456 E. Simpson Eur. J. Immunol. 1975.5: 456-461

Elizabeth Simpson Stimulation of mixed lymphocyte cultures and cytotoxic responses: evidence that T cells express SD but not LD antigens, whereas B cells express both Transplantation Biology Section, Division

of Surgical Sciences Clinical Research Centre, Harrow

By separating T and B cells from stimulator spleens it has been found that mixed lymphocyte response (MLR) and cytotoxic cells are generated in responder T cell populations by B but not by T stimulator cells in a wide variety of strain combinations.

It is suggested that MLR-stimulating or LD antigens are expressed on B but not T cells, and that their presence is necessary in order t o generate cototoxic responses t o H-2K and/or H-2D (or SD) antigens which are present on both T and B cells. The finding that B cells and macrophages stimulate MLR is con- sistent with the hypothesis that la antigens, expressed on both these cell types but not on nylon column purified peripheral T cells, are the MLR-stimulating or LD determinants.

1. Introduction

Antigens coded in the mouse major histocompatibility com- plex (MHC) are responsible for in vivo transplant rejection, such as allogeneic tumors and skin grafts [ 11, and for the T cell phenomena of mixed lymphocyte responses (MLR) and the in v i m generation of cytotoxic cells (cell-mediated lympholysis or CML) [2]. Using mouse strain combinations which differ at the whole MHC, both MLR and CML re- sponses occur, both with combinations which differ only at the Ir region, which lies between the H-2K and H-2D ends of this complex, MLR are generated but n o cytotoxic cells [3]. The MLR-stimulating determinants are, therefore, thought t o be predominantly coded in the Ir region(s), and the antigens most likely t o be responsible are the recently described la antigens [4]. From serological and immunochemi- cal evidence, it appears that these antigens are expressed pre- ferentially on B cells [4]. Cytotoxic cells, on the other hand, are directed only against H-2K and H-2D coded antigens, 2.e. the “classical” serologically detected transplantation antigens [S]. Recombinant mouse strains which differ only at these H-2K and H-2D antigens are, however, unable to develop in vifro cytotoxic responses, nor do these strain combinations stimulate each other in MLR [3]. Thus, there is a dichotomy in the MLR and CML responses, inasmuch as they are directed against different antigenic determinants in the MHC, but the MLR appears t o be in some way a prerequisite for the develop- ment of cytotoxic cells. One hypothesis t o account for this interrelation is that the MLR-stimulating antigens are acting as helper determinants, and that cells responding t o such anti- gens can “help” pre-cytotoxic cells differentiate into cytotoxic cells against H-2D and/or H-2K antigens. There is, indeed, a substantial body of evidence that two subclasses of T cells can synergize in the production of cytotoxic cells [6- lo], and that only one of these cell types is the precursor for the cyto- toxic cell [ lo ] . Recent findings using antisera directed against T cell differentiation antigens LY to separate subclasses of pheripheral T cells, show that cells which respond t o MLR antigens are a different population from those from which cytotoxic cells develop: furthermore, such MLR-reactive

[I 10511

Correspondence: Elizabeth Simpson, Transplantation Biology Section, Division of Surgical Sciences, Clinical Research Centre, Watford Road, Harrow H a l 3 UJ, Middlesex, GB

Abbreviations: MLR: Mixed lymphocyte responses CML: Cell- mediated lympholysis BSS: Balanced salt solution FCS: Fetal calf serum

MHC: Major histocompatibility complex

cells appear to be the “helpers” of the precytotoxic cells 111, 121.

Using whole spleen populations t o stimulate MLR and CML responses, it has been found that heat treatment and U/V irradiation prevent such populations being stimulatory in primary responses, although H-2K and H-2D antigens are still intact [3]. These treatments apparently preferentially destroy the expression of MLR-stimulating antigens. This paper reports a method of separating H-2K and H-2D deter- minants from MLR determinants by separation of different peripheral populations of lymphoid cells.

2. Materials and methods

2.1. Mice

The following strains of mice were obtained from the mouse breeding unit at the Clinical Research Centre (CRC):

(B10 x B10.D2)F1, BlO.Br, BlO.A, BlO.A(ZR), BlO.A(4R), CBA/He and DBA/2. BlO.S(7R) and BlO.HTT mice were kindly provided by Dr. Hilliard Festenstein.

BALB/c, C57BL/10, (BALB/c x C57BL/10)FI1 B10.D2,

2.2. Spleen cell suspensions

These were made by teasing spleens with 19 gauge needles in balanced salt solution (BSS) with 5 % fetal calf serum (BSS/ FCS, [ 131) and then passing the suspension through mono- filament nylon gauze to remove cell clumps. The cells were washed once and then the erythrocytes removed with hemo- lytic Geys [ 131. Following two further washes in BSS/FCS the spleen cells were then either ready for use as responder or stimulator “unseparated spleen” cells. For T cell enrich- ment, aliquots of unseparated spleen were loaded onto nylon wool columns, incubated for 45 min at 3 7 OC and then eluted slowly as described [ 141. For B cell enrichment, aliquots of unseparated spleen cells were treated with anti-Thy 1.2 ( 1 ml of 1.4 dilution per 10’ cells) and then guinea pig complement in BSS/FCS (2-stage treatment), followed by washing. Before setting up the cultures, the stimulator cells, unless otherwise stated, were given 2000 rads irradiation from a 6oCo source and then all cells, responder and stimulator, were suspended at 5 x 106/ml in bicarbonate buffered RPMI 1640 supple- mented with 10 % FCS, glutamine, penicillin and strepto- mycin, 1 0 mM HEPES and 5 x 1 O-’ M 2-mercaptoethanal (= complete RPMI medium).

Eur. J. Immunol. 1975.5: 456-461 Mked lymphocyte responses and cell-mediated lympholysis 457

2.3. Culture methods

MLR and CML cultures were set up in parallel using a micro- culture system described in detail elsewhere [ 131. In brief, 0.5 x lo6 responder spleen cells were mixed with the same number of 2000 rad irradiated stimulator spleen cells in 0.2 ml volumes in the wells of flat-bottomed microtest I1 plates (Falcon 3040 or Cooke M220-29 ART). The cultures were set u p in replicates of 6 (MLR) or 1 2 (CML) in com- plete RPMI medium (see above). They were incubated at 36 O C in a humidified atmosphere of 10 % CO,, 7 % 02, 83 % N,. On day 3 or day 4 of culture the MLR were pul- sed with l pCi [3H]thymidine (5 Ci/m mol) per well and then harvested 4 - 6 h later using an automated microhar- vester [ 151. Standard errors were always < 5 %. On day 5 of culture the CML cells (= attacker cells) were harvested from the plates in which they had been sensitized for the cytotoxic assay.

2.4. The cytotoxic assay

An assay was used in which 51Cr-labeled EL-4 (C57BL/10 ascites tumor) P-8 15 (a DBA/2 ascites tumor) or YAC (A strain ascites lymphoma) were used as target cells. One x 1 O5 51 Cr-labeled target cells were mixed with either 4 x 1 05, 2 x lo5 or 1 x l o 5 attacker cells in MEM/lO % FCS; each attacker: target cell ratio was in triplicate or quadruplicate. In early experiments the assay was performed in 3 5 m m dishes in quadruplicate, placed in a 1 0 % C 0 2 gassed box on a rocking platform for 4 h as already described [8], but in later experiments the assay was speeded up by incubating the same number and ratios of attacker and target cells to- gether in 0.2 volumes in the wells of microtest I1 plates in quadruplicate followed by light centrifugation before in- cubating in a humidified 1 0 % C 0 2 atmosphere for 3 h with no rocking (see ref. [ 131 for further details of the micro- method). The results of using either 35 mm or microwells for the assay were concordant, and are not distinguished in the results, but the figure legends indicate which type of assay was used in each experiment. Standard errors of % corrected lysis were < 5 %. At the end of the incubation period the cultures were spun down, either after transfer t o tubes (from t h e 35 mm dished)or by spinning the micro- plate directly. Half the culture supernatant was removed in each case for gamma counting and the % cytotoxicity calculated from the 51Cr released according t o the formula:

(Counts released by sensitized cells) - (counts released by control cells)

(maximum releasable counts) - (machine background)

Corrected % lysis = 100 x

The maximum releasable counts were taken as those released by target cells incubated with 5 % Triton. In most experiments the cytotoxicity is expressed as cytotoxic units, where cyto- toxic units = corrected % lysis a t a given attacker : target cell ratio x recovery ( x 1 O5 ) per well from sensitizing cultures. This gives a measure of the total cytotoxic activity generated per culture.

3. Results

Fig. 1 shows the results of a typical experiment using the BALB/c anti-(C57BL/ 10 x BALB/c)F, combination. In this experiment, as in all experiments unless otherwise stated, the responder cells were splenic T cells (nylon column en- riched) in order to diminish the possible effects of back

stimulation when fully allogeneic combinations were being used (see ref. [ 161) and/or amplification due to B cells of responder origin being “drawn in” t o the response, when allogeneic or semi-allogeneic cells were used as stimulators. In the lower panel on the left hand side of Fig. 1 the MLR response is shown. Unseparated 2000 rad irradiated F 1 spleen cells act as good stimulators of the response, as do B cells prepared from the same spleen cell suspension. How- ever, T cells prepared by nylon wool fractionation from this same F, spleen cell suspension failed to stimulate an MLR. The cytotoxic response shown in the upper left hand panel is concordant with the MLR, and again, splenic T cells have failed t o stimulate.

The lower right hand panel describes the mitogen responses of aliquots of the F, stimulator cell suspensions, taken before irradiation, and the upper right hand panel their mitogen re- sponses after 2000 rad irradiation. From these controls it can be seen that whereas before irradiation the unseparated F, spleen responded both t o LPS (a B cell mitogen) and PHA (a T cell mitogen), following nylon column purification, the background counts and LPS response were much diminished whereas the PHA response was unaffected, or greater. In con- trast, following treatment with anti-Thy 1.2 and complement, the LPS response was unaffected but the PHA response abrog- ated. These results provide evidence for the relative T and B cell enrichment, with concomitant depletion of the other cell type, of the F1 stimulator spleen cells used in this experiment. Such responses were monitored in almost every experiment. Following irradiation, it can be seen from the upper right hand panel of Fig. 1 that both background counts and mitogen responses are reduced t o negligible levels, suggesting that thymidine uptake on the part of the stimulator cells played no part in the MLR responses shown.

Figure 1. Comparison of unseparated T or B F1 (BALBlc x C57BL/10) cells as stimulators of BALB/c splenic T responders in MLR and CML. MLR, CML and mitogen response cultures set up as described in Section 2.3. Unseparated X, TX and BX = FI splenic preparations given 2000 rad. Mitogen responses pulsed at 48 h and harvested at 52 h. MLR pulsed at 72 h and harvested at 76 h. CML assay: attackers harvested and assayed against 51Cr-labeled EL4 at 5 days. 35 mm dish assay: cytotoxic units = corrected lysis at 2 : 1 attacker ; target cell ratios, times cell recovery (x 105) per well.

The experiment shown in Fig. 2 , again using the BALB/c anti-(CS7BL/10 x BALB/c) F l response, was performed t o show that the use of nylon column enriched splenic T cells as responders was not a reason for the failure of splenic F, T cells to stimulate an MLR or cytotoxic response.

458 E.Simpson Eur. J. Immunol. 1975.5: 456-461

CML

101 n

6 1

n

Figure 2. Comparison of unseparated T or B F1 (BALB/c X C57BL/10) lymphocytes or macrophages as stimulators of BALB/c spleen and splenic T as responders in MLR and CML. MLR, CML and mitogen response cultures set up and harvested as for Fig. 1. Ru = unseparated spleen as responder cells (BALBlc). RT = splenic T cells as responder cells (BALB/c). F1 ux = (BALB/c X C57BL/10) F1 unseparated spleen cells given 2000 rad. F l ~ x = F1 splenic T cells given 2000 rad. F l u and FIT as above, not irradiated. F l m = splenic macrophages prepared by absorption onto plastic surface of micro wells at 37 O C for 1 h followed by extensive washing to remove unattached cells. 1 x 107 spleen cells placed in each well, if m@ = 5 % of total, would leave approximately 5 x 105 m@ per well after washing. F1 method. % corrected lysis at 4 : 1 attacker : target cell ratio.

= as above given 2000 rad after washing. CML assay: micro-

In the left hand panels, the first two sets of columns show that both unseparated and splenic T responder cells make good MLR and cytotoxic responses to unseparated irradiated F1 spleen cells as stimulators. In contrast, the next two sets of columns show that neither population can make a response t o irradiated F l splenic T cells, either for MLR or for cytotoxi- city. The fifth column illustrates a further finding, that is, that splenic adherent cells, non-T and non-B according t o their mitogen responses (see panel on right hand side), stimulate both MLR and cytotoxic responses very well, suggesting that they bear both l a and H-2K and H-2D antigens. The right hand panel gives the mitogen responses, prior t o irradiation, of each of the F , stimulator cell populations.

The results obtained for the noncongenic strain combination BALB/c anti-C57BL/ 10 shown in Figs. 1 and 2 were essen- tially repeated when the B10 congenic strains were used, and a summary of the combinations tested, and results, is given in Table 1.

In lines 1 - 6 where there were K and/or D differences in ad- dition t o Ir region differences, and in which cytotoxic re- sponses could be expected, the cytotoxic results were con- cordant with t h e MLR results, and in no case did splenic T cells cause stimulation of either response. In the two strain combinations tested, involving Ir region differences alone, again T cells failed t o stimulate. When M-locus differences were also tested, either in the presence of MHC differences (CBA anti-DBA/2) or in their absence (BALB anti-DBA/2) the MLR results were the same, i.e. whereas unseparated spleen cells stimulated, splenic T cells did not. Figs.3, 4 and 5 show in greater detail individual experiments using various combinations; B 1 O.Br anti-B 1O.A (Ir ABC H-2D differences), BlO.Br anti-B10 (whole MHC), 4R anti-2R (IrB and IrC) and 7R anti-HTT (IrC).

CML tnli-fl-4

n

Figure 3. Comparison of unseparated spleen cells and splenic T cells from BIO.A and B10 to stimulate MLR and CML in B1O.Br splenic T cells. B1O.Br T = splenic T cells from B1O.Br as responders. BIO.Aux = unseparated spleen from BIO.A given 2000 rad as stimu- lators. B~O.AT, = splenic T cells from BlO.A, given 2000 rad as stimulators. BlO.,, = unseparated spleen from B10, 2000 rad as stimulators. B 1 0 . ~ ~ = splenic T cells from B10, 2000 rad as stimu- lators. MLR and CML cultures set up and harvested as for Fig. 1. CML assay: 35 mm dishassay, cytotoxicunits = % corrected lysis at 3 : 1 attacker: target cell ratio, times cell recovery ( X 105) per well. SICr-labeled EL4 and YAC (A lymphoma) as targets.

Table 1. Summary of strain combinations tested in MLR and CML, indicating results of using unseparated spleen cells, splenic T or splenic B cells (all irradiated 2000 rad) as stimulators

Differences at MLR Cl-X Strain combination MHC M Locus Unsep. B T Unsep. B T

BALB/c antiC57BL/10 K Ir S D - +++ +++ -

910 anti-BlO.D2 K I r S D - N P ) N D N D +++ N D - B1O.Br antCBlO K I r S D

BIO.A anti-BlO.DZ K I r ( A t B ) - +++ N D -

++ N D - B 10.91 anti-B 1O.A 4R anti-2R I r ( B & C ) - + N D -

-

B1O.DZ anti-BlO.A K I r ( A % B ) - +++ ++ - - Ir (C) S D

- 7R anti-HTT Ir (C) + + BALB/c anti-DBA/Z -

- CBA anti-DBA/Z K I r S D + +++ N D -

+ +++ N D -

+++ +++ +++ +++ N D + +/+ N D N D N D N D

+++ +++ N D N D ND N D N D N D N D N D

N D - N D N D N D N D a) N D = not done.

Eur. J. Immunol. 1975.5: 456-461 Mixed lymphocyte responses and cell-mediated lympholysis 459

CMIRfSPONSt n

Figure 4. BIO.A (4R) anti-BlO.A(ZR) MLR. Comparison of un- separated and splenic T cells as stimulators of splenic T responders. 4RT = splenic T cells from BlO.A(4R) spleen as responders. 2Ru, = unseparated spleen from BIO.A (2R) given 2000 rad as stimulators. ~ R T ~ = splenic T cells from BIO.A (2R) spleen, given 2000 rad as stimulators. ~ R T = splenic T cells from BIO.A (2R) spleen, as re- sponders. 4Rux = unseparated spleen BIO.A (4R) given 2000 rad as stimulators. MLR cultures set up as Section 2.3. Pulsed at 90 hand harvested at 96 h.

control

MLR RESPONSE

m-

bmo-

cam- :

rn'

- I -

Figure 6. BALB/c anti-(BALBlc x C57BL/lO)Fl MLR and CML re- sponses: effect of T : B ratio of stimulators. MLR and CML cultures set up as in Section 2.3. MLR pulsed at 72 h and harvested at 76 h. BALB/c splenic T cells as responders in all cultures. unsep, = un- separated FI spleen, 2000 rad as stimulators. T, = FI splenic T cells, 2000 rad as stimulators. Bx = F1 splenic B cells, 2000 rad as stimu- lators. Tx . B, 1. 3 etc. = ratios of 2000 rad T, + B, used as stimu- lators. CML assay on day 5 : 35 mm dish assay, 51Cr-labeled EL4 as targets. Cytotoxic units = corrected % lysis at 2 : 1 attacker : target cell ratio x cell recovery ( X 105) per well.

C 1 cnat-u BMB-U w - T

Figure 5. BlO.S(7R) anti-B1O.HTT MLR. Comparison of unseparated splenic T and splenic B cells as stimulators of splenic T responders. Unseparatedx = HTT spleen, unseparated, 2000 rad as stimulators. T, = B10 HTT splenic T 2000 rad as stimulators. B, = B10 HTT splenic B 2000 rad as stimulators. Responder cells = B1O.S (7R) splenic T. Cultures set up as in Section 2.3. Pulsed with 13 HI thymi- dine at 72 h, harvested at 96 h.

In the case of the B1O.Br anti-B10 response shown in Fig. 3 it can be seen that there is a small residual response in MLR and cytotoxicity, when B10 splenic T cells were used t o sti- mulate. This is probably due t o residual contaminating B cells in the T enriched stimulator population, since fluorescent anti-mouse Ig testing of the B10 splenic T cells used in this experiment showed a somewhat higher level of B cell con- tamination than usual (results not shown).

Fig. 6 shows that when nonstimulatory splenic T cells are titrated out against stimulatory splenic B cells, the level of MLR and cytotoxic responses falls off with decreasing numbers of B cells.

Another approach t o the question of whether T cells express MLR antigens necessary for the subsequent expression of cytotoxicity against H-2K and/or H-2D antigens is shown in the experimental results of Fig. 7.

In this experiment C57BL/10 and BALB/c unseparated and splenic T cells were used, and none of the cells were irradiated,

Figure 7. Two-way cytotoxic response CML sensitization: BALB-T = BALB/c splenic T cells. C57BL/lO-T = C57BL/10 splenic T cells BALB-U = unseparated BALB spleen cells. C57BL/U = unseparated C57BL110 spleen cells. CML assay - micromethod: s 1 Cr-labeled EL4 and P815 as targets. 7% specific cytotoxicity at attacker: target cell ratio of 2 : 1 .

therefore opening t h e possibility of a two-way reaction. How- ever, when BALB/c T cells were mixed with unseparated C57BL/10 spleen cells the cytotoxic response was only one way, BALB/c a n t i 4 3 7BL/ 10. Similarly, when C57BL/ 10 T cells were mixed with unseparated BALB/c cells, only a C57BL/10 anti-BALB/c response occurred. When the two T cell populations were mixed together, only very low levels of cytotoxic activity could be detected. This result is con- sistent with the interpretation that only B cells, present in the unseparated population but virtually absent in the T- enriched population, can stimulate an MLR, which is the necessary first step in the development of a cytotoxic re- sponse.

The above result also makes it unlikely that T cells are failing t o stimulate as a result of some change induced in them, but not in B cells, by irradiation. However, to explore this possi- bility, experiments were performed in which an irradiation dose response curve, with respect t o MLR-stimulating capacity, and t o mitogen responsiveness, was done. The results are shown in Figs. 8 and 9. The lower panel of Fig. 8 shows that

460 E. Sirnpson Eur. J . Immunol. 1975.5: 456-461

Stirnumu tells

0

1 rl Figure 8. Effect on MLR and CML of irradiation dose to stimulator cells: BALB anti-(C57BL/10 x BALB/c)F1 response. MLR and CML cultures set up and harvested as in Fig. 1. MLR results: control cpm in left hand histogram, experimental cpm in right hand histogram. CML assay: micro method. Cytotoxic units = % corrected lysis at 2 : 1 attacker : target cell ratio times cell recovery (X 105) per well.

unseparated F spleen cells given no irradiation d o stimulate the MLR, but the background of these cultures is very high When 500 rad, 1000 rad or 2000 rad irradiated F, unseparated spleen cells are used t o stimulate, the background is low and the stimulation excellent. The cytotoxic response t o unsepar- ated spleen stimulator cells is apparently better when the stimulator cells are irradiated (upper panel, Fig. 8). When non-irradiated F , splenic T cells are used, there is a small but apparent MLR which is not accompanied by a significant cytotoxic response. As soon as the FI splenic T cells are given 500 rad they fail t o stimulate either the MLR or a cytotoxic response. The results of the effects of irradiation on the mitogen responses of the same F , stimulator cells used in this experiment are shown on Fig. 9. The unseparated, non- irradiated spleen shows LPS and PHA responses; a t 500 rad the LPS response is obliterated, whereas some, a t least, of the PHA response is retained, and this is true even a t 1000 rad, and t o a lesser extent at 2000 rad. The same persistence of PHA responsiveness a t 500 rad and 1000 rad is shown by the splenic F, T cell preparation. These mitogen responses sug- gest that B cells are more sensitive t o the effects of irradiation than T cells, so that it is unlikely that the difference between T and B cells in their capacity t o stimulate the MLR lies in their radiation sensitivity. The small but apparent MLR shown when non-irradiated F , T cells were used t o stimulate (Fig. 8 lower panel, and Fig. 10) is probably the result of an amplifi- cation mechanism, which draws into the pool of cells incor- porating 3 H the F , T cells themselves. In Fig. 10 is shown an experiment comparing untreated mitomycin-treated and irradiated F I unseparated and splenic T cells as stimulators.

1m.m 1 I 1m.m 1

1

U Svlenit T cells

0

I Brqroundcpm

m LPS stimulated wpm

0 PW stimulated cpm

I Brqroundcpm

m LPS stimulated wpm

0 PW stimulated cpm

Figure 9. Effect of irradiation on mitogen responses of unseparated spleen and splenic T cells - (BALB/c X C57BL/lO)F, spleen. Mitogen cultures set up and harvested as in Section 2.3.

Again, each population of unseparated cells stimulates, al- though the backgrounds for the non-irradiated set are enor- mous, whereas neither mitomycin nor irradiated F, T cells stimulate. Non-irradiated, non-mitomycin-treated F , T cells, although they often give the small MLR responses shown in Figs. 8 and 9, never stimulate significant cytotoxic responses, so they are probably not functionally important, and the MLR responses may very well have a trivial explanation such as the one given above.

4. Discussion

There has been considerable discussion recently as t o the tissue distribution of Ia antigens coded in the Ir A , B and C regions of the MHC. Lymphoid cells, macrophages, epi- dermal cells and spermatocytes are generally agreed t o possess these antigens [ 171 and it is generally agreed that T cells, if they express Ia antigens at all, d o so at a much lower level than B cells, as far as most of the la specificities described are concerned [4]. Epidermal cells are one of the few non-lymphoid tissues t o stimulate proliferative reponses such as those of the MLR [ 181. The genetic data are con- sistent with Ia antigens being the MLR stimulating antigens, but it cannot be excluded that MLR stimulating antigens are not Ia, but are coded for by closely linked genes in the Ir region. The finding reported here that peripheral B cells and

Eur. J. Immunol. 1975.5: 456-461 Mixed lymphocyte responses and cell-mediated lympholysis 461

t r u t d stimulators

7 IPlmic 1 cells control

I rraliated sti mulatwr

Figure 10. Effect of mitomycin or irradiation on unseparated spleen and splenic T cells as stimulators of MLR - BALB anti-(BALB/c X C57BL/10)F1. MLR cultures set up and harvested as in Section 2.3. Mitomycin treatment was incubation for 20 min of cells at 50 x 106/ml with 25 pg/ml mitomycin followed by three washes in BSS/FCS.

macrophages stimulate MLR responses preferentially in com- parison with peripheral T cells is therefore of interest, and is consistent with the hypothesis that Ia antigens are the MLR- stimulating antigens. The need for two signals, one provided by the MLR (LD [3]) antigens, and the second by the H-2K and/or H-2D antigens t o generate cytotoxic cells is also con- firmed here, since splenic T cells possess H-2K and H-2D (SD [3]) antigens, but fail t o stimulate cytotoxic responses.

It could be that splenic T cells separated by the nylon wool technique [ 141 are preferentially depleted of a subpopulation of la-bearing T cells, or that the nylon wool treatment renders inactive any la determinants present o n T cells, but such T cell preparations are functionally inactive in stimulating MLR.

This has proved very useful for introducing SD determinants in the absence of LD determinants, in to the type of three cell experiments for generating CML described by Schendal e t al. [ 191, and the results of these experiments will be reported separately. Since M locus-defined MLR-stimulating antigens [20] also appear to be expressed o n B but not on peripheral T cells [21], the use both of these antigens and the MHC LD antigens as “helper” determinants for the induction of cyto- toxic responses t o SD antigens o n T cells becomes possible.

Iwould like to thank Ms. Moya Taylor for skillful technical assistance.

Received February 11,1975.

5. References

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2 Hayry, P. and Defendi, V., Science 1970. 168: 133. 3 Schendel, D.J. and Bach, F.H., J.Exp.Med. 1974.140: 1534.

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5 Alter, B.J., Schendel, D.J., Bach, M.L., Bach, F.H., Klein, J. and

6 Wagner, H., J.Exp.Med 1973.138: 1379. 7.Tigelaar, R.E. and Feldmann, M., Transplant.Proc. 1973.5: 1711.

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10 Cantor, H. and Simpson, E., Eur.J.IrnmunoL 1975. 5: 330. 11 Cantor, H. and Boyse, A.E., J.Exp.Med, in press. 12 Cantor, H. and Boyse, A.E., J.Exp.Med., in press. 13 Simpson, E., Gordon, R., Taylor, M., Mertin, J. and Chandler, P.,

14 Julius, M., Simpson, E. and Herzenberg, L.A., Eur.J.Immuno1.

15 Sanderson, C.J., Taylor, G.A. and Geary, R., J.Immunol. Method

16.Harrison, M.R. and Paul, W.E., J.Exp.Med. 1973.138: 1602. 17 McDevitt, H.O., Bechtol, K.B., Hammerling, G.J., Lonai, P. and

Stimpfling, J.H., J. Exp. Med. 1973.137: 1303.

Herzenberg, L.A., Cell Immunol. 1975. I S : 180.

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1973.3: 645.

1974.4: 17.

Delovitch, T.L., in Sercarz, E.E., Williamson, A.R. and Fox, C.F. (Eds.) The Immune System, Academic Press, New York 1974.

18 Main, R.K., Cockrum, K.C., Jones, M.J. and Koutz, S.L.,Proc. Nat.Acad.Sci. US 1971. 68: 1165.

19 Schendel, D.J., Alter, B.J. and Bach, F.H., Transplant.Proc. 1973.5: 1651.

20 Festenstein, H., Transplant.Rev. 1973. 15: 62.

21 von Boehmer, H. and Sprent,J., Nature 1974. 249: 363.