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lpr mice: Evidence for decreased helper-T-cell and increased suppressor-T-cell function with age
CELLULAR
IMMUNOLOGY
74, 72-85 (1982)
lmmunoregulation in MRL/Mp-lpr/lpr Mice: Evidence for Decreased Helper-T-Cell and Increased Suppressor-T-Cell Function with Age’ DEBORAHA. WILSON*,*
AND
HELEN BRALEY-MULLEN*T~-,~
*Division of Immunology-Rheumatology, Department of Medicine, and TDepartment of Microbiology. University of Missouri Health Sciences Center, Columbia, Missouri 65212 Received May 24, 1982; accepted August 30, 1982 In vivo and in vitro plaque-forming cell (PFC) responsivenessto sheep erythrocytes (SRBC) was used to assess immunoregulatory function in the autoimmune MRL mouse strain. MRL/Mp-lpr/lpr (MRL/l) mice had good primary and secondary IgM and IgG responses in vivo compared to MRL/Mg+/+ (MRL/n) mice when young, but with age the MRL/l responses declined markedly. In vitro primary SRBC-specific PFC responsesin MRL/l mice declined at the same time as in vivo responses,indicating that the in vivo autoimmune environment could not account for cellular dysfunction. When varied mixtures of T and B cells from MRL/l and MRL/n mice were cultured, abnormalities in MRL/l T-cell function became apparent. Thelper-cell (Tn) function declined rapidly with age, beginning by 2 to 2% months of age. T cells from MRL/l mice 2 months of age and older also had increased suppressor activity when cultured with B cells and MRL/n T.cells. The degree of suppressor activity increased with age. The correlation of these findings with results of previous studies by others and with autoimmune disease is discussed.
INTRODUCTION MRL/Mplpr/lpr (MRL/1)4 mice spontaneously develop autoimmune disease characterized by high levels of serum immunoglobulin and immune complexes ( 1) and numerous autoantibodies (l-3). They die at an early age with severe glomerulonephritis and arteritis (1). The lpr gene, which causes marked proliferation of Lyt “null” (4) or Lyt-l f (5) T cells, appears to be responsible for acceleration and severity of autoimmune disease because the MRL/Mp-+/+ (MRL/n) strain which is congenic except for the lpr gene develops autoimmune disease more slowly and dies at a much later age (1). Numerous studies have been done in an attempt to determine the immunoregulatory abnormality responsible for autoimmune disease in MRL mice. Thus MRL/l mice have been reported to have both normal (6) and hyperactive (7) B-cell function ’ Supported by NIH Grants CA 25054 and AM 20305. 2 Recipient of an Arthritis Foundation Post-Doctoral Research Fellowship. To whom all correspondence should be addressed. 3 Recipient of NIH Research Career Development Award AI 100322. 4 Abbreviations used: MRL/l, MRL/Mp-lpr/lpr; MRL/n, MRL/Mp+/+; Ts, suppressor T cell; Tn, helper T cell; IL-2, interleukin 2; TD, thymus dependent; SRBC, sheep erythrocytes; PFC, plaque-forming cell; Ig, immunoglobulin. 72 0008-87491821I70072-14$02.00/O Copyright 0 1982 by Academic Press, Inc. All righti of mpmductiw in any form reserved.
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as well as an age-related decreasedresponsivenessto a B-cell mitogen (8). SuppressorT-cell (T,) function has been found to be either increased (9) or normal (6, 8) and the ability to develop tolerance to exogenous (6, 10) or modified self ( 11) antigens has been reported to be normal (6, 11) and abnormal (10). Becausethe proliferating T cells are probably Lyt- 1+ (5), studies of helper-T-cell (Tu) function (5) and feedback suppressor inducer function (5, 12) may be very relevant to determining the immunoregulatory abnormalities in MRL/l mice. Indeed, both normal (6) and increased (8, 9, 13) Tu function have been reported in different studies. Decreased responsivenesspresumably of Lyt- 1+ T cells to feedback suppressor signals has been suggested(14), and the syngeneic mixed lymphocyte response which requires Lyt1’ T cell function is impaired in MRL/l mice (15). Recent studies have shown that MRL/l mice become unable to produce and to respond to interleukin-2 (IL-2) formerly called T-cell growth factor ( 16- 18). These results are consistent with our findings of decreased TH function in MRL/l mice with age. We have also found an age-related decrease in B-cell (or macrophage) function and an increase in suppressor cell activity with age. The system we have used to study T- and B-cell function is that of in vivo and in vitro plaque-forming cell responses to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC) (19). Because MRL/l and MRL/n mice are congenic except for the lpr gene, in vitro cell mixing experiments provide a means of determining which populations of cells function abnormally in MRL/l mice. The correlation of these cellular dysfunctions with autoimmunity is discussed. MATERIALS
AND METHODS
Mice. MRL/Mp-lpr/lpr (MRL/l) and MRL/Mp-+/+ (MRL/n) mice were obtained from The Jackson Laboratory, Bar Harbor, Maine and subsequently maintained by brother-sister matings in our colony. New breeding pairs of both strains were obtained annually to avoid genetic drift. The immunopathologic characteristics and mortality of MRL mice have been described elsewhere (l-4). The MRL/l mice used in the following studies ranged in age from 1 to 7 months and the MRL/n mice from 1 to 10 months. All mice were in good condition when used aside from the expected autoimmune disease manifestations. Both male and female mice were used in these experiments since preliminary studies showed that the results were essentially the same for both sexesof both substrains except that the abnormalities occurred slightly earlier in MRL/l females than in MRL/l males. Antigens and immunizations. SRBC was used as the antigen for all experiments. In vivo primary IgM and IgG SRBC responses were determined following an ip injection of 2 X lo* SRBC. Secondary IgM and IgG in vivo responses were elicited with a primary injection of 2 X lo8 SRBC ip at 1, 2, or 3 months and a secondary challenge at 4 months of age. In vitro spleen cell cultures were immunized with 1 X lo6 SRBC per culture. In most in vitro experiments the mice were given 2 X lo5 SRBC iv 4 days prior to culture in order to carrier-prime T cells (20) and thus increase the magnitude of the SRBC-specific plaque-forming cell (PFC) responses. Spleen cell cultures. Spleen cell suspensions were prepared and cultured according to the method of Mishell and Dutton (21). RPM1 1640 culture medium (KC. Biological, Inc., Lenexa, Kan.) was supplemented with 10% heat-inactivated fetal calf serum, nonessential amino acids, L-glutamine, sodium pyruvate, and vitamins
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(all from Grand Island Biological Co., Grand Island, N.Y.) and with 5 X lop5 M 2-mercaptoethanol. Spleen cells, l-2 X lo7 were cultured at 37°C in 35 X lo-mm culture dishes (Costar 3035) which were gently rocked; cultures were fed nutritional cocktail daily. Preparation of T- and B-cell-enriched populations. B-cell-enriched spleen cells were obtained by treatment with anti-Thy- 1.2 (AKR anti-C3H antiserum and complement (22)). This antiserum has been shown to be effective in eliminating T cells but to have no effect on B cells (22). Forty to fifty percent of the original spleen cells were eliminated by this technique. In most experiments T cells were obtained by passing spleen cells through nylon wool according to the method of Julius et al. (23). This method yielded approximately 30% of the original spleen cells from MRL/n mice and 30-40s of original MRL/l spleen cells, with the highest yields obtained from old MRL/l mice where massive splenomegaly was present. Functional purity of the B- and T-cell-enriched populations was assessedby PFC responses to SRBC and in some experiments by proliferative responses to concanavalin A, phytohemagglutinin, and lipopolysaccharide. In some experiments nylon wool-passed spleen cells were treated with anti-Thy-l.2 and complement, anti-la serum (A.Th anti-A.TL, which has been shown previously in our laboratory to kill B cells and macrophages but not T cells and to lack I-J specificity (unpublished results)) and complement, or 1500 rad. The 6oCo facility at the Dalton Research Center, University of Missouri, was used to irradiate the cells. Mixed lymphocyte reaction (MLR). In order to ensure that alloreactivity could not account for the responsiveness of MRL/l and MRL/n spleen cells when mixed in culture, two-way MLR studies were performed using young and old MRL/l, young and old MRL/n, C3H and C57B1/6 mice in all possible combinations. No alloreactivity between young and old MRL/l or MRL/n mice was detected (not shown). RESULTS In vivo primary and secondary SRBC responsesin MRL mice. In order to indirectly assessT- and B-cell function in MRL mice in vivo MRL/l and MRl/n female mice of varied ages were immunized with SRBC. The PFC responses 4 and 5 days after immunization are shown in Fig. 1. On Day 4, young MRL/I mice had predominantly IgM PFC responseswhich were lower than those of MRL/n mice at all agestested. The IgM PFC response fell markedly in MRL/l mice by 4 months of age. The Day 4 IgG PFC response was low in all groups except for 7-week-old MRL/n mice. The IgM PFC responsesin all groups varied little from Day 4 to Day 5. The IgG PFC responsesincreased markedly in young MRL/l mice and in 7-week-old and 4-monthold MRL/n mice between Days 4 and 5. Four-month-old MRL/l and ten-monthold MRL/n mice had essentially no IgG PFC on either day. Other in vivo studies not shown here indicated that the IgM PFC responsein MRL/l mice usually declined between 2 and 3 months of age and the age at which the MRL/n response fell was variable but generally began to occur by 10 months of age or later. Secondary IgM and IgG SRBC responses in MRL mice were similar to primary responses in that I- to 2-month-old MRL/l and all MRL/n mice tested up to age 8 months responded well whereas MRL/l mice responded poorly at 3-4 months (not shown). Actually, as shown in Table 1, it is the age at which MRL/l mice are
IMMUNE
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7wk 4mo ML/I a IgM
7wk
4mo 1Omo ML/n
75
IN MRL MICE
7wk 4mo MRLA
7wk
4mo 1Omo MRL/n
0 IgG
FIG. 1. Primary IgM and IgG SRBC responses in MRL mice of varied ages in vivo. Female MRL/l and MRL/n mice were given 2 X lo* SRBC ip and PFC responseswere determined 4 and 5 days later. Results shown are mean IgM and IgG PFC/spleen + SEM (four mice/group).
primed rather than the age at which they are given a secondary challenge (and thus the age at which PFC responsesare determined) which determines whether they are capable of producing secondary IgM and IgG responses. In this experiment secondary IgM and IgG responses in MRL/l and MRL/n mice were determined at 4 months of age, 5 days after the secondary challenge. The mice were primed at 1, 2, or 3 months of age or not at all. The MRL/l mice which were primed at 1 or 2 months of age had considerably higher responsesthan those which were primed at 3 months or which received only a primary injection 5 days prior to PFC deterTABLE 1 Secondary in vivo SRBC Responses in MRL Mice: Importance of Age of Priming Secondary SRBC PFC/spleenb Age of priming” (months)
Mice MRL/l
kM
w
P
1 2 3 None
25,267 + 33,975 -t 8,667 f 7,200 +
10,992 12,243 4,758 3,354
112,733 -c 53,769 279,167 2 88,704 14,500 rt 14,500 840 + 705
MRL/n 0
1 2 3 None
6,080 + 5,960 f 5,260 + 34,300 f
2,187 1,087 1,818 10,388
47,020 k 7,695 43,740 ?I 9,2 13 60,140 + 10,310 65,000 f 23,43 1
p All mice were given 2 X lo8 SRBC ip as primary injection. Secondary challenge (2 X 108SRBC ip) was given to all mice at 4 months of age. PFC were determined 5 days later. ’ Mean PFC/spleen f SEM, 3-5 mice/group.
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mination. The responsesof MRL/l mice primed at 2 months were also much greater than those of MRL/n mice. Each group of MRL/n mice responded similarly regardless of the age at which they were primed and the MRL/n mice which received only one injection of SRBC had high IgM and IgG primary responses. Thus the ability of MRL/l mice to be primed declines with age. However, if the mice are primed when young, a secondary response may be elicited at 4 months of age, even though MRL/l mice do not develop good primary responses at this age. Thus young MRL/l mice produced similar and in some experiments even greater primary and secondary SRBC PFC responses compared to MRL/n mice but with age their ability to respond to SRBC decreasedmarkedly. These results thus suggest that either (1) T and/or B cells or accessory cells in MRL/l mice become dysfunctional with age, (2) that abnormal proportions of splenic B and T cells may cause altered feedback and regulatory cell activity, or (3) that the autoimmune environment may prevent otherwise normal cells from functioning well. In vitro primary IgM SRBC responsesin MRL mice. In order to eliminate the autoimmune environment of MRL/l mice as a possible factor causing hyporesponsivenessto SRBC, spleen cells were cultured in vitro with SRBC. The primary IgM SRBC PFC responsesin MRL/l and MRL/n mice at varied agesdetermined on Day 4 of culture are shown in Fig. 2. Spleen cells from l- and 2-month-old MRL/l mice responded well to SRBC in vitro but by 4 months the responseshad declined markedly. By contrast, spleen cells from MRL/n mice responded well at all ages up to 10 months. The lack of responsivenesswas not due to a switch to IgG PFC because all of the MRL/l spleen cell cultures had less than 100 IgG PFC per culture on Day 4 or Day 5 of culture (not shown). Thus the decline in responsiveness to SRBC in MRL/l mice occurred between 2 and 4 months of age both in vivo (Fig. 1) and in vitro (Fig. 2) suggesting that the decreased responsiveness was not due to the autoimmune environment of MRL/I mice. Culture of fractionated spleen cellsfrom young MRL mice. In order to provide a means of comparing B- and T-cell function of these mice more directly and to lmo 2mo 4mo 6mo lmo 2mo 4mo 6mo
3000
5000
7000
IgM SRBC PFWculture FIG. 2. Primary IgM SRBC responsesin vitro in MRL mice. MRL/l and MRL/n male mice of indicated ageswere primed on Day -4 with 2 X 1O*SRBC iv. On Day 0 spleen cells ( 1 X 1O’/cuIture) were cultured with 1 x lo6 SRBC. PFC responseswere determined on Day 4. Results shown are the means of triplicate cultures + SEM.
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preclude the problem of the excessive T cell to B cell ratio in MRL/l mice, enriched populations of T cells and B cells were cultured in various combinations. The 9-week MRL/l spleen cell response was much lower than the 9-week MRL/ n spleen cell response (Fig. 3). When MRL/l B cells were cultured with MRL/l T cells, the response was slightly lower than the spleen cell response. However, when MRL/l B cells were cultured with MRL/n T cells the response was markedly increased, indicating that MRL/l B cells could function well when provided adequate T-cell help. MRL/n B cells cultured with their own T cells also had good responses, but the response of MRL/n B cells was much lower when MRL/l T cells were substituted. This experiment thus illustrates that shortly after 2 months of age decreased T-cell helper function is present in MRL/l as compared to MRL/n mice, whereas B cells and accessory cells of MRL/l mice appear to function normally. Similar experiments have been done using the panning technique for enrichment of T cells (24,25) and the results were the same, indicating that the defect in MRL/ 1T cells is not due to loss of a nylon wool-adherent TH cell population (26). Because of the facility of obtaining larger numbers of T cells by nylon wool passage,this method was used for all other experiments described below. T-Cell Function in old MRL/l mice. That T-cell dysfunction also contributes to the SRBC hyporesponsiveness of older MRL/l mice is shown in Fig. 4. Spleen cells from 2- and 4-month-old MRL/n mice produced approximately 6000 PFC/culture, but 4-month MRL/l spleen cells again produced few PFC. Whereas T cells from the older 4-month MRL/n mice helped 2-month MRL/n B cells to produce responses similar to those of unfractionated spleen cells, 4-month MRL/l T cells provided no helper activity for 2-month MRL/n B cells. Thus MRL/l mice continue to demonstrate decreased T-cell helper activity even though the spleens in these older mice are presumably overpopulated with Lyt 1 + T cells (5). B-Cell function in MRL mice. In order to determine whether B-cell and/or accessory cell function in MRL/l mice might also decline with age, B cells from MRL/ 1and MRL/n mice of various ageswere cultured with T cells from 9-week-old MRL/ ML/I
Spleen
MRL/n Spleen ML/I
B+T
IgM SRBC PFCXulture
FIG. 3. In vitro SRBC responsesin young MRL mice: B- and T-cell function. Nine week MRL/l and MRL/n male mice were primed with 2 X lo5 SRBC iv on Day -4. On Day 0, 1 X 10’ spleen cells, 1X 10’ B cells + 5 X lo6 T cells, or 1 X 10’ B cells as indicated above were cultured with 1 X IO6SRBC per culture. PFC responses were determined on Day 4. Results indicate means of two to three pooled duplicate cultures 2 SEM. MRL/l B cells and MRL/n T cells alone had 0 PFC/culture; MRL/l T cells alone had 10 PFC/culture.
78
WILSON AND BRALEY-MULLEN MRLhI~een MRL/nq$tleen MRL/l4s&8en 2mo 4mo 2mo 4mo
MRL/n MRL/n MRL/n MRLA
Bt 1 Bt 1
2mo MRL/n B
1000 2000
4000
7000
IgM SRBC PFCXulture FIG. 4. In vitro SRBC responses in MRL mice: T-cell function. Two- and four-month-old MRL/n male and 4-month-old MRL/l male mice were primed on Day -4 with 2 X 10’ SRBC iv. On Day 0, 1 X 10’ spleen cells or 1 X lo7 B cells plus 5 X lo6 T cells from indicated mice were cultured with SRBC. Results shown are mean PFC -+ SEM of two pooled duplicate cultures. MRL/n T cells alone had 30 PFC/culture, MRL/l T cells alone had 2 PFC/culture.
n mice (Fig. 5). B-cell and/or macrophage function in MRL/l mice increased between 4 and 7 weeks and the responses at 7 and 10 weeks were very high. MRL/I B cell function declined markedly at 20 weeks compared with IO-week B cells although the responses were similar to 4-week MRL/l mice and to MRL/n mice at all ages. MRL/n mice maintained similar B-cell SRBC responsiveness from 9 to 40 weeks of age. The markedly disproportionate response of MRL/l spleen cells versus that of MRLA
MRL/n
30 20 15 IgM SRBC PFCxlO3 Culture ,.
5
5wk 0
Spleen cells
a
B cellstgwk
9wk
24wk
40wk
MRL/n 1 cells
Frc. 5. In vitro SRBC responsesin MRL mice: B-cell function. MRL/I and MRL/n female mice of indicated ages were primed on Day -4 with 2 X lo5 SRBC iv. On Day 0, I X 10’ of their B cells plus 5 X lo6 T cells from 9-week-old MRL/n mice were cultured with SRBC. Results shown are mean PFC t SEM of duplicate (spleen cell) or triplicate (B + T cell) cultures. Results of B-cell cultures were as follows (PFC ? SEM/culture): 4-week-old MRL/I B, 115 + 75; 7-week-old B, 155 + 115; IO-week-old MRL/l B, 45 f 25; 20-week-old MRL/l B, 5 + 5; 5-week-old MRL/n B, 165 & 55; 9-week-old MRL/n B, 415 ? 145; ZCweek-old MRL/n B, 150 f 0; 40-week-old MRL/n B, 80 + 60. 9-week-old MRL/n T cells had 180 + 20 PFC/culture.
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1000
3000 IgM SRBC
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5000
7000
9000
PFCXulture
FIG. 6. Suppression of MRL SRBC responsesby MRL/l T cells. Two-month-old MRL/l and MRL/ n female mice were primed on Day -4 with 2 X 10’ SRBC iv. On Day 0, I X 10’ spleen ceils, 1 X 10’ B cells + 5 X lo6 T cells, or 1 X IO’ B cells plus 5 X lo6 of each type of T cells were cultured with SRBC. Results shown are means of two to six cultures f SEM. MRL/n B cells plus 1 X 10’ MRL/n T cells had 2650 PFC/culture; MRL/I B cells plus 1 X 10’ MRL/I T cells had 620 PFC/culture. MRL/I B cells had 57 f 3, MRL/n B cells had 213 + 12, MRL/I T cells had 0, and MRL/n T cells had 145 PFC/culture.
MRL/l B cells mixed with MRL/n T cells was most apparent at 10 weeks, indicating that the decreased spleen cell responsesof MRL/l mice at 10 weeks was largely due to T-cell dysfunction. The results of this experiment also suggest that young MRL/l B cells and/or accessory cells may be hyperactive compared to MRL/n B cells although this was not evident in all experiments (e.g., Fig. 3). Decreased helper versus increased suppressor-T-cell function in MRL/l mice. The above studies indicated that MRL/l mice have decreased helper T-cell function by 2’12months of age. Because studies by others have shown evidence for increased suppression in MRL/l mice (9), we asked whether MRL/l T cells might suppress a SRBC response which was helped by MRL/n T cells (Fig. 6). MRL/l B + T cells from 2-month-old female mice produced approximately 1000 PFC. The addition of MRL/n T cells to MRL/I B cells again resulted in a marked increase in PFC (ca. 10,000 PFC). However, if MRL/l B cells were cultured with equal numbers of T cells from both strains, the response was suppressedby approximately 50% (Fig. 6, line 3). Similar results occurred with B cells from MRL/n mice. Addition of MRL/ 1 T cells to the MRL/n B + T cell combination suppressed the response by approximately 70% (line 4 vs line 6). To determine whether these results were due to the in vivo priming with a low dose of SRBC, the experiment shown in Fig. 7 was done using spleen cells from unprimed mice. All of the PFC responses were considerably lower but the overall results were the same: that is, MRL/l T cells suppressed the responses of MRL/l B- + MRL/n T-cell and of MRL/n B- + T-cell combinations. Cell crowding was not responsible for the suppressive effect since both MRL/l and MRL/n spleen cell responsesincreased in proportion to the number of cells per culture. Moreover in both of the above experiments, MRL/n B cells were also cultured with 1 X 10’ MRL/n T cells. The responses were greater than those with 5 X lo6 T cells (Figs. 6 and 7) which also indicates that the suppressive effect was not due to the presence of increased numbers of T cells in the cultures containing both MRL/n and MRL/l T cells. The approximate age at which this suppressor effect appears in MRL/l mice is shown in Table 2. T cells from l-month-old MRL/l mice were able to help B cells from l-month-old MRL/l mice as well as T cells from the 2-month-old MRL/n mice but T cells from 2- and 3-month-old MRL/l mice provided essentially no help.
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WILSON AND BRALEY-MULLEN
MRLA B+T 2 MRLA BtMRLh T . MRLA BtTtMRLh T 1
w I *
MRL/n BtT MRL/n BtMRLA T .% MRL/n BtTtMRLA T B 6
200
50 100
300
400
600
IgM SRBC PFCXulture FIG. 7. Suppression of SRBC responsesin MRL mice by MRL/l T cells. Spleen cells, B cells, and T cells from 2-month-old MRL/I and MRL/n male mice were cultured on Day 0 with SRBC as in Fig. 6. PFC responses on Day 4 are indicated as mean PFC + SEM of two to six cultures. MRL/n B cells plus 1 X IO’ MRL/n T cells had 310 PFC/culture and MRL/l B plus 1 X 10’ MRL/I T cells had 25 PFC/culture. PFC results of enriched cell populations cultured alone were as follows: MRL/I B, 8 k 8 PFC/culture; MRL/n B, 50 PFC/culture; MRL/l T, 10 PFC culture; MRL/n T, 5 PFC/culture.
When MRL/ 1 T cells were added to the MRL/ 1 B + MRL/n T cell combination, l-month MRL/l T cells did not suppress the PFC response whereas T cells from 2-month MRL/l mice suppressed the response by approximately one-third, and 3month MRL/l T cells by greater than 50%. These results suggest that the loss of helper activity of MRL/l T cells occurs concommitantly with the appearance of Tcell suppressor activity and that both changes become more pronounced with age. They also show no evidence of increased TH activity in young or old MRL/l mice. TABLE 2 Age of Appearance of Suppression by MRL/l T Cells IgM SRBC PFC/culture”. ’
Cells cultured’, h l-month-old 2-month-old 3-month-old 2-month-old l-month-old
MRL/ 1 spleen MRL/l spleen MRL/l spleen MRL/n spleen MRL/I B + l-month-old + 2-month-old + 3-month-old + 2-month-old
MRL/I MRL/l MRL/I MRL/n
T T T T + l-month-old MRL/l T + 2-month-old MRL/I T + 3-month-old MRL/l T
5000 + 653 250 f 50 10 f 10 2417 + 142 2750 + 250 197 -c 33 23 + 9 2000 * 100 2540 f 716 1330 f 153 933 f 289
“All female mice; 1.5 X 10’ spleen cells, 1 X 10’ B + 5 X lo6 T cells, or 1 X 10’ B + 5 X lo6 MRL/I T + 5 X 106MRL/n T cells were cultured 4 days after mice were primed with 2 X lo5 SRBC iv. b MRL/l B cells alone had 55 f 5 PFC/culture; all cultures of T cells alone had I 25 PFC/culture. ’ Mean + SEM of duplicate or triplicate cultures. d MRL/n B + 1 X 10’ MRL/n T cells had 4550 f 1050 PFC/culture.
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TABLE 3 Elimination of MRL/ I Ts Activity with Anti-Thy- 1.2 and Irradiation Cells cultured”~’
SRBC IgM PFC/cuIture’
MRL/ 1 spleen MRL/n spleen MRL/n B + MRL/n T + MRL/l T + MRL/n T + MRL/I T + MRL/I T (a Thy l.2)d + MRL/l T (a Ia)d + MRL/l T (I 500 rads)“ + MRL/l T (C’ only) + MRL/n T + MRL/I T + MRL/I T + MRL/n T (01Thy 1.2) MRL/n B
43 -c 12 3600 2 539 1130 f 225 21 f 9 1-U f 17 1010 + 277 240 + 80 1947 + 399 243 f 44 3500 f 450 10 f 0 90 f 40 45 f 25
’ 1.5 X 10’ spleen cells, 1 X 10’ B cells, 5 X lo6 T cells cultured with 0.1% SRBC. h 3%month-old MRL/l females and 2-month-old MRL/n females primed 4 days prior to culture with 2 X IO5 SRBC iv. ’ Mean 2 SEM of duplicate or triplicate cultures. PFC assay, Day 4 of culture. ’ 5 x IO6T cells (nylon wool-passed spleen) treated with anti-Thy-l.2 + C’, anti-Ia + C’, C’ alone, or 1500 rad. Residual cells cultured as shown.
These experiments have been repeated using Ig-coated plates for T-cell enrichment and the results are the same (not shown). Elimination of MRL/I T, activity with anti-Thy-l.2 and irradiation. In order to determine whether suppressor cells were T cells, nylon wool-passed spleen cells from 3%month-old MRL/l mice were treated with anti-Thy- 1.2 or anti-Ia sera and complement, or they were exposed to 1500 rads. The residual cells were cultured as shown in Table 3 with MRL/n B + T cells. Whereas the untreated or complementtreated MRL/l T cells suppressedthe MRL/n B + T response, treatment with antiThy-l .2 and with irradiation abrogated the suppression, while anti-Ia serum had no effect on the suppressor activity. The slight increase in the response of the cultures with irradiated MRL/l T cells was not due to the uncovering of T-cell help because irradiated MRL/l T cells cultured with B cells alone produced very low PFC responses (data not shown). The results in Table 3 also demonstrate that additional MRL/n T cells had no suppressive activity. DISCUSSION The studies described above were done to assessB- and T-cell function in MRL mice of varied agesin order to determine whether immunoregulatory abnormalities might explain the onset of autoimmune disease. Evaluation of immune responses to exogenous antigens is, of course, indirect, becausethe same mechanisms may not apply to autoantibody production and it is difficult to determine whether immunoregulatory abnormalities precede or result from autoimmune disease. Our results show that MRL/l mice respond well to SRBC in vivo and in vitro when younger than 2 months of age but that between 2 and 3 months with some variation among individual mice, the response declines rapidly and becomes very low by age
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3 to 4 months (Figs. 1 and 2). The fact that the in vivo splenic PFC responsesdeclined at the same time as in vitro PFC responsessuggeststhat the internal environment of the mice as they develop autoimmune disease is not the reason for their hyporesponsivenessand that one or more cell populations were defective prior to and after removal from the mice. Our studies show evidence for decreased TH function and increased T-cell suppressor activity in aging MRL/l mice. Although young MRL/l mice appeared to have normal Tu responsiveness, the most striking abnormality which we observed in our experiments was a rapid loss of Tn function in MRL/l mice. This loss of TH function occurred as early as 2-3 months of age (Figs. 2 and 3, Tables 1 and 2) and persisted in older mice (Fig. 4) despite the fact that the proliferating T cells in MRL/l mice have been reported to be of the Tn phenotype, Lyt-l+ (5). These results are consistent with recent studies which indicate that MRL/l mice have a markedly decreased response with age to the TD antigen rabbit gamma globulin (27) and decreasedresponsiveness to the Tcell mitogens concanavalin A and phytohemagglutinin (9). In addition, MRL/l mice are unable to produce normal amounts of IL-2 (a product of Lyt- 1+ T cells) as early as 4-6 weeks of age ( 16- 18) and by 4-6 months of age their inability to produce IL-2 is very marked (16-18). MRL/ 1 T cells are also incapable of responding to exogenous IL-2 (16, 17). Although our results and those discussed above indicate that MRL/l mice lose Tn function with age, other studies have suggestedthat Tu function is either normal or increased (6, 8, 9, 13, 28). Thus T cells from old MRL/l mice were capable of providing help to B cells for responses to a hapten-protein conjugate if cells were transferred to young syngeneic recipients (6). Moreover, young MRL/l spleen cells had extremely high responses to phytohemagglutinin (9) (although these declined in older mice), T cells from MRL/l mice provided increased help for anti-DNA responses in vitro (13), and T cells were required for MRL/l in vitro responses to. the nuclear antigen Sm (28). In another report (8) T cells from older MRL/l mice were found to be hyperactive in a system in which immunoglobulin production by B cells was determined. Since most of the immunoglobulin produced by older MRL/ 1 B cells was found to have anti-DNA activity (8), it is possible that the hyperactive T cells were specific for DNA or other autoantigens. It is difficult to explain the reasons for all of the discrepant findings regarding Tu activity in MRL/l mice. The systems used to assessT- and B-cell function were different and this may be partially responsible. We have no direct evidence for increased TH activity at any age although we have not yet selectively eliminated Ts in order to determine whether Tn activity is increased when unopposed by Ts. Furthermore, since the secondary responses in MRL/l mice were very high if the mice were primed at an early age (Table l), it is possible that memory Tn are normal or hyperactive. Furthermore, in most of the studies described above in which Tn activity has been found to be increased, the response has either been antigen nonspecific or the T-cell responses have been specific for self-antigens. The possible significance of these findings will be discussed below. Our results also indicate that MRL/l T cells have increased suppressor activity which develops with age since the addition of MRL/l T cells to MRL/l or MRL/n B cells + MRL/n T cells suppresses the response (Figs. 6 and 7; Table 2). The suppressor cell is presumably a T cell becauseit is nylon wool (and anti-Ig)-nonadherent and its activity is eliminated by anti-Thy- 1.2 and complement but not by an
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anti-Ia antiserum which kills B cells and macrophages but lacks I-J activity (Table 3). The Lyt phenotype has not yet been determined. The results in Table 2 indicate that the loss of T-cell helper function occurs concommitantly with the appearance of T-cell suppressor function. Studies are currently in progress to isolate the T-cell subsets in order to determine exactly what degree of “loss of help” is actually due to increased suppression, to determine the phase of the immune response in which these suppressor cells are active, and to determine the effect of adding fewer or greater numbers of T cells. The fact that the suppressor activity is present in cells from unprimed mice (Fig. 7) suggeststhat the suppressor cells may not be Lyt-l+ feedback suppressor inducer cells which have been described previously (12). Our finding of increased suppressor T-cell activity in MRL/l mice is consistent with results described in previous studies. Thus, Gershon et al. ( 14) found increased feedback suppressor activity in MRL/l T cells which was thought to be preceded by the inability of Lyt-l+ cells to respond to feedback suppressor signals. Theophilopoulos et al. (4) found a decreased proportion of Lyt-2,3+ and Lyt-1,2,3+ T cells in lymph nodes of old MRL/l mice but also noted a disproportionately large number of Thy-l+ cells bearing Ia antigens controlled by the I-J subregion which could be Ts (29). Increased suppressor activity by old MRL/l T cells was also noted by Ruppert et al. (9). The suppressor cell was radiation sensitive, as was ours. Santoro et al. (11) have also shown that MRL/l and MRL/n mice presumably have intact suppressor cell activity since they develop tolerance and suppression with hapten-coupled spleen cells and Creighton et al. (6) found normal Ts function in older MRL/l mice in an adoptive transfer system. A more recent study has shown, however, that older MRL/l mice are resistant to tolerance induction (27). Further studies are needed to determine the mechanism of the increased suppressor activity noted in these studies and how increased Ts activity can be present concommitantly with autoimmune disease. Our findings of an age-related decline in Tn activity and increased Ts activity in MRL/l mice by 2-2Y2 months of age suggeststhat these mice may be a model of accelerated aging, since decreased IL-2 production (30) and decreased Tn function (3 1, 32) as well as increased Ts activity (33, 34) have been reported in aging normal mice. Further studies are needed to determine how these findings correlate with the presence of autoimmune disease. From our studies of B-cell function in MRL/l mice it appears that young MLR/ B cells may often be hyperactive (Fig. 5, 6, and 7) and that B-cell function becomes more normal with age (Fig. 5). Certainly the possibilities remain that macrophage function may account for these findings or that the apparent return to normal Bcell activity with age is actually due to the presence of MRL/l Thy- 1.2-resistant Ts. Studies are in progress to test these possibilities. The B-cell hyperresponsiveness we have noted in young mice is consistent with some results of others (7, 8) although B-cell function did not appear to decline in older mice in those studies (7, 8) or in those studies in which B-cell function in young mice was normal (6). However, hyporesponsiveness to the polyclonal B-cell activator lipopolysaccharide has been noted in old MRL/l spleen cells (8) and this may be indicative of B-cell hyporesponsiveness. We have thus found that MRL/l mice develop decreased TH function and increasedTs activity with agealthough it still remains to be shown whether the decrease in TH function is only apparent and is actually due to increased Ts activity. While
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these findings may seem inconsistent with a T-cell role in the development of autoimmune disease,they are concordant with findings by others including decreased IL-2 production (16- 18), decreased responsiveness with age to a TD antigen (27), and increased suppressor cell activity (9, 14). Our finding that mice which are primed with SRBC at a young age are still responsive to SRBC when old, despite the inability of T and/or B cells in old MRL/l mice to be primed (Table 1) may provide an explanation of how these abnormalities can exist concurrently with autoimmunity. Apparently T and B cells which recognize an antigen as foreign early in life remain functional with aging; thus the MRL/l mice may become sensitized to the autoantigens at an early age when T- and/or B-cell function is normal or possibly hyperactive. Further studies in MRL/l and MRL/n mice are needed in order to determine whether the immunoregulatory abnormalities described here are related to the autoimmune disease, to a quantitative and/or functional imbalance of regulatory T cells associated with lymphoproliferation or to other causes. ACKNOWLEDGMENT The authors thank Jean Rinacke for typing the manuscript.
REFERENCES 1. Andrews, B. S., Eisenberg, R. A., Theophilopoulos, A. N., Izui, S., Wilson, C. B., McConahey, P. S., Murphy, E. D., Roths, J. B., and Dixon, F. J., J. Exp. Med. 148, 1198, 1978. 2. Eisenberg, R. A., Tan, E. M., and Dixon, F. J., J. Exp. Med. 147, 582, 1978. 3. Benage, D., Olive, P., Takano, M., Treadwell, E., Winn, D., Singsen, B., and Sharp, G. C., Clin. Res. 28, 138A (abstract), 1980. 4. Theophilopoulos, A. N., Eisenberg, R. A., Bourdon, M., Crowell, J. S., Jr., and Dixon, F. J., J. Exp. Med. 149, 5 16, 1979. 5. Cantor, H., and Gershon, R. K., Fed. Proc. 38, 2058, 1979. 6. Creighton, W. D., Katz, D. H., and Dixon, F. J., J. Immunol. 123, 2627, 1979. 7. Izui, S., McConahey, P. J., and Dixon, F. J., J. Immunol. 121, 2213, 1978. 8. Theophilopoulos, A. N., Shawler, D. L., Eisenberg, R. A., and Dixon, F. J., J. Exp. Med. 151,446, 1980. 9. Ruppet-t, B. L., Ford, R. J., and Maize], A. L., Cell. Immunol. 61, 40, 1981. 10. Laskin, C. A., Taurog, J. D., Smathers, P. A., and Steinberg, A. D., J. Immunol. 127, 1743, 1981. 11. Santoro, T. J., Scott, D. W., and Pisetsky, D. S., J. Zmmunol. 127, 690, 1981. 12. Eardley, D. D., Hugenberger, J., McVay-Boudreau, L., Shen, F. W., Gershon, R. K., and Cantor, H., J. Exp. Med. 147, 1106, 1978. 13. Sawada, S., and Talal, N., Arthritis Rheum. 22, 655 (abstract), 1979. 14. Gershon, R. K., Horowitz, M., Kemp, J. D., Murphy, D. B., and Murphy, E. D., In “Genetic Control of Autoimmune Disease” (N. Rose, P. Bigazzi and N. Warner, Eds.), p. 223. Elsevier-North Holland, New York. 15. Glimcher, L. H., Steinberg, A. D., House, S. B., and Green, I., J. Immunol. 125, 1832, 1980. 16. Altman, A., Theophilopoulos, A. N., Weiner, R., Katz, D. H., and Dixon, F. J., J. Exp. Med. 154, 791, 1981. 17. Wofsy, D., Murphy, E. D., Roths, J. B., Dauphinee, M. S., Kipper, S. B., and Talal, N., J. Exp. Med. 154, 1671, 1981. 18. Dauphin& M. J., Kipper, S. B., Wofsy, D., and Talal, N., J. Immunol. 127, 2483, 1981. 19. Claman, H. N., Chaperon, E. A., and Triplett, R. F., Proc. Sot. Exp. Biol. Med. 122, 1167, 1966. 20. Falkoff, R., and Kettman, J., J. Immunol. 108, 54, 1972. 21. Mishell, R., and Dutton, R., J. Exp. Med. 126, 423, 1967. 22. Braley-Mullen, H., Eur. J. Immunol. 7, 775, 1977. 23. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 24. Mage, M. G., McHugh, L., and Rothstein, T., J. Immunol. Methods 15, 47, 1977. 25. Wysocki, L. J., and Sato, V. L., Proc. Nat. Acad. Sci. USA 75, 2844, 1978.
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26. Tada, T., Takemori, T., O’Kumura, K., Nonaka, M., and Tokuhisa, T., J. Exp. Med. 147, 446, 1978. 21. Amagai, T., and Cinader, B., Eur. .I. Immunol. 11,923, 1981. 28. Cohen, P. L., and Eisenberg, R. A., Fed. Proc. 40,975 (abstract), 1981. 29. Murphy, D. B., Herzenberg, L. A., Okumura, K., Herzenberg, L. A., and McDevitt, H. 0.. J. Exp. Med. 144, 699, 1976.
30. 3 I. 32. 33. 34.
Thoman, M. L., and Weigle, W. O., J. Immunol. 127, 2 102, 198 I. Doria, G., D’Agostaro, G., and Garatini, M., Cell. Immunol. 53, 195, 1980. Krosgrud, R., and Perkins, E., J. Immunol. 118, 1607, 1977. Goidl, E. A., Innes, J. B. and Weksler, M. E., J. Exp. Med. 144, 1037, 1976. Segre, D., and Segre. M., J. Immunol. 116, 735, 1976.
IMMUNOLOGY
74, 72-85 (1982)
lmmunoregulation in MRL/Mp-lpr/lpr Mice: Evidence for Decreased Helper-T-Cell and Increased Suppressor-T-Cell Function with Age’ DEBORAHA. WILSON*,*
AND
HELEN BRALEY-MULLEN*T~-,~
*Division of Immunology-Rheumatology, Department of Medicine, and TDepartment of Microbiology. University of Missouri Health Sciences Center, Columbia, Missouri 65212 Received May 24, 1982; accepted August 30, 1982 In vivo and in vitro plaque-forming cell (PFC) responsivenessto sheep erythrocytes (SRBC) was used to assess immunoregulatory function in the autoimmune MRL mouse strain. MRL/Mp-lpr/lpr (MRL/l) mice had good primary and secondary IgM and IgG responses in vivo compared to MRL/Mg+/+ (MRL/n) mice when young, but with age the MRL/l responses declined markedly. In vitro primary SRBC-specific PFC responsesin MRL/l mice declined at the same time as in vivo responses,indicating that the in vivo autoimmune environment could not account for cellular dysfunction. When varied mixtures of T and B cells from MRL/l and MRL/n mice were cultured, abnormalities in MRL/l T-cell function became apparent. Thelper-cell (Tn) function declined rapidly with age, beginning by 2 to 2% months of age. T cells from MRL/l mice 2 months of age and older also had increased suppressor activity when cultured with B cells and MRL/n T.cells. The degree of suppressor activity increased with age. The correlation of these findings with results of previous studies by others and with autoimmune disease is discussed.
INTRODUCTION MRL/Mplpr/lpr (MRL/1)4 mice spontaneously develop autoimmune disease characterized by high levels of serum immunoglobulin and immune complexes ( 1) and numerous autoantibodies (l-3). They die at an early age with severe glomerulonephritis and arteritis (1). The lpr gene, which causes marked proliferation of Lyt “null” (4) or Lyt-l f (5) T cells, appears to be responsible for acceleration and severity of autoimmune disease because the MRL/Mp-+/+ (MRL/n) strain which is congenic except for the lpr gene develops autoimmune disease more slowly and dies at a much later age (1). Numerous studies have been done in an attempt to determine the immunoregulatory abnormality responsible for autoimmune disease in MRL mice. Thus MRL/l mice have been reported to have both normal (6) and hyperactive (7) B-cell function ’ Supported by NIH Grants CA 25054 and AM 20305. 2 Recipient of an Arthritis Foundation Post-Doctoral Research Fellowship. To whom all correspondence should be addressed. 3 Recipient of NIH Research Career Development Award AI 100322. 4 Abbreviations used: MRL/l, MRL/Mp-lpr/lpr; MRL/n, MRL/Mp+/+; Ts, suppressor T cell; Tn, helper T cell; IL-2, interleukin 2; TD, thymus dependent; SRBC, sheep erythrocytes; PFC, plaque-forming cell; Ig, immunoglobulin. 72 0008-87491821I70072-14$02.00/O Copyright 0 1982 by Academic Press, Inc. All righti of mpmductiw in any form reserved.
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as well as an age-related decreasedresponsivenessto a B-cell mitogen (8). SuppressorT-cell (T,) function has been found to be either increased (9) or normal (6, 8) and the ability to develop tolerance to exogenous (6, 10) or modified self ( 11) antigens has been reported to be normal (6, 11) and abnormal (10). Becausethe proliferating T cells are probably Lyt- 1+ (5), studies of helper-T-cell (Tu) function (5) and feedback suppressor inducer function (5, 12) may be very relevant to determining the immunoregulatory abnormalities in MRL/l mice. Indeed, both normal (6) and increased (8, 9, 13) Tu function have been reported in different studies. Decreased responsivenesspresumably of Lyt- 1+ T cells to feedback suppressor signals has been suggested(14), and the syngeneic mixed lymphocyte response which requires Lyt1’ T cell function is impaired in MRL/l mice (15). Recent studies have shown that MRL/l mice become unable to produce and to respond to interleukin-2 (IL-2) formerly called T-cell growth factor ( 16- 18). These results are consistent with our findings of decreased TH function in MRL/l mice with age. We have also found an age-related decrease in B-cell (or macrophage) function and an increase in suppressor cell activity with age. The system we have used to study T- and B-cell function is that of in vivo and in vitro plaque-forming cell responses to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC) (19). Because MRL/l and MRL/n mice are congenic except for the lpr gene, in vitro cell mixing experiments provide a means of determining which populations of cells function abnormally in MRL/l mice. The correlation of these cellular dysfunctions with autoimmunity is discussed. MATERIALS
AND METHODS
Mice. MRL/Mp-lpr/lpr (MRL/l) and MRL/Mp-+/+ (MRL/n) mice were obtained from The Jackson Laboratory, Bar Harbor, Maine and subsequently maintained by brother-sister matings in our colony. New breeding pairs of both strains were obtained annually to avoid genetic drift. The immunopathologic characteristics and mortality of MRL mice have been described elsewhere (l-4). The MRL/l mice used in the following studies ranged in age from 1 to 7 months and the MRL/n mice from 1 to 10 months. All mice were in good condition when used aside from the expected autoimmune disease manifestations. Both male and female mice were used in these experiments since preliminary studies showed that the results were essentially the same for both sexesof both substrains except that the abnormalities occurred slightly earlier in MRL/l females than in MRL/l males. Antigens and immunizations. SRBC was used as the antigen for all experiments. In vivo primary IgM and IgG SRBC responses were determined following an ip injection of 2 X lo* SRBC. Secondary IgM and IgG in vivo responses were elicited with a primary injection of 2 X lo8 SRBC ip at 1, 2, or 3 months and a secondary challenge at 4 months of age. In vitro spleen cell cultures were immunized with 1 X lo6 SRBC per culture. In most in vitro experiments the mice were given 2 X lo5 SRBC iv 4 days prior to culture in order to carrier-prime T cells (20) and thus increase the magnitude of the SRBC-specific plaque-forming cell (PFC) responses. Spleen cell cultures. Spleen cell suspensions were prepared and cultured according to the method of Mishell and Dutton (21). RPM1 1640 culture medium (KC. Biological, Inc., Lenexa, Kan.) was supplemented with 10% heat-inactivated fetal calf serum, nonessential amino acids, L-glutamine, sodium pyruvate, and vitamins
74
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(all from Grand Island Biological Co., Grand Island, N.Y.) and with 5 X lop5 M 2-mercaptoethanol. Spleen cells, l-2 X lo7 were cultured at 37°C in 35 X lo-mm culture dishes (Costar 3035) which were gently rocked; cultures were fed nutritional cocktail daily. Preparation of T- and B-cell-enriched populations. B-cell-enriched spleen cells were obtained by treatment with anti-Thy- 1.2 (AKR anti-C3H antiserum and complement (22)). This antiserum has been shown to be effective in eliminating T cells but to have no effect on B cells (22). Forty to fifty percent of the original spleen cells were eliminated by this technique. In most experiments T cells were obtained by passing spleen cells through nylon wool according to the method of Julius et al. (23). This method yielded approximately 30% of the original spleen cells from MRL/n mice and 30-40s of original MRL/l spleen cells, with the highest yields obtained from old MRL/l mice where massive splenomegaly was present. Functional purity of the B- and T-cell-enriched populations was assessedby PFC responses to SRBC and in some experiments by proliferative responses to concanavalin A, phytohemagglutinin, and lipopolysaccharide. In some experiments nylon wool-passed spleen cells were treated with anti-Thy-l.2 and complement, anti-la serum (A.Th anti-A.TL, which has been shown previously in our laboratory to kill B cells and macrophages but not T cells and to lack I-J specificity (unpublished results)) and complement, or 1500 rad. The 6oCo facility at the Dalton Research Center, University of Missouri, was used to irradiate the cells. Mixed lymphocyte reaction (MLR). In order to ensure that alloreactivity could not account for the responsiveness of MRL/l and MRL/n spleen cells when mixed in culture, two-way MLR studies were performed using young and old MRL/l, young and old MRL/n, C3H and C57B1/6 mice in all possible combinations. No alloreactivity between young and old MRL/l or MRL/n mice was detected (not shown). RESULTS In vivo primary and secondary SRBC responsesin MRL mice. In order to indirectly assessT- and B-cell function in MRL mice in vivo MRL/l and MRl/n female mice of varied ages were immunized with SRBC. The PFC responses 4 and 5 days after immunization are shown in Fig. 1. On Day 4, young MRL/I mice had predominantly IgM PFC responseswhich were lower than those of MRL/n mice at all agestested. The IgM PFC response fell markedly in MRL/l mice by 4 months of age. The Day 4 IgG PFC response was low in all groups except for 7-week-old MRL/n mice. The IgM PFC responsesin all groups varied little from Day 4 to Day 5. The IgG PFC responsesincreased markedly in young MRL/l mice and in 7-week-old and 4-monthold MRL/n mice between Days 4 and 5. Four-month-old MRL/l and ten-monthold MRL/n mice had essentially no IgG PFC on either day. Other in vivo studies not shown here indicated that the IgM PFC responsein MRL/l mice usually declined between 2 and 3 months of age and the age at which the MRL/n response fell was variable but generally began to occur by 10 months of age or later. Secondary IgM and IgG SRBC responses in MRL mice were similar to primary responses in that I- to 2-month-old MRL/l and all MRL/n mice tested up to age 8 months responded well whereas MRL/l mice responded poorly at 3-4 months (not shown). Actually, as shown in Table 1, it is the age at which MRL/l mice are
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7wk 4mo ML/I a IgM
7wk
4mo 1Omo ML/n
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IN MRL MICE
7wk 4mo MRLA
7wk
4mo 1Omo MRL/n
0 IgG
FIG. 1. Primary IgM and IgG SRBC responses in MRL mice of varied ages in vivo. Female MRL/l and MRL/n mice were given 2 X lo* SRBC ip and PFC responseswere determined 4 and 5 days later. Results shown are mean IgM and IgG PFC/spleen + SEM (four mice/group).
primed rather than the age at which they are given a secondary challenge (and thus the age at which PFC responsesare determined) which determines whether they are capable of producing secondary IgM and IgG responses. In this experiment secondary IgM and IgG responses in MRL/l and MRL/n mice were determined at 4 months of age, 5 days after the secondary challenge. The mice were primed at 1, 2, or 3 months of age or not at all. The MRL/l mice which were primed at 1 or 2 months of age had considerably higher responsesthan those which were primed at 3 months or which received only a primary injection 5 days prior to PFC deterTABLE 1 Secondary in vivo SRBC Responses in MRL Mice: Importance of Age of Priming Secondary SRBC PFC/spleenb Age of priming” (months)
Mice MRL/l
kM
w
P
1 2 3 None
25,267 + 33,975 -t 8,667 f 7,200 +
10,992 12,243 4,758 3,354
112,733 -c 53,769 279,167 2 88,704 14,500 rt 14,500 840 + 705
MRL/n 0
1 2 3 None
6,080 + 5,960 f 5,260 + 34,300 f
2,187 1,087 1,818 10,388
47,020 k 7,695 43,740 ?I 9,2 13 60,140 + 10,310 65,000 f 23,43 1
p All mice were given 2 X lo8 SRBC ip as primary injection. Secondary challenge (2 X 108SRBC ip) was given to all mice at 4 months of age. PFC were determined 5 days later. ’ Mean PFC/spleen f SEM, 3-5 mice/group.
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mination. The responsesof MRL/l mice primed at 2 months were also much greater than those of MRL/n mice. Each group of MRL/n mice responded similarly regardless of the age at which they were primed and the MRL/n mice which received only one injection of SRBC had high IgM and IgG primary responses. Thus the ability of MRL/l mice to be primed declines with age. However, if the mice are primed when young, a secondary response may be elicited at 4 months of age, even though MRL/l mice do not develop good primary responses at this age. Thus young MRL/l mice produced similar and in some experiments even greater primary and secondary SRBC PFC responses compared to MRL/n mice but with age their ability to respond to SRBC decreasedmarkedly. These results thus suggest that either (1) T and/or B cells or accessory cells in MRL/l mice become dysfunctional with age, (2) that abnormal proportions of splenic B and T cells may cause altered feedback and regulatory cell activity, or (3) that the autoimmune environment may prevent otherwise normal cells from functioning well. In vitro primary IgM SRBC responsesin MRL mice. In order to eliminate the autoimmune environment of MRL/l mice as a possible factor causing hyporesponsivenessto SRBC, spleen cells were cultured in vitro with SRBC. The primary IgM SRBC PFC responsesin MRL/l and MRL/n mice at varied agesdetermined on Day 4 of culture are shown in Fig. 2. Spleen cells from l- and 2-month-old MRL/l mice responded well to SRBC in vitro but by 4 months the responseshad declined markedly. By contrast, spleen cells from MRL/n mice responded well at all ages up to 10 months. The lack of responsivenesswas not due to a switch to IgG PFC because all of the MRL/l spleen cell cultures had less than 100 IgG PFC per culture on Day 4 or Day 5 of culture (not shown). Thus the decline in responsiveness to SRBC in MRL/l mice occurred between 2 and 4 months of age both in vivo (Fig. 1) and in vitro (Fig. 2) suggesting that the decreased responsiveness was not due to the autoimmune environment of MRL/I mice. Culture of fractionated spleen cellsfrom young MRL mice. In order to provide a means of comparing B- and T-cell function of these mice more directly and to lmo 2mo 4mo 6mo lmo 2mo 4mo 6mo
3000
5000
7000
IgM SRBC PFWculture FIG. 2. Primary IgM SRBC responsesin vitro in MRL mice. MRL/l and MRL/n male mice of indicated ageswere primed on Day -4 with 2 X 1O*SRBC iv. On Day 0 spleen cells ( 1 X 1O’/cuIture) were cultured with 1 x lo6 SRBC. PFC responseswere determined on Day 4. Results shown are the means of triplicate cultures + SEM.
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preclude the problem of the excessive T cell to B cell ratio in MRL/l mice, enriched populations of T cells and B cells were cultured in various combinations. The 9-week MRL/l spleen cell response was much lower than the 9-week MRL/ n spleen cell response (Fig. 3). When MRL/l B cells were cultured with MRL/l T cells, the response was slightly lower than the spleen cell response. However, when MRL/l B cells were cultured with MRL/n T cells the response was markedly increased, indicating that MRL/l B cells could function well when provided adequate T-cell help. MRL/n B cells cultured with their own T cells also had good responses, but the response of MRL/n B cells was much lower when MRL/l T cells were substituted. This experiment thus illustrates that shortly after 2 months of age decreased T-cell helper function is present in MRL/l as compared to MRL/n mice, whereas B cells and accessory cells of MRL/l mice appear to function normally. Similar experiments have been done using the panning technique for enrichment of T cells (24,25) and the results were the same, indicating that the defect in MRL/ 1T cells is not due to loss of a nylon wool-adherent TH cell population (26). Because of the facility of obtaining larger numbers of T cells by nylon wool passage,this method was used for all other experiments described below. T-Cell Function in old MRL/l mice. That T-cell dysfunction also contributes to the SRBC hyporesponsiveness of older MRL/l mice is shown in Fig. 4. Spleen cells from 2- and 4-month-old MRL/n mice produced approximately 6000 PFC/culture, but 4-month MRL/l spleen cells again produced few PFC. Whereas T cells from the older 4-month MRL/n mice helped 2-month MRL/n B cells to produce responses similar to those of unfractionated spleen cells, 4-month MRL/l T cells provided no helper activity for 2-month MRL/n B cells. Thus MRL/l mice continue to demonstrate decreased T-cell helper activity even though the spleens in these older mice are presumably overpopulated with Lyt 1 + T cells (5). B-Cell function in MRL mice. In order to determine whether B-cell and/or accessory cell function in MRL/l mice might also decline with age, B cells from MRL/ 1and MRL/n mice of various ageswere cultured with T cells from 9-week-old MRL/ ML/I
Spleen
MRL/n Spleen ML/I
B+T
IgM SRBC PFCXulture
FIG. 3. In vitro SRBC responsesin young MRL mice: B- and T-cell function. Nine week MRL/l and MRL/n male mice were primed with 2 X lo5 SRBC iv on Day -4. On Day 0, 1 X 10’ spleen cells, 1X 10’ B cells + 5 X lo6 T cells, or 1 X 10’ B cells as indicated above were cultured with 1 X IO6SRBC per culture. PFC responses were determined on Day 4. Results indicate means of two to three pooled duplicate cultures 2 SEM. MRL/l B cells and MRL/n T cells alone had 0 PFC/culture; MRL/l T cells alone had 10 PFC/culture.
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WILSON AND BRALEY-MULLEN MRLhI~een MRL/nq$tleen MRL/l4s&8en 2mo 4mo 2mo 4mo
MRL/n MRL/n MRL/n MRLA
Bt 1 Bt 1
2mo MRL/n B
1000 2000
4000
7000
IgM SRBC PFCXulture FIG. 4. In vitro SRBC responses in MRL mice: T-cell function. Two- and four-month-old MRL/n male and 4-month-old MRL/l male mice were primed on Day -4 with 2 X 10’ SRBC iv. On Day 0, 1 X 10’ spleen cells or 1 X lo7 B cells plus 5 X lo6 T cells from indicated mice were cultured with SRBC. Results shown are mean PFC -+ SEM of two pooled duplicate cultures. MRL/n T cells alone had 30 PFC/culture, MRL/l T cells alone had 2 PFC/culture.
n mice (Fig. 5). B-cell and/or macrophage function in MRL/l mice increased between 4 and 7 weeks and the responses at 7 and 10 weeks were very high. MRL/I B cell function declined markedly at 20 weeks compared with IO-week B cells although the responses were similar to 4-week MRL/l mice and to MRL/n mice at all ages. MRL/n mice maintained similar B-cell SRBC responsiveness from 9 to 40 weeks of age. The markedly disproportionate response of MRL/l spleen cells versus that of MRLA
MRL/n
30 20 15 IgM SRBC PFCxlO3 Culture ,.
5
5wk 0
Spleen cells
a
B cellstgwk
9wk
24wk
40wk
MRL/n 1 cells
Frc. 5. In vitro SRBC responsesin MRL mice: B-cell function. MRL/I and MRL/n female mice of indicated ages were primed on Day -4 with 2 X lo5 SRBC iv. On Day 0, I X 10’ of their B cells plus 5 X lo6 T cells from 9-week-old MRL/n mice were cultured with SRBC. Results shown are mean PFC t SEM of duplicate (spleen cell) or triplicate (B + T cell) cultures. Results of B-cell cultures were as follows (PFC ? SEM/culture): 4-week-old MRL/I B, 115 + 75; 7-week-old B, 155 + 115; IO-week-old MRL/l B, 45 f 25; 20-week-old MRL/l B, 5 + 5; 5-week-old MRL/n B, 165 & 55; 9-week-old MRL/n B, 415 ? 145; ZCweek-old MRL/n B, 150 f 0; 40-week-old MRL/n B, 80 + 60. 9-week-old MRL/n T cells had 180 + 20 PFC/culture.
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1000
3000 IgM SRBC
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IN MRL MICE
5000
7000
9000
PFCXulture
FIG. 6. Suppression of MRL SRBC responsesby MRL/l T cells. Two-month-old MRL/l and MRL/ n female mice were primed on Day -4 with 2 X 10’ SRBC iv. On Day 0, I X 10’ spleen ceils, 1 X 10’ B cells + 5 X lo6 T cells, or 1 X IO’ B cells plus 5 X lo6 of each type of T cells were cultured with SRBC. Results shown are means of two to six cultures f SEM. MRL/n B cells plus 1 X 10’ MRL/n T cells had 2650 PFC/culture; MRL/I B cells plus 1 X 10’ MRL/I T cells had 620 PFC/culture. MRL/I B cells had 57 f 3, MRL/n B cells had 213 + 12, MRL/I T cells had 0, and MRL/n T cells had 145 PFC/culture.
MRL/l B cells mixed with MRL/n T cells was most apparent at 10 weeks, indicating that the decreased spleen cell responsesof MRL/l mice at 10 weeks was largely due to T-cell dysfunction. The results of this experiment also suggest that young MRL/l B cells and/or accessory cells may be hyperactive compared to MRL/n B cells although this was not evident in all experiments (e.g., Fig. 3). Decreased helper versus increased suppressor-T-cell function in MRL/l mice. The above studies indicated that MRL/l mice have decreased helper T-cell function by 2’12months of age. Because studies by others have shown evidence for increased suppression in MRL/l mice (9), we asked whether MRL/l T cells might suppress a SRBC response which was helped by MRL/n T cells (Fig. 6). MRL/l B + T cells from 2-month-old female mice produced approximately 1000 PFC. The addition of MRL/n T cells to MRL/I B cells again resulted in a marked increase in PFC (ca. 10,000 PFC). However, if MRL/l B cells were cultured with equal numbers of T cells from both strains, the response was suppressedby approximately 50% (Fig. 6, line 3). Similar results occurred with B cells from MRL/n mice. Addition of MRL/ 1 T cells to the MRL/n B + T cell combination suppressed the response by approximately 70% (line 4 vs line 6). To determine whether these results were due to the in vivo priming with a low dose of SRBC, the experiment shown in Fig. 7 was done using spleen cells from unprimed mice. All of the PFC responses were considerably lower but the overall results were the same: that is, MRL/l T cells suppressed the responses of MRL/l B- + MRL/n T-cell and of MRL/n B- + T-cell combinations. Cell crowding was not responsible for the suppressive effect since both MRL/l and MRL/n spleen cell responsesincreased in proportion to the number of cells per culture. Moreover in both of the above experiments, MRL/n B cells were also cultured with 1 X 10’ MRL/n T cells. The responses were greater than those with 5 X lo6 T cells (Figs. 6 and 7) which also indicates that the suppressive effect was not due to the presence of increased numbers of T cells in the cultures containing both MRL/n and MRL/l T cells. The approximate age at which this suppressor effect appears in MRL/l mice is shown in Table 2. T cells from l-month-old MRL/l mice were able to help B cells from l-month-old MRL/l mice as well as T cells from the 2-month-old MRL/n mice but T cells from 2- and 3-month-old MRL/l mice provided essentially no help.
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WILSON AND BRALEY-MULLEN
MRLA B+T 2 MRLA BtMRLh T . MRLA BtTtMRLh T 1
w I *
MRL/n BtT MRL/n BtMRLA T .% MRL/n BtTtMRLA T B 6
200
50 100
300
400
600
IgM SRBC PFCXulture FIG. 7. Suppression of SRBC responsesin MRL mice by MRL/l T cells. Spleen cells, B cells, and T cells from 2-month-old MRL/I and MRL/n male mice were cultured on Day 0 with SRBC as in Fig. 6. PFC responses on Day 4 are indicated as mean PFC + SEM of two to six cultures. MRL/n B cells plus 1 X IO’ MRL/n T cells had 310 PFC/culture and MRL/l B plus 1 X 10’ MRL/I T cells had 25 PFC/culture. PFC results of enriched cell populations cultured alone were as follows: MRL/I B, 8 k 8 PFC/culture; MRL/n B, 50 PFC/culture; MRL/l T, 10 PFC culture; MRL/n T, 5 PFC/culture.
When MRL/ 1 T cells were added to the MRL/ 1 B + MRL/n T cell combination, l-month MRL/l T cells did not suppress the PFC response whereas T cells from 2-month MRL/l mice suppressed the response by approximately one-third, and 3month MRL/l T cells by greater than 50%. These results suggest that the loss of helper activity of MRL/l T cells occurs concommitantly with the appearance of Tcell suppressor activity and that both changes become more pronounced with age. They also show no evidence of increased TH activity in young or old MRL/l mice. TABLE 2 Age of Appearance of Suppression by MRL/l T Cells IgM SRBC PFC/culture”. ’
Cells cultured’, h l-month-old 2-month-old 3-month-old 2-month-old l-month-old
MRL/ 1 spleen MRL/l spleen MRL/l spleen MRL/n spleen MRL/I B + l-month-old + 2-month-old + 3-month-old + 2-month-old
MRL/I MRL/l MRL/I MRL/n
T T T T + l-month-old MRL/l T + 2-month-old MRL/I T + 3-month-old MRL/l T
5000 + 653 250 f 50 10 f 10 2417 + 142 2750 + 250 197 -c 33 23 + 9 2000 * 100 2540 f 716 1330 f 153 933 f 289
“All female mice; 1.5 X 10’ spleen cells, 1 X 10’ B + 5 X lo6 T cells, or 1 X 10’ B + 5 X lo6 MRL/I T + 5 X 106MRL/n T cells were cultured 4 days after mice were primed with 2 X lo5 SRBC iv. b MRL/l B cells alone had 55 f 5 PFC/culture; all cultures of T cells alone had I 25 PFC/culture. ’ Mean + SEM of duplicate or triplicate cultures. d MRL/n B + 1 X 10’ MRL/n T cells had 4550 f 1050 PFC/culture.
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TABLE 3 Elimination of MRL/ I Ts Activity with Anti-Thy- 1.2 and Irradiation Cells cultured”~’
SRBC IgM PFC/cuIture’
MRL/ 1 spleen MRL/n spleen MRL/n B + MRL/n T + MRL/l T + MRL/n T + MRL/I T + MRL/I T (a Thy l.2)d + MRL/l T (a Ia)d + MRL/l T (I 500 rads)“ + MRL/l T (C’ only) + MRL/n T + MRL/I T + MRL/I T + MRL/n T (01Thy 1.2) MRL/n B
43 -c 12 3600 2 539 1130 f 225 21 f 9 1-U f 17 1010 + 277 240 + 80 1947 + 399 243 f 44 3500 f 450 10 f 0 90 f 40 45 f 25
’ 1.5 X 10’ spleen cells, 1 X 10’ B cells, 5 X lo6 T cells cultured with 0.1% SRBC. h 3%month-old MRL/l females and 2-month-old MRL/n females primed 4 days prior to culture with 2 X IO5 SRBC iv. ’ Mean 2 SEM of duplicate or triplicate cultures. PFC assay, Day 4 of culture. ’ 5 x IO6T cells (nylon wool-passed spleen) treated with anti-Thy-l.2 + C’, anti-Ia + C’, C’ alone, or 1500 rad. Residual cells cultured as shown.
These experiments have been repeated using Ig-coated plates for T-cell enrichment and the results are the same (not shown). Elimination of MRL/I T, activity with anti-Thy-l.2 and irradiation. In order to determine whether suppressor cells were T cells, nylon wool-passed spleen cells from 3%month-old MRL/l mice were treated with anti-Thy- 1.2 or anti-Ia sera and complement, or they were exposed to 1500 rads. The residual cells were cultured as shown in Table 3 with MRL/n B + T cells. Whereas the untreated or complementtreated MRL/l T cells suppressedthe MRL/n B + T response, treatment with antiThy-l .2 and with irradiation abrogated the suppression, while anti-Ia serum had no effect on the suppressor activity. The slight increase in the response of the cultures with irradiated MRL/l T cells was not due to the uncovering of T-cell help because irradiated MRL/l T cells cultured with B cells alone produced very low PFC responses (data not shown). The results in Table 3 also demonstrate that additional MRL/n T cells had no suppressive activity. DISCUSSION The studies described above were done to assessB- and T-cell function in MRL mice of varied agesin order to determine whether immunoregulatory abnormalities might explain the onset of autoimmune disease. Evaluation of immune responses to exogenous antigens is, of course, indirect, becausethe same mechanisms may not apply to autoantibody production and it is difficult to determine whether immunoregulatory abnormalities precede or result from autoimmune disease. Our results show that MRL/l mice respond well to SRBC in vivo and in vitro when younger than 2 months of age but that between 2 and 3 months with some variation among individual mice, the response declines rapidly and becomes very low by age
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WILSON AND BRALEY-MULLEN
3 to 4 months (Figs. 1 and 2). The fact that the in vivo splenic PFC responsesdeclined at the same time as in vitro PFC responsessuggeststhat the internal environment of the mice as they develop autoimmune disease is not the reason for their hyporesponsivenessand that one or more cell populations were defective prior to and after removal from the mice. Our studies show evidence for decreased TH function and increased T-cell suppressor activity in aging MRL/l mice. Although young MRL/l mice appeared to have normal Tu responsiveness, the most striking abnormality which we observed in our experiments was a rapid loss of Tn function in MRL/l mice. This loss of TH function occurred as early as 2-3 months of age (Figs. 2 and 3, Tables 1 and 2) and persisted in older mice (Fig. 4) despite the fact that the proliferating T cells in MRL/l mice have been reported to be of the Tn phenotype, Lyt-l+ (5). These results are consistent with recent studies which indicate that MRL/l mice have a markedly decreased response with age to the TD antigen rabbit gamma globulin (27) and decreasedresponsiveness to the Tcell mitogens concanavalin A and phytohemagglutinin (9). In addition, MRL/l mice are unable to produce normal amounts of IL-2 (a product of Lyt- 1+ T cells) as early as 4-6 weeks of age ( 16- 18) and by 4-6 months of age their inability to produce IL-2 is very marked (16-18). MRL/ 1 T cells are also incapable of responding to exogenous IL-2 (16, 17). Although our results and those discussed above indicate that MRL/l mice lose Tn function with age, other studies have suggestedthat Tu function is either normal or increased (6, 8, 9, 13, 28). Thus T cells from old MRL/l mice were capable of providing help to B cells for responses to a hapten-protein conjugate if cells were transferred to young syngeneic recipients (6). Moreover, young MRL/l spleen cells had extremely high responses to phytohemagglutinin (9) (although these declined in older mice), T cells from MRL/l mice provided increased help for anti-DNA responses in vitro (13), and T cells were required for MRL/l in vitro responses to. the nuclear antigen Sm (28). In another report (8) T cells from older MRL/l mice were found to be hyperactive in a system in which immunoglobulin production by B cells was determined. Since most of the immunoglobulin produced by older MRL/ 1 B cells was found to have anti-DNA activity (8), it is possible that the hyperactive T cells were specific for DNA or other autoantigens. It is difficult to explain the reasons for all of the discrepant findings regarding Tu activity in MRL/l mice. The systems used to assessT- and B-cell function were different and this may be partially responsible. We have no direct evidence for increased TH activity at any age although we have not yet selectively eliminated Ts in order to determine whether Tn activity is increased when unopposed by Ts. Furthermore, since the secondary responses in MRL/l mice were very high if the mice were primed at an early age (Table l), it is possible that memory Tn are normal or hyperactive. Furthermore, in most of the studies described above in which Tn activity has been found to be increased, the response has either been antigen nonspecific or the T-cell responses have been specific for self-antigens. The possible significance of these findings will be discussed below. Our results also indicate that MRL/l T cells have increased suppressor activity which develops with age since the addition of MRL/l T cells to MRL/l or MRL/n B cells + MRL/n T cells suppresses the response (Figs. 6 and 7; Table 2). The suppressor cell is presumably a T cell becauseit is nylon wool (and anti-Ig)-nonadherent and its activity is eliminated by anti-Thy- 1.2 and complement but not by an
IMMUNE
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anti-Ia antiserum which kills B cells and macrophages but lacks I-J activity (Table 3). The Lyt phenotype has not yet been determined. The results in Table 2 indicate that the loss of T-cell helper function occurs concommitantly with the appearance of T-cell suppressor function. Studies are currently in progress to isolate the T-cell subsets in order to determine exactly what degree of “loss of help” is actually due to increased suppression, to determine the phase of the immune response in which these suppressor cells are active, and to determine the effect of adding fewer or greater numbers of T cells. The fact that the suppressor activity is present in cells from unprimed mice (Fig. 7) suggeststhat the suppressor cells may not be Lyt-l+ feedback suppressor inducer cells which have been described previously (12). Our finding of increased suppressor T-cell activity in MRL/l mice is consistent with results described in previous studies. Thus, Gershon et al. ( 14) found increased feedback suppressor activity in MRL/l T cells which was thought to be preceded by the inability of Lyt-l+ cells to respond to feedback suppressor signals. Theophilopoulos et al. (4) found a decreased proportion of Lyt-2,3+ and Lyt-1,2,3+ T cells in lymph nodes of old MRL/l mice but also noted a disproportionately large number of Thy-l+ cells bearing Ia antigens controlled by the I-J subregion which could be Ts (29). Increased suppressor activity by old MRL/l T cells was also noted by Ruppert et al. (9). The suppressor cell was radiation sensitive, as was ours. Santoro et al. (11) have also shown that MRL/l and MRL/n mice presumably have intact suppressor cell activity since they develop tolerance and suppression with hapten-coupled spleen cells and Creighton et al. (6) found normal Ts function in older MRL/l mice in an adoptive transfer system. A more recent study has shown, however, that older MRL/l mice are resistant to tolerance induction (27). Further studies are needed to determine the mechanism of the increased suppressor activity noted in these studies and how increased Ts activity can be present concommitantly with autoimmune disease. Our findings of an age-related decline in Tn activity and increased Ts activity in MRL/l mice by 2-2Y2 months of age suggeststhat these mice may be a model of accelerated aging, since decreased IL-2 production (30) and decreased Tn function (3 1, 32) as well as increased Ts activity (33, 34) have been reported in aging normal mice. Further studies are needed to determine how these findings correlate with the presence of autoimmune disease. From our studies of B-cell function in MRL/l mice it appears that young MLR/ B cells may often be hyperactive (Fig. 5, 6, and 7) and that B-cell function becomes more normal with age (Fig. 5). Certainly the possibilities remain that macrophage function may account for these findings or that the apparent return to normal Bcell activity with age is actually due to the presence of MRL/l Thy- 1.2-resistant Ts. Studies are in progress to test these possibilities. The B-cell hyperresponsiveness we have noted in young mice is consistent with some results of others (7, 8) although B-cell function did not appear to decline in older mice in those studies (7, 8) or in those studies in which B-cell function in young mice was normal (6). However, hyporesponsiveness to the polyclonal B-cell activator lipopolysaccharide has been noted in old MRL/l spleen cells (8) and this may be indicative of B-cell hyporesponsiveness. We have thus found that MRL/l mice develop decreased TH function and increasedTs activity with agealthough it still remains to be shown whether the decrease in TH function is only apparent and is actually due to increased Ts activity. While
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these findings may seem inconsistent with a T-cell role in the development of autoimmune disease,they are concordant with findings by others including decreased IL-2 production (16- 18), decreased responsiveness with age to a TD antigen (27), and increased suppressor cell activity (9, 14). Our finding that mice which are primed with SRBC at a young age are still responsive to SRBC when old, despite the inability of T and/or B cells in old MRL/l mice to be primed (Table 1) may provide an explanation of how these abnormalities can exist concurrently with autoimmunity. Apparently T and B cells which recognize an antigen as foreign early in life remain functional with aging; thus the MRL/l mice may become sensitized to the autoantigens at an early age when T- and/or B-cell function is normal or possibly hyperactive. Further studies in MRL/l and MRL/n mice are needed in order to determine whether the immunoregulatory abnormalities described here are related to the autoimmune disease, to a quantitative and/or functional imbalance of regulatory T cells associated with lymphoproliferation or to other causes. ACKNOWLEDGMENT The authors thank Jean Rinacke for typing the manuscript.
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26. Tada, T., Takemori, T., O’Kumura, K., Nonaka, M., and Tokuhisa, T., J. Exp. Med. 147, 446, 1978. 21. Amagai, T., and Cinader, B., Eur. .I. Immunol. 11,923, 1981. 28. Cohen, P. L., and Eisenberg, R. A., Fed. Proc. 40,975 (abstract), 1981. 29. Murphy, D. B., Herzenberg, L. A., Okumura, K., Herzenberg, L. A., and McDevitt, H. 0.. J. Exp. Med. 144, 699, 1976.
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Thoman, M. L., and Weigle, W. O., J. Immunol. 127, 2 102, 198 I. Doria, G., D’Agostaro, G., and Garatini, M., Cell. Immunol. 53, 195, 1980. Krosgrud, R., and Perkins, E., J. Immunol. 118, 1607, 1977. Goidl, E. A., Innes, J. B. and Weksler, M. E., J. Exp. Med. 144, 1037, 1976. Segre, D., and Segre. M., J. Immunol. 116, 735, 1976.