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PHA) responsiveness to phytohemagglutinin
CELLULAR
IMMUNOLOGY
44,
29-38
(1979)
Mixed Lymphocyte Reaction in Mice Genetically Selected High (HVPHA) or Low (Lo/PHA) Responsiveness to Phytohemagglutininl M. LIACOPOULOS-BRIOT,”
C. STIFFEL,?
F. LAMBERT,*
for
AND
C. DECREUSEFOND~ “Institut d’lmmuno-Biologic~, INSERM U 20. CNRS LA 143 Hdpital Broussais, 96, rue Didot, 75674 Paris Cedex 14, and tLaboratoire d’lmmuno-GhnCtique, INSERM lJ 125. CNRS ER 70, lnstitut Curie, 26. rue d’Ulm, 75005 Paris. France Received
July 27, 1978
Lymph node cells from Hi/PHA and LoiPHA mice were evaluated for proliferative response after stimulation by allogeneic lymphocytes (MLR) originating from four inbred strains of different H-2 haplotype (C57B1/6, DBAI2, CBA, A). Reactivity to MLR and PHA were compared in these two lines and in the four inbred strains. The high and low responder status of Hi/PHA and Lo/PHA, as determined by T mitogens lymphocyte responsiveness, was also observed when one measured T responsiveness after MLR. Values obtained with the four inbred strains are included in the range of those measured in Hi/PHA and LoiPHA cells when stimulated by PHA as well as by allogeneic cells. In contrast, when used as stimulator cells, Hi/PHA or Lo/PHA lymphocytes induce an equivalent proliferative response versus every responder inbred strain studied. These experiments support the hypothesis of a common genetic control of proliferative response following PHA or MLR stimulation. The genes implicated would be different from those coding for I region associated antigens.
INTRODUCTION Subpopulations of T lymphocytes are usually characterized according to their surface markers or their functional properties. Attempts have also been made to associate their in vitro responsiveness to specific mitogens and their immunological characteristics (1). Most of the immune functions of T cells can be elicited by phytomitogens, namely, amplification (2) or suppression (3), cytotoxicity (4), and liberation of factors (skin reactive factors, MIF) (5). It was thought that information obtained from an in vitro system with mitogens might increase our understanding of immunological responses. As a practical means of approaching this problem, mice have been genetically selected according to the character “intensity of responsiveness of lymphocytes to PHA.” Two lines were obtained: one (Hi/PHA) with a high response to both PHA and Con A, the other (Lo/PHA) with a low response to these mitogens (6). At 1 Supported by a grant (76-7-1456) from the “Delegation Technique.”
GCnCrale a la Recherche Scientifique et
29 000%8749/79/050029-10$02.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
30
LIACOPOULOS-BRIOT
ET AL.
the maximal interline separation (generation F,,), lymphocytes from Hi/PHA incorporate 39 times higher levels of tritiated thymidine following optimal PHA stimulation than do Lo/PHA lymphocytes. Another characteristic of these two lines is their similar responsiveness to B mitogens: PPD and LPS. A first attempt to evaluate T-cell immune functions of these two lines was made by studying the in vitro response of their lymphocytes, in mixed lymphocyte culture, to various allogeneic stimuli provided by cells from four different inbred strains of mice. MATERIALS
AND METHODS
Mice. Mice genetically equivalent to F,-F,, generations of selective breeding were obtained by a second mating of the same parents. The foundation population of the selection consisted of random-bred albino mice and brother-sister mating was avoided in the course of the selection. So that mice of each selected line were probably not identical at the major histocompatibility locus. Then, each experiment was performed with an individual mouse tested versus the four inbred strains and results from several mice were pooled. Inbred strains were provided by the “Centre de Selection et d’Elevage d’Animaux de Laboratoire” of the C.N.R.S., Orleans, France (A and DBA/2, Jackson origin), Iffa Credo, 69210 L’Arbresle, France (C57B1/6, Jackson origin), and from our own breeding colony (CBA, origin: Radiobiological Research Unit, Harwell, Great Britain). Mitogen and medium. PHA (freeze-dried reagent grade PHA, batch K 3473, Wellcome Lab., Beckenham, Great Britain) was reconstituted in 5 ml distilled water and aliquots of this solution were stored at -20°C. RPMI-1640 (Gibco Biocult., Glasgow, Scotland) supplemented with glutamine (2 mM), penicillin (100 III/ml), and streptomycin (100 pg/ml) was used with or without addition of 10% fetal calf serum (FCS) (Gibco Biocult., from two batches: 7691018 (No. 76) and 86101s (No. 86). h4LR test. Isolated cell suspensions from lymph nodes (inguinal, axillary, and mesenteric) or spleen of one individual mouse were prepared as previously described (6) and resuspended in RPMI-1640 supplemented with 10% FCS. The percentage of viable cells was determined using the trypan blue exclusion method. One-way MLR was performed by mixing equal numbers (4 x 105) of responder cells from lymph node and stimulator cells from spleen or lymph node. Stimulator cells were irradiated with 1750 rads from a 6oCo source. For autologous controls, cells from the same mouse were used as responder and as stimulator. Cell mixtures were set up, in two to three replicates, in round-bottom tissue culture microplates (Cooke Microtiter M-24-l-l 10) in a final volume of 80 ~1 and incubated in a humidified atmosphere of 10% CO, in air at 37°C for 3 or 4 days. In some experiments, 2-mercaptoethanol(2-ME) was added to the culture at a final concentration of 5.10-5 M. PHA response. Various mitogen concentrations (10 to 100 pg/ml) in 25 ~1 of medium were added to 4 x lo5 lymphocytes in 55 ~1 of medium. Cultures were incubated 2 or 3 days under the conditions described above for MLR. MLR and PHA responses were carried out in parallel for each individual mouse of the two lines and of the four inbred strains. Measure of [3H]thymidine incorporation. Cultures were harvested on Days 2,
MLR IN MICE OF HIGH AND LOW RESPONDER
31
LINES TO PHA
3, or 4 after 23 hr labeling with 0.4 +i of [methyl-3H]thymidine (TMM 79, specific activity 2 Ci/mM, C.E.A. Saclay, France) in 25 ~1 of RPMI-1640 and processed according to the technique of Williams (7) as previously described (6). The mean count per minute (cpm) of two or three replicates was calculated. Results were expressed as the net increase in thymidine uptake, i.e., cpm in experimental stimulated cultures minus cpm in control unstimulated cultures (for PHA assays) or minus cpm in autologous control cultures (for MLC assays). Reported values are the means of results obtained with 4 to 10 mice for each experiment. RESULTS PHA Responsiveness
in Cultures Supplemented
with FCS
For several reasons previously explained (6), the usual method for testing the proliferative effect of PHA was performed in FCS-free medium. Attempts to obtain MLR in serum-free medium were unsuccessful under our microculture conditions. So, first of all, we studied the effect of the addition of FCS to the medium on the reactivity of Hi/PHA and Lo/PHA lymphocytes to PHA. Results are reported in Table 1. The maximal net increase in cpm was similar in Hi/PHA mice of F, generation whether or not FCS was added to the medium. A slight decrease in 3H incorporation was observed in cells from mice of F,,, generation when they were cultured during 2 days with FCS, but this effect disappeared in 3-day cultures. In Lo/PHA, the addition of FCS increased the amount of thymidine incorporation in 2-day cultures, but not in 3-day cultures. Higher doses of PHA were required for maximal stimulation with lymphocytes from both lines when the medium was supplemented with FCS. Similar findings were obtained with the two batches of FCS. This first series of experiments demonstrates that the interline difference remains important when PHA responsiveness is measured in the presence of FCS: the ratio (Hi/PHA)/(Lo/PHA) for F,,, is 15.5 to 19 and 33.6 to 40 for 2- and 3-day cultures, respectively. TABLE
1
Effect of Fetal Calf Serum (FCS) on PHA Maximal Responsiveness of Hi/PHA and Lo/PHA Lymphocytes (Mice of F, and F,, Generations of Selective Breeding) Generation of mice and culture time F,” 2 days
FM0 2 days
F,Ob 3 days
Batch of FCS
PHA (&ml)
HiiPHA L”Hlthymidine uptake (cpm)
76 86
20 50 50
49.6Ow 46.100 50,250
76 86
IO 20 20
104.000 78,000 90,900
76 86
IO 15 75
74,000 85,600 84,150
PHA k3W
LoiPHA IIHlthymidine uptake (cm)
(HiIPHA)/(Lo/PHA) 1.3 4.35 2.7
HiiPHA
LoiPHA
50 50 50
6.800 IO.600 18,600
z 8,500 2 2,300 2 4,600
IO 50 20
2,000 2 1,250 4.100 ? 850 5,850 2 1,250
52 19 15.5
102,OOil 74,oM) 85,050
2 7,200 2 7,200 + 5,900
IO 50 50
3,500 e 1,550 2.100 t !ml 2,500 c 750
21 40 33.6
70,500 83,500 81,650
n Mean of two mice in each experiment. b Mean of four to seven mice in each experiment. ( Net increase: experimental cpm - control cpm (?SE)
42,800 35.500 31,600
32
LIACOPOULOS-BRIOT TABLE
ET AL. 2
PHA Responsiveness in Inbred Strains of Mice [:‘H]Thymidine uptake (cpm)
Strains C57BLi6” (4) CBA (4) A (5) DBAi2 (4) HilPHA” (8) Lo/PHA (8)
Controls 1,800 1,560 2,100 1,850 1,300 1,300
2 ? 2 + * 5
260 150 220 100 130 200
50 20,708 35,400 35,650 42,700 81,150 4,175
t f + + -t+
75 6,000* 7,500 5,000 3,800 10,100 1,900
19,300 29,000 35,300 46,000 80,875 3,950
n Number of mice is indicated in parentheses. b Net increase in 2-day cultures (10% FCS supplemented medium): experimental cpm + SE. c Hi/PHA and Lo/PHA are mice of F,, generation.
2 f 2 + 5 2
5,300 6,200 4,800 4,500 11,350 2,900
cpm - control
We have compared the PHA response of both lines to those of the four inbred strains of mice used in MLR experiments (Table 2). Maximal values obtained with these strains are included in the range of those measured in Hi/PHA and Lo/PHA mice [(Hi/PHA)/(Lo/PHA): 19.41. The highest values are found in DBAR and the lowest in C57B1/6. On the other hand, thymidine incorporation values in unstimulated cultures were similar in the two selected lines and slightly lower than in the four inbred strains. Mixed Lymphocyte Cultures
The response of Hi/PHA and Lo/PHA cells to stimulation by irradiated lymph node cells from the four inbred strains and by cells from other Hi/PHA and Lo/ PHA mice are reported in Table 3. Whatever the origin of the stimulator cells, the largest thymidine uptake is always obtained with Hi/PHA cells as indicated both by the ratio (Hi/PHA)/(Lo/PHA) (from 1.9 to 17.4) and by the difference Hi/PHA - Lo/PHA (from 990 to 5770 cpm). Similar results were obtained in mice of F, and F,, generation when the data were expressed as the difference in cpm between Hi/ PHA and Lo/PHA mice. Ratios (Hi/PHA)/(Lo/PHA) fluctuated from F, to F,,. A decrease of interline difference was observed after 4-day culture compared with results of 3-day culture, but the difference of response between Hi/PHA and Lo/ PHA mice still remained important. Cells from C57B l/6 and DBA/2 mice were the most efficient stimulators. The weakest response of Hi/PHA was induced when stimulator cells were provided either by a Lo/PHA or by another Hi/PHA mouse. The same observation was made with regard to Lo/PHA response. As for PHA responsiveness, it is useful to compare response to allogeneic stimulation of Hi/PHA and Lo/PHA with that of the four strains. Three- or four-day MLRs were established with two by two reciprocal mixtures of cells from these four strains (Table 4). No striking difference was observed in any strain combination. None of the four strains showed the character of good responder versus any of the other three strains used as stimulators. Cells from DBA/2 mice provoked the
2200
5400
8300
1200
2500
CBA
A
DBA/2
Hi/PHA’
LolPHA
150
90
3300
2840
160
230
Lo/PHA
16.6
13.3
2350
1110
5000
2560
1.9
2.5
2040
3770
Hi/PHA - Lo/PHA
13.4
17.4
(Hi/PHA)/ (Lo/PHA)
1300
1700
6700
2880
3730
4430
Hi/PHA
130
280
930
290
690
780
Lo/PHA
10
6.1
7.2
9.9
5.4
5.6
(Hi/PHA)/ (Lo/PHA)
F 10
1170
1420
5770
2590
3000
3620
HUPHA - Lo/PHA
uptake by responder cells (cpm)
and Lo/PHa Mice
3
1150
1600
5300
160
250
1890
1335
635
2600 3425
1520
Lo/PHA
7.2
6.4
2.8
2.5
4.1
3.5
(Hi/PHA)/ (LolPHA)
4-day cultureC F,
5300
Hi/PHA
a Irradiated lymph node cells. b Four or five mice per experiment. c Mean of two experiments. d Net increase: stimulated value - autologous control value. p HilPHA and LoiPHA stimulator cells are provided by mice different from those used as responders.
4OOOd
Hi/PHA
C57BV6
Stimulator cells”
F,
3-day cultureb
[3H]Thymidine
MLR in HiiPHA
TABLE
990
1350
3410
2090
1965
3780
Hi/PHA - Lo/PHA
w w
34
LIACOPOULOS-BRIOT
ET AL.
TABLE Two
by Two
MLR
4
in Inbred
L”H]Thymidlne
uptake
Strains
of Mice
by responder
cells (cpm)
3.day cultweb Stimulator cells”
C57BLi6
C57BW6
CBA
2910 i 435
DBAI2
2930 -rltxnl
3300 5900
1.24
2650 1100
2165 k 450
1.3
340
I270 t 400
3.36
950
1200
1.2
1300
3200
t
A
I850 -r 650
550 z 350
DBA/Z
3200 ?600
3850 ? 1300
Highest value/ lowest value
A
CBA 265W I ! 600
4.day
f
3700 1200
u Irradiated lymph node cells. D Four or five mice per experiment. ’ Mean of two experiments. d Net increase _f SE: stimulated value - autologous 1750 t 375; A, 1900 t- 145: DBAIZ, 2200 2 375).
control
value
C57BU6
(autologous
control
culture’ value/ value
A
1745
I550
1515
I.15
750
IO50
3.1
2600
2.7
values
DBAi2
Highest lowest
CBA
2900
? SE: C57BLi6,
2.46
I800 5 200; CBA,
highest and cells from A mice the lowest stimulating effect. When the culture time was prolonged from 3 to 4 days, [3H]thymidine incorporation values decreased. Nevertheless the results have essentially the same meaning. From a comparison of the results reported in Tables 3 and 4, it is obvious that, on the whole, responses obtained in the different inbred strain combinations tested are lower or higher than those observed with Hi/PHA and Lo/PHA mice, respectively. For a given stimuc57B16 *
$1
CBA +j t
A 44 .
VPHA
DBAz *
$4
HiIPHA
Hi/PHA
HI/PHA
Hn/PHA - LdPHA
= 17,600
Hr/PHn:5g LolPHA HI/PHA -Lo,T’HA’33,200
H!!=7,15
LO/P&4 HalPHA -Lo/PHA=22,600
HIIPHA=q * Lo/PHA Hn/PHA -Lo/PHA=24,6m
FIG. 1. Effect of 2-ME on MLR responsiveness of Hi/PHA, Lo/PHA (Flo generation), and strains of mice. Cells were cultured during 3 days in 5 x 10m5 M 2-ME-supplemented medium. lator cells were irradiated spleen cells. Each column represents the mean of four experiments crease: experimental value - control value). SE is not indicated but it is comprised between 30% of experimental values.
inbred Stimu(net in10 and
MLR IN MICE OF HIGH AND LOW RESPONDER
35
LINES TO PHA
lator cell, the ratio highest value/lowest value (Table 4), is, on the whole, lower than the ratio (Hi/PHA)/(Lo/PHA) (Table 3). When culture time is prolonged from 3 to 4 days the incorporation values decreased. Nevertheless the results have essentially the same meaning. Effect of 2-ME on the Proliferative
Response
Since 2-ME greatly enhances the proliferation of responder cells in MLR (8,9), the response of the different strain combinations was studied in a culture medium supplemented with 2-ME (Fig. 1). Again the Hi/PHA lymphocytes proved to be the best responders and the Lo/PHA lymphocytes, the poorest responders against every stimulator strain (irradiated spleen cells). Increased values of thymidine incorporation were obtained in every case: 4.6 to 11 times higher for Hi/PHA and 3.6 to 12.4 times higher for Lo/PHA. Consequently the ratios (Hi/PHA)/(Lo/PHA) did not differ much from those obtained in medium free of 2-ME (Table 3, F,, generation). In view of the intensity of the stimulating effect, it appears that the enhancement of the response to 2-ME is also dependent on the origin of the stimulator cells: in this medium CBA mice provided the best stimulator cells. If cell responses of the three inbred strains are compared, A mice gave the highest and DBA/2 mice the lowest response against each of the other strains. Stimulator
Effect of Irradiated
HilPHA
and LoIPHA
Lymphocytes
Hi/PHA and Lo/PHA mice were compared with regard to their ability to stimulate lymphocytes from inbred strains as well as from other Hi/PHA and Lo/PHA mice. Experiments were carried out in medium with or without 2-ME and with irradiated spleen cells as stimulator cells (Fig. 2). It is remarkable that each of the strains was stimulated to the same extent by either Hi/PHA or Lo/PHA cells. Presence of 2-ME in the medium does not modify these results. Among the inbred strains, magnitude of the responses to Hi/PHA and Lo/PHA cells decreases as
-ME
WPHA
#
tME
i
6
A
CBA
HIPHA
Lo/PM
A
CBA
DBAz
CwBl6
WPHA
Lo/PHI
FIG. 2. Stimulatory effects of irradiated Hi/PHA and Lo/PHA spleen cells (F,, generation) on MLR responsiveness of inbred strains (3-day culture without or with 2-ME). Each colum represents the mean of four experiments (net increase and SE as in Fig. 1).
36
LIACOPOULOS-BRIOT
ET AL.
follows: A > CBA > DBA/2 > C57B1/6. The response of Hi/PHA higher than that of Lo/PHA, even toward cells of its own line.
is always
DISCUSSION The overall differences in the proliferative responses to PHA between Hi/PHA and Lo/PHA lymphocytes is not dependent on conditions of culture. Cultures were incubated for 2 or 3 days with or without FCS. In each case, Hi/PHA lymph node cells incorporated a significantly higher amount of thymidine as compared to Lo/ PHA cells. The ratio (Hi/PHA)/(Lo/PHA) decreases in 2-day cultures but increases in 3-day cultures in an FCS-supplemented medium. If interline separation is expressed as the differences of [3H]thymidine incorporation values, then no great variation is observed between results of culture with or without FCS. Increasing numbers of Lo/PHA cells were tested under identical conditions: 1.6 x lo6 Lo/PHA lymph node cells showed a PHA-stimulated DNA synthetic response that was still much lower than that of 4 x lo5 Hi/PHA cells (to be published). The progress in interline separation during selective breeding (F, to F,, generation) is evidenced by the increased ratio as well as by the greater difference between maximal [3H]thymidine uptake of Hi/PHA and Lo/PHA lymphocytes [ratio: 7.3 to 52 and difference 42,800 to 102,000 from F, to F,, mice, respectively, after optimal PHA stimulation (Table l)]. Compared with the four strains studied, cells from Hi/PHA mice always proved to be better responders and cells from Lo/PHA mice worse responders to PHA than cells from any of the four strains (Table 2). In MLR experiments, lymphocytes from Hi/PHA and Lo/PHA differ also in the magnitude of the in vitro proliferative response to allogeneic stimulation. The ratio (Hi/PHA/Lo/PHA) for MLR varies from 1.9 to 17.4 depending on the origin of the stimulator cells. The increase in the interline divergence observed for PHA responsiveness between the F, and the F,, generations is not brought to light in the responsiveness to allogeneic cells. Some changes are observed in the ratio (Hi/PHA)/ (Lo/PHA) between F, and F,, but the overall differences Hi/PHA - Lo/PHA are similar. In all events it appears that, as was the case for mitogen stimulation, Hi/PHA cells are, on the whole, better responders and Lo/PHA cells worse responders to alloantigens than any of the other strains studied. Addition of 2-ME or use of spleen cells instead of lymph node cells as stimulator cells did not modify this conclusion. A correlation between intensity of T lymphocyte responses to mitogens and to allogeneic stimulation has been observed by several authors in inbred strains of rats (lo- 12). Inbred strains of mice have been compared with regard to their sensitivity to PHA stimulation (13- 18). Discrepancies in the results appear which are due to the variation in culture conditions, in the origin of the lymphocytes or of the sera used and in the ways of expressing the results (ratio or increment). On the other hand, various allogeneic combinations have been tested in mixed lymphocyte cultures and these experiments demonstrated a great variability in the intensity of the stimulation (19, 20) depending principally on the pairs of inbred strains used. However, to our knowledge, no comparison has been made between reactivity to PHA and to alloantigens in inbred strains of mice. In our experiments, DBA/2 mice
MLR
IN MICE
OF HIGH
AND
LOW
RESPONDER
LINES
TO PHA
37
were the best and C57B1/6 were the worst responders to PHA (Table 1). In fact, in MLR, DBA/2 mice were not always good responders and C57B1/6 not always bad responders (Table 4, Fig. 1). Similarly, the response of A mice was intermediate to PHA, but to MLR it was either intermediate (Table 4) or higher than those of the other inbred strains (Figs. 1 and 2). An evident correlation appears only in the two selected lines. However, the divergence, as far as MLR is concerned [maximum (Hi/PHA/Lo/PHA): 17.41, is not so high as the divergence for PHA reactivity (20 to 40). It has been reported that the synergy of two T-cell subpopulations [reviewed by Katz (21)] and that the presence of macrophages (22,23) are necessary to induce cell proliferation by MLR as well as by T mitogens. Suppressive T cells [reviewed by Katz (21)] and macrophages or adherent cells (24-27) have been shown to play a part in the regulation of both types of proliferation. Studies have been carried out which show the similitude (28-31) or the discrepancies (32,33) of the characteristics and functions of T-cell subpopulations activated by T mitogens or immunogens. A possible explanation of the differential response to PHA and to MLR of the two lines (Hi/PHA and Lo/PHA) might be a dissimilarity in T-cell subpopulations or a difference in the cooperation between lymphocytes and macrophages. The point of control of phenotypic expression of genetic divergence between the two lines remains to be checked. The question is to find out whether it is due to a higher density of receptors on the cell surface or to a better recognition of antigen or mitogen by cells of Hi/PHA rather than by cells of Lo/PHA. However, it appears that cells of Lo/PHA have retained the capacity of selective recognition: the worst response was observed when they were stimulated by another Lo/PHA or a Hi/PHA. This might be expected in view of the common foundation population of the two lines. In spite of outbreeding, some similitude may be retained at the level of the Ia molecule critical for the process of stimulation in MLR. It should be emphasized that, when used as stimulator cells, Hi/PHA or Lo/PHA lymphocytes induce an equivalent proliferative response versus every responder inbred strain studied. So, the divergence in the ability to respond is not reflected in the ability to stimulate. This fact indicates that MLR is controlled independently of antigenic specificity as has been suggested by others (12, 34, 35). It must be remembered that responsiveness to PHA is submitted to polygenic regulation as shown in our previous work (6). Consequently, it is possible to infer that some of these genes are also implicated in regulation of MLR. The responsiveness to MLR is due to the disparity between the Ia antigens of two individuals but, in addition, the intensity of response is governed by a genetic control dependent on either the same genes as (or on genes linked to) those controlling PHA responsiveness. The results obtained when Hi/PHA and Lo/PHA cells are used as stimulant suggest that these genes would be different from those coding for I region-associated antigens. REFERENCES 1. 2. 3. 4.
Stobo, J. D., Transplant. Rev. 11, 60, 1972. Andersson, J., Sjoberg, O., and Mailer, G., Transplant. Rev. Rich, R. R., and Pierce, C. W., J. Exp. Med. 137, 649, 1973. Pearlmann, P., and Holm, G., Adv. Immunol. 11, 117, 1969.
11, 160, 1972.
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LIACOPOULOS-BRIOT
ET AL.
5. Pick, E., Brostoff, J., Kresjci, J., and Turk, J. L., Cell. Immunol. 1, 92, 1970. 6. Stiffel, C., Liacopoulos-Briot, M., Decreusefond, C., and Lambert, F., Eur. J. Immunol. 7, 291, 1977. 7. Williams, R. M., Cell. Immuno[. 9, 435, 1973. 8. Soulillou, J. P., Carpenter, C. B., Lundin, A. P., and Strom, T. B., J. Immunol. 115, 1566, 1975. 5, 410, 1972. 9. Heber-Katz, E., and Click, R. E., Cell. Immunol. 111, 1579, 1973. 10. Williams, R. M., Moore, M. J., and Benacerraf, B., J. Immunol. 11. Nielsen, H. E., and Koch, C., Stand. J. Immunol. 4, 31, 1975. 12. Raff, H. V., and Hinrichs, D. J., Cell. Immunol. 29, 96, 1977. 13. Adler, W. H., Takiguchi, T., Marsch, B., and Smith, R. T., J. Exp. Med. 131, 1049, 1970. 14. Williams, R. M., and Benacerraf, B., J. Exp. Med. 135, 1279, 1972. 15. Gery, I., Gershon, R. K., and Waksman, B. H., J. Exp. Med. 136, 128, 1972. 16. Donner, M., Vallier, D., and Burg, C., Eur. J. Immunol. 3, 424, 1973. 17. Strong, D. M., Ahmed, A. A., Thurman, G. B., and Sell, K. W., J. Immunol. Met/z. 2, 279, 1973. 18. Heiniger, H. J., Taylor, B. A., Hards, E. J., and Meier, H., Cancer Res. 35, 825, 1975. 19. Phillips, S. M., Carpenter, C. B., and Merrill, J. P., Cell. Immunol. 5, 235, 1972. 20. Bach, F. H., Bach, M. L., Sondel, P. M., and Gnanasigamoni Sundharadas, Transplant Rev. 12, 30, 1972. 21. Katz, D. H., “Lymphocyte Differentiation, Recognition and Regulation,” pp. 467,472. Academic Press, New York, 1977. 22. Alter, B. J., and Bach, F. H., Cell. Immunol. 1, 207, 1970. 23. Rosenstreich, D. L., and Mizel, S. B., Immunol. Rev. 40, 102, 1978. 24. Folch, H., and Waksman, B. H., J. Immunol. 113, 127, 1974. 25. Folch, H., and Waksman, B. H., J. Immunol. 113, 140, 1974. 26. Raff, H. V., and Hinrichs, D. J., Cell. Immunol. 29, 109, 1977. 27. Gillette, R. W., Cell. Immunol. 33, 309, 1977. 28. Dicke, K. A., Tridente, G., and van Bekkum, D. W., Transplantation 8, 422, 1969. 29. Smith, R. T., Transplant. Rev. 11, 178, 1972. 30. Colley, D. G., Shih Wu, A. Y., and Waksman, B. H., J. Exp. Med. 132, 1107, 1970. 31. Watanabe, T., Fathman, C. G., and Coutinho, A., Eur. J. Immunol. 7, 603, 1977. 32. Andersson, L. C., and Hayry, P., Eur. J. Immunol. 3, 595, 1973. 33. Dennert, G., and De Rose, M., J. Immunol. 116, 1601, 1976. 34. Yunis, E. J., Plate, J. M., Ward, F. E., Seigler, H. F., and Amos, D. B., Transplant. Proc. 3, 118, 1971. 35. Marchalonis, J. J., Morris, P. J., and Harris, A. W., J. Immunogenet. 1, 63, 1974.
IMMUNOLOGY
44,
29-38
(1979)
Mixed Lymphocyte Reaction in Mice Genetically Selected High (HVPHA) or Low (Lo/PHA) Responsiveness to Phytohemagglutininl M. LIACOPOULOS-BRIOT,”
C. STIFFEL,?
F. LAMBERT,*
for
AND
C. DECREUSEFOND~ “Institut d’lmmuno-Biologic~, INSERM U 20. CNRS LA 143 Hdpital Broussais, 96, rue Didot, 75674 Paris Cedex 14, and tLaboratoire d’lmmuno-GhnCtique, INSERM lJ 125. CNRS ER 70, lnstitut Curie, 26. rue d’Ulm, 75005 Paris. France Received
July 27, 1978
Lymph node cells from Hi/PHA and LoiPHA mice were evaluated for proliferative response after stimulation by allogeneic lymphocytes (MLR) originating from four inbred strains of different H-2 haplotype (C57B1/6, DBAI2, CBA, A). Reactivity to MLR and PHA were compared in these two lines and in the four inbred strains. The high and low responder status of Hi/PHA and Lo/PHA, as determined by T mitogens lymphocyte responsiveness, was also observed when one measured T responsiveness after MLR. Values obtained with the four inbred strains are included in the range of those measured in Hi/PHA and LoiPHA cells when stimulated by PHA as well as by allogeneic cells. In contrast, when used as stimulator cells, Hi/PHA or Lo/PHA lymphocytes induce an equivalent proliferative response versus every responder inbred strain studied. These experiments support the hypothesis of a common genetic control of proliferative response following PHA or MLR stimulation. The genes implicated would be different from those coding for I region associated antigens.
INTRODUCTION Subpopulations of T lymphocytes are usually characterized according to their surface markers or their functional properties. Attempts have also been made to associate their in vitro responsiveness to specific mitogens and their immunological characteristics (1). Most of the immune functions of T cells can be elicited by phytomitogens, namely, amplification (2) or suppression (3), cytotoxicity (4), and liberation of factors (skin reactive factors, MIF) (5). It was thought that information obtained from an in vitro system with mitogens might increase our understanding of immunological responses. As a practical means of approaching this problem, mice have been genetically selected according to the character “intensity of responsiveness of lymphocytes to PHA.” Two lines were obtained: one (Hi/PHA) with a high response to both PHA and Con A, the other (Lo/PHA) with a low response to these mitogens (6). At 1 Supported by a grant (76-7-1456) from the “Delegation Technique.”
GCnCrale a la Recherche Scientifique et
29 000%8749/79/050029-10$02.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
30
LIACOPOULOS-BRIOT
ET AL.
the maximal interline separation (generation F,,), lymphocytes from Hi/PHA incorporate 39 times higher levels of tritiated thymidine following optimal PHA stimulation than do Lo/PHA lymphocytes. Another characteristic of these two lines is their similar responsiveness to B mitogens: PPD and LPS. A first attempt to evaluate T-cell immune functions of these two lines was made by studying the in vitro response of their lymphocytes, in mixed lymphocyte culture, to various allogeneic stimuli provided by cells from four different inbred strains of mice. MATERIALS
AND METHODS
Mice. Mice genetically equivalent to F,-F,, generations of selective breeding were obtained by a second mating of the same parents. The foundation population of the selection consisted of random-bred albino mice and brother-sister mating was avoided in the course of the selection. So that mice of each selected line were probably not identical at the major histocompatibility locus. Then, each experiment was performed with an individual mouse tested versus the four inbred strains and results from several mice were pooled. Inbred strains were provided by the “Centre de Selection et d’Elevage d’Animaux de Laboratoire” of the C.N.R.S., Orleans, France (A and DBA/2, Jackson origin), Iffa Credo, 69210 L’Arbresle, France (C57B1/6, Jackson origin), and from our own breeding colony (CBA, origin: Radiobiological Research Unit, Harwell, Great Britain). Mitogen and medium. PHA (freeze-dried reagent grade PHA, batch K 3473, Wellcome Lab., Beckenham, Great Britain) was reconstituted in 5 ml distilled water and aliquots of this solution were stored at -20°C. RPMI-1640 (Gibco Biocult., Glasgow, Scotland) supplemented with glutamine (2 mM), penicillin (100 III/ml), and streptomycin (100 pg/ml) was used with or without addition of 10% fetal calf serum (FCS) (Gibco Biocult., from two batches: 7691018 (No. 76) and 86101s (No. 86). h4LR test. Isolated cell suspensions from lymph nodes (inguinal, axillary, and mesenteric) or spleen of one individual mouse were prepared as previously described (6) and resuspended in RPMI-1640 supplemented with 10% FCS. The percentage of viable cells was determined using the trypan blue exclusion method. One-way MLR was performed by mixing equal numbers (4 x 105) of responder cells from lymph node and stimulator cells from spleen or lymph node. Stimulator cells were irradiated with 1750 rads from a 6oCo source. For autologous controls, cells from the same mouse were used as responder and as stimulator. Cell mixtures were set up, in two to three replicates, in round-bottom tissue culture microplates (Cooke Microtiter M-24-l-l 10) in a final volume of 80 ~1 and incubated in a humidified atmosphere of 10% CO, in air at 37°C for 3 or 4 days. In some experiments, 2-mercaptoethanol(2-ME) was added to the culture at a final concentration of 5.10-5 M. PHA response. Various mitogen concentrations (10 to 100 pg/ml) in 25 ~1 of medium were added to 4 x lo5 lymphocytes in 55 ~1 of medium. Cultures were incubated 2 or 3 days under the conditions described above for MLR. MLR and PHA responses were carried out in parallel for each individual mouse of the two lines and of the four inbred strains. Measure of [3H]thymidine incorporation. Cultures were harvested on Days 2,
MLR IN MICE OF HIGH AND LOW RESPONDER
31
LINES TO PHA
3, or 4 after 23 hr labeling with 0.4 +i of [methyl-3H]thymidine (TMM 79, specific activity 2 Ci/mM, C.E.A. Saclay, France) in 25 ~1 of RPMI-1640 and processed according to the technique of Williams (7) as previously described (6). The mean count per minute (cpm) of two or three replicates was calculated. Results were expressed as the net increase in thymidine uptake, i.e., cpm in experimental stimulated cultures minus cpm in control unstimulated cultures (for PHA assays) or minus cpm in autologous control cultures (for MLC assays). Reported values are the means of results obtained with 4 to 10 mice for each experiment. RESULTS PHA Responsiveness
in Cultures Supplemented
with FCS
For several reasons previously explained (6), the usual method for testing the proliferative effect of PHA was performed in FCS-free medium. Attempts to obtain MLR in serum-free medium were unsuccessful under our microculture conditions. So, first of all, we studied the effect of the addition of FCS to the medium on the reactivity of Hi/PHA and Lo/PHA lymphocytes to PHA. Results are reported in Table 1. The maximal net increase in cpm was similar in Hi/PHA mice of F, generation whether or not FCS was added to the medium. A slight decrease in 3H incorporation was observed in cells from mice of F,,, generation when they were cultured during 2 days with FCS, but this effect disappeared in 3-day cultures. In Lo/PHA, the addition of FCS increased the amount of thymidine incorporation in 2-day cultures, but not in 3-day cultures. Higher doses of PHA were required for maximal stimulation with lymphocytes from both lines when the medium was supplemented with FCS. Similar findings were obtained with the two batches of FCS. This first series of experiments demonstrates that the interline difference remains important when PHA responsiveness is measured in the presence of FCS: the ratio (Hi/PHA)/(Lo/PHA) for F,,, is 15.5 to 19 and 33.6 to 40 for 2- and 3-day cultures, respectively. TABLE
1
Effect of Fetal Calf Serum (FCS) on PHA Maximal Responsiveness of Hi/PHA and Lo/PHA Lymphocytes (Mice of F, and F,, Generations of Selective Breeding) Generation of mice and culture time F,” 2 days
FM0 2 days
F,Ob 3 days
Batch of FCS
PHA (&ml)
HiiPHA L”Hlthymidine uptake (cpm)
76 86
20 50 50
49.6Ow 46.100 50,250
76 86
IO 20 20
104.000 78,000 90,900
76 86
IO 15 75
74,000 85,600 84,150
PHA k3W
LoiPHA IIHlthymidine uptake (cm)
(HiIPHA)/(Lo/PHA) 1.3 4.35 2.7
HiiPHA
LoiPHA
50 50 50
6.800 IO.600 18,600
z 8,500 2 2,300 2 4,600
IO 50 20
2,000 2 1,250 4.100 ? 850 5,850 2 1,250
52 19 15.5
102,OOil 74,oM) 85,050
2 7,200 2 7,200 + 5,900
IO 50 50
3,500 e 1,550 2.100 t !ml 2,500 c 750
21 40 33.6
70,500 83,500 81,650
n Mean of two mice in each experiment. b Mean of four to seven mice in each experiment. ( Net increase: experimental cpm - control cpm (?SE)
42,800 35.500 31,600
32
LIACOPOULOS-BRIOT TABLE
ET AL. 2
PHA Responsiveness in Inbred Strains of Mice [:‘H]Thymidine uptake (cpm)
Strains C57BLi6” (4) CBA (4) A (5) DBAi2 (4) HilPHA” (8) Lo/PHA (8)
Controls 1,800 1,560 2,100 1,850 1,300 1,300
2 ? 2 + * 5
260 150 220 100 130 200
50 20,708 35,400 35,650 42,700 81,150 4,175
t f + + -t+
75 6,000* 7,500 5,000 3,800 10,100 1,900
19,300 29,000 35,300 46,000 80,875 3,950
n Number of mice is indicated in parentheses. b Net increase in 2-day cultures (10% FCS supplemented medium): experimental cpm + SE. c Hi/PHA and Lo/PHA are mice of F,, generation.
2 f 2 + 5 2
5,300 6,200 4,800 4,500 11,350 2,900
cpm - control
We have compared the PHA response of both lines to those of the four inbred strains of mice used in MLR experiments (Table 2). Maximal values obtained with these strains are included in the range of those measured in Hi/PHA and Lo/PHA mice [(Hi/PHA)/(Lo/PHA): 19.41. The highest values are found in DBAR and the lowest in C57B1/6. On the other hand, thymidine incorporation values in unstimulated cultures were similar in the two selected lines and slightly lower than in the four inbred strains. Mixed Lymphocyte Cultures
The response of Hi/PHA and Lo/PHA cells to stimulation by irradiated lymph node cells from the four inbred strains and by cells from other Hi/PHA and Lo/ PHA mice are reported in Table 3. Whatever the origin of the stimulator cells, the largest thymidine uptake is always obtained with Hi/PHA cells as indicated both by the ratio (Hi/PHA)/(Lo/PHA) (from 1.9 to 17.4) and by the difference Hi/PHA - Lo/PHA (from 990 to 5770 cpm). Similar results were obtained in mice of F, and F,, generation when the data were expressed as the difference in cpm between Hi/ PHA and Lo/PHA mice. Ratios (Hi/PHA)/(Lo/PHA) fluctuated from F, to F,,. A decrease of interline difference was observed after 4-day culture compared with results of 3-day culture, but the difference of response between Hi/PHA and Lo/ PHA mice still remained important. Cells from C57B l/6 and DBA/2 mice were the most efficient stimulators. The weakest response of Hi/PHA was induced when stimulator cells were provided either by a Lo/PHA or by another Hi/PHA mouse. The same observation was made with regard to Lo/PHA response. As for PHA responsiveness, it is useful to compare response to allogeneic stimulation of Hi/PHA and Lo/PHA with that of the four strains. Three- or four-day MLRs were established with two by two reciprocal mixtures of cells from these four strains (Table 4). No striking difference was observed in any strain combination. None of the four strains showed the character of good responder versus any of the other three strains used as stimulators. Cells from DBA/2 mice provoked the
2200
5400
8300
1200
2500
CBA
A
DBA/2
Hi/PHA’
LolPHA
150
90
3300
2840
160
230
Lo/PHA
16.6
13.3
2350
1110
5000
2560
1.9
2.5
2040
3770
Hi/PHA - Lo/PHA
13.4
17.4
(Hi/PHA)/ (Lo/PHA)
1300
1700
6700
2880
3730
4430
Hi/PHA
130
280
930
290
690
780
Lo/PHA
10
6.1
7.2
9.9
5.4
5.6
(Hi/PHA)/ (Lo/PHA)
F 10
1170
1420
5770
2590
3000
3620
HUPHA - Lo/PHA
uptake by responder cells (cpm)
and Lo/PHa Mice
3
1150
1600
5300
160
250
1890
1335
635
2600 3425
1520
Lo/PHA
7.2
6.4
2.8
2.5
4.1
3.5
(Hi/PHA)/ (LolPHA)
4-day cultureC F,
5300
Hi/PHA
a Irradiated lymph node cells. b Four or five mice per experiment. c Mean of two experiments. d Net increase: stimulated value - autologous control value. p HilPHA and LoiPHA stimulator cells are provided by mice different from those used as responders.
4OOOd
Hi/PHA
C57BV6
Stimulator cells”
F,
3-day cultureb
[3H]Thymidine
MLR in HiiPHA
TABLE
990
1350
3410
2090
1965
3780
Hi/PHA - Lo/PHA
w w
34
LIACOPOULOS-BRIOT
ET AL.
TABLE Two
by Two
MLR
4
in Inbred
L”H]Thymidlne
uptake
Strains
of Mice
by responder
cells (cpm)
3.day cultweb Stimulator cells”
C57BLi6
C57BW6
CBA
2910 i 435
DBAI2
2930 -rltxnl
3300 5900
1.24
2650 1100
2165 k 450
1.3
340
I270 t 400
3.36
950
1200
1.2
1300
3200
t
A
I850 -r 650
550 z 350
DBA/Z
3200 ?600
3850 ? 1300
Highest value/ lowest value
A
CBA 265W I ! 600
4.day
f
3700 1200
u Irradiated lymph node cells. D Four or five mice per experiment. ’ Mean of two experiments. d Net increase _f SE: stimulated value - autologous 1750 t 375; A, 1900 t- 145: DBAIZ, 2200 2 375).
control
value
C57BU6
(autologous
control
culture’ value/ value
A
1745
I550
1515
I.15
750
IO50
3.1
2600
2.7
values
DBAi2
Highest lowest
CBA
2900
? SE: C57BLi6,
2.46
I800 5 200; CBA,
highest and cells from A mice the lowest stimulating effect. When the culture time was prolonged from 3 to 4 days, [3H]thymidine incorporation values decreased. Nevertheless the results have essentially the same meaning. From a comparison of the results reported in Tables 3 and 4, it is obvious that, on the whole, responses obtained in the different inbred strain combinations tested are lower or higher than those observed with Hi/PHA and Lo/PHA mice, respectively. For a given stimuc57B16 *
$1
CBA +j t
A 44 .
VPHA
DBAz *
$4
HiIPHA
Hi/PHA
HI/PHA
Hn/PHA - LdPHA
= 17,600
Hr/PHn:5g LolPHA HI/PHA -Lo,T’HA’33,200
H!!=7,15
LO/P&4 HalPHA -Lo/PHA=22,600
HIIPHA=q * Lo/PHA Hn/PHA -Lo/PHA=24,6m
FIG. 1. Effect of 2-ME on MLR responsiveness of Hi/PHA, Lo/PHA (Flo generation), and strains of mice. Cells were cultured during 3 days in 5 x 10m5 M 2-ME-supplemented medium. lator cells were irradiated spleen cells. Each column represents the mean of four experiments crease: experimental value - control value). SE is not indicated but it is comprised between 30% of experimental values.
inbred Stimu(net in10 and
MLR IN MICE OF HIGH AND LOW RESPONDER
35
LINES TO PHA
lator cell, the ratio highest value/lowest value (Table 4), is, on the whole, lower than the ratio (Hi/PHA)/(Lo/PHA) (Table 3). When culture time is prolonged from 3 to 4 days the incorporation values decreased. Nevertheless the results have essentially the same meaning. Effect of 2-ME on the Proliferative
Response
Since 2-ME greatly enhances the proliferation of responder cells in MLR (8,9), the response of the different strain combinations was studied in a culture medium supplemented with 2-ME (Fig. 1). Again the Hi/PHA lymphocytes proved to be the best responders and the Lo/PHA lymphocytes, the poorest responders against every stimulator strain (irradiated spleen cells). Increased values of thymidine incorporation were obtained in every case: 4.6 to 11 times higher for Hi/PHA and 3.6 to 12.4 times higher for Lo/PHA. Consequently the ratios (Hi/PHA)/(Lo/PHA) did not differ much from those obtained in medium free of 2-ME (Table 3, F,, generation). In view of the intensity of the stimulating effect, it appears that the enhancement of the response to 2-ME is also dependent on the origin of the stimulator cells: in this medium CBA mice provided the best stimulator cells. If cell responses of the three inbred strains are compared, A mice gave the highest and DBA/2 mice the lowest response against each of the other strains. Stimulator
Effect of Irradiated
HilPHA
and LoIPHA
Lymphocytes
Hi/PHA and Lo/PHA mice were compared with regard to their ability to stimulate lymphocytes from inbred strains as well as from other Hi/PHA and Lo/PHA mice. Experiments were carried out in medium with or without 2-ME and with irradiated spleen cells as stimulator cells (Fig. 2). It is remarkable that each of the strains was stimulated to the same extent by either Hi/PHA or Lo/PHA cells. Presence of 2-ME in the medium does not modify these results. Among the inbred strains, magnitude of the responses to Hi/PHA and Lo/PHA cells decreases as
-ME
WPHA
#
tME
i
6
A
CBA
HIPHA
Lo/PM
A
CBA
DBAz
CwBl6
WPHA
Lo/PHI
FIG. 2. Stimulatory effects of irradiated Hi/PHA and Lo/PHA spleen cells (F,, generation) on MLR responsiveness of inbred strains (3-day culture without or with 2-ME). Each colum represents the mean of four experiments (net increase and SE as in Fig. 1).
36
LIACOPOULOS-BRIOT
ET AL.
follows: A > CBA > DBA/2 > C57B1/6. The response of Hi/PHA higher than that of Lo/PHA, even toward cells of its own line.
is always
DISCUSSION The overall differences in the proliferative responses to PHA between Hi/PHA and Lo/PHA lymphocytes is not dependent on conditions of culture. Cultures were incubated for 2 or 3 days with or without FCS. In each case, Hi/PHA lymph node cells incorporated a significantly higher amount of thymidine as compared to Lo/ PHA cells. The ratio (Hi/PHA)/(Lo/PHA) decreases in 2-day cultures but increases in 3-day cultures in an FCS-supplemented medium. If interline separation is expressed as the differences of [3H]thymidine incorporation values, then no great variation is observed between results of culture with or without FCS. Increasing numbers of Lo/PHA cells were tested under identical conditions: 1.6 x lo6 Lo/PHA lymph node cells showed a PHA-stimulated DNA synthetic response that was still much lower than that of 4 x lo5 Hi/PHA cells (to be published). The progress in interline separation during selective breeding (F, to F,, generation) is evidenced by the increased ratio as well as by the greater difference between maximal [3H]thymidine uptake of Hi/PHA and Lo/PHA lymphocytes [ratio: 7.3 to 52 and difference 42,800 to 102,000 from F, to F,, mice, respectively, after optimal PHA stimulation (Table l)]. Compared with the four strains studied, cells from Hi/PHA mice always proved to be better responders and cells from Lo/PHA mice worse responders to PHA than cells from any of the four strains (Table 2). In MLR experiments, lymphocytes from Hi/PHA and Lo/PHA differ also in the magnitude of the in vitro proliferative response to allogeneic stimulation. The ratio (Hi/PHA/Lo/PHA) for MLR varies from 1.9 to 17.4 depending on the origin of the stimulator cells. The increase in the interline divergence observed for PHA responsiveness between the F, and the F,, generations is not brought to light in the responsiveness to allogeneic cells. Some changes are observed in the ratio (Hi/PHA)/ (Lo/PHA) between F, and F,, but the overall differences Hi/PHA - Lo/PHA are similar. In all events it appears that, as was the case for mitogen stimulation, Hi/PHA cells are, on the whole, better responders and Lo/PHA cells worse responders to alloantigens than any of the other strains studied. Addition of 2-ME or use of spleen cells instead of lymph node cells as stimulator cells did not modify this conclusion. A correlation between intensity of T lymphocyte responses to mitogens and to allogeneic stimulation has been observed by several authors in inbred strains of rats (lo- 12). Inbred strains of mice have been compared with regard to their sensitivity to PHA stimulation (13- 18). Discrepancies in the results appear which are due to the variation in culture conditions, in the origin of the lymphocytes or of the sera used and in the ways of expressing the results (ratio or increment). On the other hand, various allogeneic combinations have been tested in mixed lymphocyte cultures and these experiments demonstrated a great variability in the intensity of the stimulation (19, 20) depending principally on the pairs of inbred strains used. However, to our knowledge, no comparison has been made between reactivity to PHA and to alloantigens in inbred strains of mice. In our experiments, DBA/2 mice
MLR
IN MICE
OF HIGH
AND
LOW
RESPONDER
LINES
TO PHA
37
were the best and C57B1/6 were the worst responders to PHA (Table 1). In fact, in MLR, DBA/2 mice were not always good responders and C57B1/6 not always bad responders (Table 4, Fig. 1). Similarly, the response of A mice was intermediate to PHA, but to MLR it was either intermediate (Table 4) or higher than those of the other inbred strains (Figs. 1 and 2). An evident correlation appears only in the two selected lines. However, the divergence, as far as MLR is concerned [maximum (Hi/PHA/Lo/PHA): 17.41, is not so high as the divergence for PHA reactivity (20 to 40). It has been reported that the synergy of two T-cell subpopulations [reviewed by Katz (21)] and that the presence of macrophages (22,23) are necessary to induce cell proliferation by MLR as well as by T mitogens. Suppressive T cells [reviewed by Katz (21)] and macrophages or adherent cells (24-27) have been shown to play a part in the regulation of both types of proliferation. Studies have been carried out which show the similitude (28-31) or the discrepancies (32,33) of the characteristics and functions of T-cell subpopulations activated by T mitogens or immunogens. A possible explanation of the differential response to PHA and to MLR of the two lines (Hi/PHA and Lo/PHA) might be a dissimilarity in T-cell subpopulations or a difference in the cooperation between lymphocytes and macrophages. The point of control of phenotypic expression of genetic divergence between the two lines remains to be checked. The question is to find out whether it is due to a higher density of receptors on the cell surface or to a better recognition of antigen or mitogen by cells of Hi/PHA rather than by cells of Lo/PHA. However, it appears that cells of Lo/PHA have retained the capacity of selective recognition: the worst response was observed when they were stimulated by another Lo/PHA or a Hi/PHA. This might be expected in view of the common foundation population of the two lines. In spite of outbreeding, some similitude may be retained at the level of the Ia molecule critical for the process of stimulation in MLR. It should be emphasized that, when used as stimulator cells, Hi/PHA or Lo/PHA lymphocytes induce an equivalent proliferative response versus every responder inbred strain studied. So, the divergence in the ability to respond is not reflected in the ability to stimulate. This fact indicates that MLR is controlled independently of antigenic specificity as has been suggested by others (12, 34, 35). It must be remembered that responsiveness to PHA is submitted to polygenic regulation as shown in our previous work (6). Consequently, it is possible to infer that some of these genes are also implicated in regulation of MLR. The responsiveness to MLR is due to the disparity between the Ia antigens of two individuals but, in addition, the intensity of response is governed by a genetic control dependent on either the same genes as (or on genes linked to) those controlling PHA responsiveness. The results obtained when Hi/PHA and Lo/PHA cells are used as stimulant suggest that these genes would be different from those coding for I region-associated antigens. REFERENCES 1. 2. 3. 4.
Stobo, J. D., Transplant. Rev. 11, 60, 1972. Andersson, J., Sjoberg, O., and Mailer, G., Transplant. Rev. Rich, R. R., and Pierce, C. W., J. Exp. Med. 137, 649, 1973. Pearlmann, P., and Holm, G., Adv. Immunol. 11, 117, 1969.
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LIACOPOULOS-BRIOT
ET AL.
5. Pick, E., Brostoff, J., Kresjci, J., and Turk, J. L., Cell. Immunol. 1, 92, 1970. 6. Stiffel, C., Liacopoulos-Briot, M., Decreusefond, C., and Lambert, F., Eur. J. Immunol. 7, 291, 1977. 7. Williams, R. M., Cell. Immuno[. 9, 435, 1973. 8. Soulillou, J. P., Carpenter, C. B., Lundin, A. P., and Strom, T. B., J. Immunol. 115, 1566, 1975. 5, 410, 1972. 9. Heber-Katz, E., and Click, R. E., Cell. Immunol. 111, 1579, 1973. 10. Williams, R. M., Moore, M. J., and Benacerraf, B., J. Immunol. 11. Nielsen, H. E., and Koch, C., Stand. J. Immunol. 4, 31, 1975. 12. Raff, H. V., and Hinrichs, D. J., Cell. Immunol. 29, 96, 1977. 13. Adler, W. H., Takiguchi, T., Marsch, B., and Smith, R. T., J. Exp. Med. 131, 1049, 1970. 14. Williams, R. M., and Benacerraf, B., J. Exp. Med. 135, 1279, 1972. 15. Gery, I., Gershon, R. K., and Waksman, B. H., J. Exp. Med. 136, 128, 1972. 16. Donner, M., Vallier, D., and Burg, C., Eur. J. Immunol. 3, 424, 1973. 17. Strong, D. M., Ahmed, A. A., Thurman, G. B., and Sell, K. W., J. Immunol. Met/z. 2, 279, 1973. 18. Heiniger, H. J., Taylor, B. A., Hards, E. J., and Meier, H., Cancer Res. 35, 825, 1975. 19. Phillips, S. M., Carpenter, C. B., and Merrill, J. P., Cell. Immunol. 5, 235, 1972. 20. Bach, F. H., Bach, M. L., Sondel, P. M., and Gnanasigamoni Sundharadas, Transplant Rev. 12, 30, 1972. 21. Katz, D. H., “Lymphocyte Differentiation, Recognition and Regulation,” pp. 467,472. Academic Press, New York, 1977. 22. Alter, B. J., and Bach, F. H., Cell. Immunol. 1, 207, 1970. 23. Rosenstreich, D. L., and Mizel, S. B., Immunol. Rev. 40, 102, 1978. 24. Folch, H., and Waksman, B. H., J. Immunol. 113, 127, 1974. 25. Folch, H., and Waksman, B. H., J. Immunol. 113, 140, 1974. 26. Raff, H. V., and Hinrichs, D. J., Cell. Immunol. 29, 109, 1977. 27. Gillette, R. W., Cell. Immunol. 33, 309, 1977. 28. Dicke, K. A., Tridente, G., and van Bekkum, D. W., Transplantation 8, 422, 1969. 29. Smith, R. T., Transplant. Rev. 11, 178, 1972. 30. Colley, D. G., Shih Wu, A. Y., and Waksman, B. H., J. Exp. Med. 132, 1107, 1970. 31. Watanabe, T., Fathman, C. G., and Coutinho, A., Eur. J. Immunol. 7, 603, 1977. 32. Andersson, L. C., and Hayry, P., Eur. J. Immunol. 3, 595, 1973. 33. Dennert, G., and De Rose, M., J. Immunol. 116, 1601, 1976. 34. Yunis, E. J., Plate, J. M., Ward, F. E., Seigler, H. F., and Amos, D. B., Transplant. Proc. 3, 118, 1971. 35. Marchalonis, J. J., Morris, P. J., and Harris, A. W., J. Immunogenet. 1, 63, 1974.