147) 96 (91-100) NT NT
” The data are expressed as percentage of control, calculated from the peak anti-SRBC PFCiculture values determined for each group in two to four experiments. ‘The control responses of I x IO’ adult NZB and C57BLi6 spleen cells were 383 i 56 and 4311 i 919 PFCiculture. respectively. ’ Number of donor spleen cells added to adult cell cultures. ” Not tested.
422
SHORTCOMMUNICATIONS
When spleen cells obtained from 14-day-old NZB mice were added to adult cell cultures, the number of the young spleen cells required for optimal suppression appeared altered. The addition of either 1 or 5 X lo6 cells from the 14-day-old mice resulted in suppression but the more potent effect was consistently noted with the addition of 1 x lo6 young cells (Table 1). Spleen cells from 21-day-old NZB and lCday-old C57BL/6 mice no longer had the capacity to suppress the antibody responses of the syngeneic adult spleen cells. DISCUSSION These data demonstrate that young NZB mice have splenic suppressor cells which inhibit the in vitro antibody response of adult cells, and that in this study this suppressor cell population was as active as that of C57BL/6 mice (Fig. 1). Furthermore this suppressor cell activity was still strong in NZB spleen cell populations at 7 days of age, a time when it was inconsistently detected or present at a low level in C57BL/6 spleen cell populations (Table 1). Little or no suppressor cell activity was detected in the 14-day-old C57BL/6 spleen cell population while consistent suppression was observed with lCday-old NZB spleen cells (Table 1). It is unclear why at this age the degree of suppression was increased with a decrease in the number of NZB cells, however, similar observations have been made in other suppressor cell systems (10, 11). It has been postulated that this results from conflicting effects of helper and suppressor cells with the activity of the former being diluted out more rapidly. It is possible that a similar situation develops in the maturing mouse spleen. In any event, it is clear that the suppression of the adult responses was caused by some activity of the young cells and not by cell crowding in the cultures, since suppression could be obtained with the addition of very few young cells (Fig. 1) and additional adult cells in the cultures, even at the higher cell numbers, did not result in suppression (Table 1). Defects in immune regulation have been described in adult NZB mice in several different systems (2). There is evidence suggesting both that adult NZB mice are lacking active suppressor cells (12) and that these mice have suppressor cells but are insensitive to their suppressive signals (13, 14). The experiments presented here suggest that the suppressor cells observed in the first weeks of life in several normal strains of mice are also present in NZB mice of this age and that adult spleen cells are sensitive to their suppressive activity. The thymocytotoxic antibody which has been shown to be toxic to suppressor cells (15) is either not present in 7- to 14-day-old mice or has no in viva effect on the activity of the young suppressor cells demonstrated in this system. NZB mice throughout life seem to have a lower percentage of Lyt 1,2,3+ cells in the spleen than other strains of mice and as adults have a deficiency in at least one suppressor cell function attributed to Lyt 1,2,3+ lymphocytes (16). The experiments of Mosier et al. (17) have implicated thymocytes bearing the Lyt 1,2,3 markers as the active suppressor cell in young mice. Our present finding that spleen cells from young NZB mice are no less active in suppression than those from a “normal” strain of mice may indicate that the young suppressor cell is only a subpopulation of Lyt 1,2,3+ lymphocytes or that the splenic suppressor cells bear different surface markers from those in the young thymus. While young NZB spleen cells function well in suppressing adult antibody
SHORT COMMUNICATIONS
423
responses in vitro, the possibility exists that the suppressor cells are blocked in their in biro activity in NZB mice or that lymphoid cells of young NZB mice, unlike those of the adults tested, are resistant to the effects of the suppressor cells. Either condition could lead to the abrogation of any normal in rir-o function these cells might have while leaving them functional in the in ~zitro system used here. The data presented here do indicate, however, that in young NZB mice there is not a defect inherent in the splenic suppressor cell itself and that the defects which have been described in in vitro suppressor cell activities in these mice (2) arise later than 14 days after birth or in a different population of suppressor cells than that shown to be active in newborn and young mice (7). REFERENCES 1. 2. 3. 4. 5. 6. 7. X. 9. IO. I I. I?. 13. 14. IS. 16. 17.
Weigle. W. 0.. Sieckmann. D. G.. Doyle, M. V.. and Chiller. J. M.. P’rtr,r\p/trr~r. Kc,\,. 26, 186. 1975. Talal. N.. Trcrnsplont. Ret,. 31, 240. 1976. Shirai. T.. and Mellors, R. C.. Proc,. Xf~f. Ac,tril. SC.;. (/.~A 68, 1412. 1971. Playfair. J. H. L.. Immrrnolop~ 15, 35. 1968. Moutsopoulos. H. M.. Boehm-Truitt. M.. Kansan. S. S.. and Chused. T. M.. J. Itr~munol. 119. 1639. 1977. Staples. P. J.. and Talal, N., S~.ie!z(~’ 163. 215. 1969. Mosier. D. E.. and Johnson, B. M.. J. ttp. .Mctl. 141, 216. 1975. Mishell. R. I., and Dutton. R. W.. ./. b:.t/). .Metl. 126, 475. 1967. McCombs, C., Horn, J.. Talal. N.. and Mishell, R. I.. ./. I,~zmrtn~~/. 112, 376. 1974. Haskill. J. S.. and Axelrad. M. A., :Vnl/rrc .Vcrr, Rir~i. 237. 251. 1972. Durkin, H. G., Bash, J. A.. and Waksman. B. H.. Proc .‘Vtrt. i\(.(lcl. Sr,i. (/.~A 72, 5090, 1975. Krakauer, R. S.. Waldmann. T. A.. and Strober, W., J. &-up. .Mcd. 144. 662, 1976. Klassen, L. W., Zarate. A.. Gelfand. M. C.. and Steinberg. A. D.. J. /r,~,rlrr,rr,/. 119, 2067. 1977. Dauphinee. M. J.. and Talal. N.. ./. frnrtrr~rzoi. 122, 936, 1979. Shirai, T., Hayakawa. K.. Okumura. K., and Tada. T.. ./. /nrmr,n~,/. 120, 1924. 197X. Cantor, H.. McVay-Boudreau, L.., Hugenberger. J.. Naidorf, K.. Shen. F. W.. and Gershon. R. K., J. hp. ‘Ifcd. 147, I 116, 197X. Mosier, D. E., Mathieson. B. J.. and Campbell. P. S.. ./. E.ry~. Me
OGY 51, 419-423
Demonstration
(1980)
of Active Suppressor Cells in Spleens Young NZB Mice’
of
D. A. LERMAN .&ND C. E. CALKINS
NZB mice. a strain prone to the development ofautoimmune disease, have during the first 2 weeks of life suppressor cells in their spleens which can in coculture with adult spleen cells \uppres\ the antibody response to sheep red blood cells (SRBC) generated in culture by the adult cells. The suppressive activity of spleen cells from NZB mice in the first week after birth i5 similar to that of spleen cells from 4-day-old C57BL/6 mice. a strain which does not strains of mice, suppressor cell spontaneously develop autoimmune disease. As in “normal“ activity in NZB mice is diminished at 2 weeks and undetectable at 3 weeks of age. The data indicate that there is no defect inherent In the suppressor cells detected in the spleens of newhorn and young NZB mice and suggest that the development of autoimmune responses doe\ not result from a lack of suppressor cells in the young animals.
INTRODUCTION Suppressor cells have been demonstrated in states of tolerance to a number of non-self-antigens and may play a role in the induction and/or maintenance of tolerance (1). The possibility that suppressor cells may also be important in selftolerance has led to a consideration of suppressor cell activity in autoimmune responses, Several suppressor cell deficiencies have been demonstrated in adult NZB mice (3). a strain of mice in which antibodies to autologous thymocytes INTAY have been demonstrated as early as I month of age (3) and to autologous erythrocytes somewhat later (2). There is considerable evidence that immunoregulatory deficiencies occur early in life in these mice. At birth NZB mice, unlike other strains of mice. respond to SRBC i/l \si\.o (4) and their lymphocytes produce high levels of IgM i/l I‘iftw (5). In addition, weanling NZB mice cannot sustain experimentally induced tolerance for the long periods of time characteristic of tolerance induced in mice of other strains (6). If a defect in suppressor cell activity is responsible for the abnormal responsiveness of young NZB mice and for the development of autoimmune responses, then this defect should be detected in NZB mice earlier than 1 month of age. the time of onset of the first detectable autoimmune response. Other strains of i Supported In part by Grant IM-139 from the American Cancer National Institutes of Health, and by a Grant from the Pew Pennsylvania Chapter of the Arthriti\ Foundation. ” .4bhreviation\ used: NT. not tested: NTA. naturally occurring plaque-forming cell\: SRBC. sheep red blood cell\.
Society. by Grant AI-l.5789 from Foundation through the Eastern thymocytotoxlc
antihody:
PFC,
4 I9 0008.8749/80/060419-05$02.00/O Copyright All rights
(. 19X0 by Academic of reproduction in
Press.
any form
Inc. rexwed.
420
SHORT COMMUNICATIONS
NEWBORN
CELLS
1x706)
FIG. 1. Ability of young spleen cells to suppress adult anti-SRBC antibody responses in rzitro. 0.5 x lo6 to 5.0 x lo6 spleen cells from 7-day-old NZB (0 -.- 0) or 4-day-old C57BLl6 (0 0) mice were added with SRBC to cultures of 1 x lo7 syngeneic adult spleen cells. PFC responses of cultures of adult cells which received syngeneic young cells were compared with those of control cultures which received no young cells. Mean control responses were 311 5 69 and 4850 t 538 PFC/culture for NZB and C57BL/6 cell cultures, respectively. Brackets represent the range of values in two to four experiments.
mice have active suppressor cells present in their spleens in the first few weeks of life which act in vitro to prevent the cells of these mice from responding to Tdependent antigens (7). Since these suppressor cells may function to suppress antiself-responses in young mice, it is important to compare suppressor cell activity in the spleen cell populations of young NZB mice and of a strain which is not prone to autoimmune disease. MATERIALS
AND METHODS
Animals. Adult NZB and C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, Maine). Additional NZB mice were generously donated by Dr. J. Bruce Smith (Institute for Cancer Research, Phila., Pa.). Newborn NZB and C57BL/6J mice were bred in the animal colony at Thomas Jefferson University. In each experiment newborn and adult mice were matched according to age, sex, and source of origin. Adult mice were used at 8- 16 weeks of age and young mice from 4 to 21 * 1 days of age. Cell culture and assay. Adult spleen cells were cultured according to the method of Mishell and Dutton (8). Varying numbers of newborn spleen cells were added to duplicate adult spleen cell cultures containing lo7 adult cells and 3 x lo6 sheep erythrocytes (SRBC, obtained from a single sheep by Colorado Serum Co.,
421
SHORT COMMUNICATIONS
Denver, Cola.). Direct IgM plaque-forming cells (PFC) to SRBC were enumerated 4 and 5 days after the initiation of culture using a slide modification of the Jerne plaque assay (8). The peak response which occurred on Day 4 or on Day 5 is reported. NZB mice are low responders to SRBC in \litr’o (9). When the peak response was less than 150 PFCiculture (in approximately 20%, of the experiments), the number of plaques counted in unsuppressed control cultures were too low to accurately evaluate the degree of suppression in cultures to which suppressor cells had been added. Therefore, only those experiments where control responses were greater than 150 PFCiculture are included in the present communication. The data are expressed as percentage of control, with the control response being the number of PFC per culture in cultures of 1 x IO’ syngeneic adult spleen cells + SRBC. RESULTS To determine the presence and activity of suppressor cells in spleens of young NZB and C57BLi6 mice, varying numbers of spleen cells from mice of each strain aged 4-21 days were cultured with 1 x 10’ adult spleen cells of the same strain in the presence of SRBC. Suppressor cells were detectable in the spleens of both strains in the first week after birth (Fig. I). The degree of suppression of the adult response was dependent upon the number of young spleen cells added. As few as 0.5 x 10” NZB spleen cells obtained from 7-day-old mice reduced the adult PFC response to 60% of the control response; 5.0 x 10” young spleen cells reduced the adult response to 15% of control. At 4 days of age, C57BLi6 mice had suppressor cells in their spleens capable of suppressing the homologous adult response to 19% of control when 5.0 x IO” young cells were added to the adult cultures. The addition of fewer young C57BLi6 cells produced no apparent suppression. By 7 days of age, the suppressive capacity of the C57BLi6 spleen cells was less strong than either that of younger C57BLi6 cells or that of 7-day-old NZB cells (Table I). TABLE
1
Suppression of Antibody Responses in Cultures of Adult NZB or C57BL;6 Spleen Cells by Addition of Spleen Cells from Syngeneic Mice of Varying Ages” Percentage of control Age of suppressor cell donors Days 4 7 I4 21 Adult
PFC response (range)”
NZB ( x IO”’ NT” I6 (O-29) 64 (44- 103) 108 (98- 118) I48 (77-242)
C57BL;6 I x IO” NT 54 (21-74) 37 (266.51) I21 (91-151) NT
5 x IO”
I x 10”
19 (3-36) 61 (21-86) 76 (63-88) NT ! 16 (X3- 153)
86 (74-98) 86 (52- 147) 96 (91-100) NT NT
” The data are expressed as percentage of control, calculated from the peak anti-SRBC PFCiculture values determined for each group in two to four experiments. ‘The control responses of I x IO’ adult NZB and C57BLi6 spleen cells were 383 i 56 and 4311 i 919 PFCiculture. respectively. ’ Number of donor spleen cells added to adult cell cultures. ” Not tested.
422
SHORTCOMMUNICATIONS
When spleen cells obtained from 14-day-old NZB mice were added to adult cell cultures, the number of the young spleen cells required for optimal suppression appeared altered. The addition of either 1 or 5 X lo6 cells from the 14-day-old mice resulted in suppression but the more potent effect was consistently noted with the addition of 1 x lo6 young cells (Table 1). Spleen cells from 21-day-old NZB and lCday-old C57BL/6 mice no longer had the capacity to suppress the antibody responses of the syngeneic adult spleen cells. DISCUSSION These data demonstrate that young NZB mice have splenic suppressor cells which inhibit the in vitro antibody response of adult cells, and that in this study this suppressor cell population was as active as that of C57BL/6 mice (Fig. 1). Furthermore this suppressor cell activity was still strong in NZB spleen cell populations at 7 days of age, a time when it was inconsistently detected or present at a low level in C57BL/6 spleen cell populations (Table 1). Little or no suppressor cell activity was detected in the 14-day-old C57BL/6 spleen cell population while consistent suppression was observed with lCday-old NZB spleen cells (Table 1). It is unclear why at this age the degree of suppression was increased with a decrease in the number of NZB cells, however, similar observations have been made in other suppressor cell systems (10, 11). It has been postulated that this results from conflicting effects of helper and suppressor cells with the activity of the former being diluted out more rapidly. It is possible that a similar situation develops in the maturing mouse spleen. In any event, it is clear that the suppression of the adult responses was caused by some activity of the young cells and not by cell crowding in the cultures, since suppression could be obtained with the addition of very few young cells (Fig. 1) and additional adult cells in the cultures, even at the higher cell numbers, did not result in suppression (Table 1). Defects in immune regulation have been described in adult NZB mice in several different systems (2). There is evidence suggesting both that adult NZB mice are lacking active suppressor cells (12) and that these mice have suppressor cells but are insensitive to their suppressive signals (13, 14). The experiments presented here suggest that the suppressor cells observed in the first weeks of life in several normal strains of mice are also present in NZB mice of this age and that adult spleen cells are sensitive to their suppressive activity. The thymocytotoxic antibody which has been shown to be toxic to suppressor cells (15) is either not present in 7- to 14-day-old mice or has no in viva effect on the activity of the young suppressor cells demonstrated in this system. NZB mice throughout life seem to have a lower percentage of Lyt 1,2,3+ cells in the spleen than other strains of mice and as adults have a deficiency in at least one suppressor cell function attributed to Lyt 1,2,3+ lymphocytes (16). The experiments of Mosier et al. (17) have implicated thymocytes bearing the Lyt 1,2,3 markers as the active suppressor cell in young mice. Our present finding that spleen cells from young NZB mice are no less active in suppression than those from a “normal” strain of mice may indicate that the young suppressor cell is only a subpopulation of Lyt 1,2,3+ lymphocytes or that the splenic suppressor cells bear different surface markers from those in the young thymus. While young NZB spleen cells function well in suppressing adult antibody
SHORT COMMUNICATIONS
423
responses in vitro, the possibility exists that the suppressor cells are blocked in their in biro activity in NZB mice or that lymphoid cells of young NZB mice, unlike those of the adults tested, are resistant to the effects of the suppressor cells. Either condition could lead to the abrogation of any normal in rir-o function these cells might have while leaving them functional in the in ~zitro system used here. The data presented here do indicate, however, that in young NZB mice there is not a defect inherent in the splenic suppressor cell itself and that the defects which have been described in in vitro suppressor cell activities in these mice (2) arise later than 14 days after birth or in a different population of suppressor cells than that shown to be active in newborn and young mice (7). REFERENCES 1. 2. 3. 4. 5. 6. 7. X. 9. IO. I I. I?. 13. 14. IS. 16. 17.
Weigle. W. 0.. Sieckmann. D. G.. Doyle, M. V.. and Chiller. J. M.. P’rtr,r\p/trr~r. Kc,\,. 26, 186. 1975. Talal. N.. Trcrnsplont. Ret,. 31, 240. 1976. Shirai. T.. and Mellors, R. C.. Proc,. Xf~f. Ac,tril. SC.;. (/.~A 68, 1412. 1971. Playfair. J. H. L.. Immrrnolop~ 15, 35. 1968. Moutsopoulos. H. M.. Boehm-Truitt. M.. Kansan. S. S.. and Chused. T. M.. J. Itr~munol. 119. 1639. 1977. Staples. P. J.. and Talal, N., S~.ie!z(~’ 163. 215. 1969. Mosier. D. E.. and Johnson, B. M.. J. ttp. .Mctl. 141, 216. 1975. Mishell. R. I., and Dutton. R. W.. ./. b:.t/). .Metl. 126, 475. 1967. McCombs, C., Horn, J.. Talal. N.. and Mishell, R. I.. ./. I,~zmrtn~~/. 112, 376. 1974. Haskill. J. S.. and Axelrad. M. A., :Vnl/rrc .Vcrr, Rir~i. 237. 251. 1972. Durkin, H. G., Bash, J. A.. and Waksman. B. H.. Proc .‘Vtrt. i\(.(lcl. Sr,i. (/.~A 72, 5090, 1975. Krakauer, R. S.. Waldmann. T. A.. and Strober, W., J. &-up. .Mcd. 144. 662, 1976. Klassen, L. W., Zarate. A.. Gelfand. M. C.. and Steinberg. A. D.. J. /r,~,rlrr,rr,/. 119, 2067. 1977. Dauphinee. M. J.. and Talal. N.. ./. frnrtrr~rzoi. 122, 936, 1979. Shirai, T., Hayakawa. K.. Okumura. K., and Tada. T.. ./. /nrmr,n~,/. 120, 1924. 197X. Cantor, H.. McVay-Boudreau, L.., Hugenberger. J.. Naidorf, K.. Shen. F. W.. and Gershon. R. K., J. hp. ‘Ifcd. 147, I 116, 197X. Mosier, D. E., Mathieson. B. J.. and Campbell. P. S.. ./. E.ry~. Me