CELLULAR
37, 86-95 (1978)
IMMUNOLOGY
Influence
of Killer
Assisting
Factor
of Cytotoxic
T Cells
CHARLES G.OROSZANDJAMES Dept.
(KAF) on Generation
H. FINKED
of Biology and Health Sciences, Cleveland State University, Cleveland, Ohio 44115 and 1 Department of Immunology, Research Division, The Cleveland Clinic Fozlndation, Cleveland, Ohio 44106 Received
October 11.1977
Generation of killer T cells in murine mixed lymphocyte culture required that prekiller cells be presented with both a killer assisting factor(s) (KAF) and certain cellular alloantigens (presumably H-2K or H-2D) early in the sensitization process. Late addition of either KAF or alloantigen-bearing cells to pre-killer cell cultures decreased the levels of cytotoxicity seen by the fifth day of incubation. In both cases this decrease reflected a delay in the development of cytotoxicity. Periodic removal of KAF from mixed lymphocyte cultures showed that maximum cytotoxicity was obtained when KAF was present along with the alloantigen for the full S-day sensitization period. However, a significant cytotoxicity could be detected when contact with KAF, in the presence of alloantigen, was allowed for as few as 48 hr of culture. These observations suggest that both alloantigen and KAF must be experienced together to initiate cell mediated lympholysis (CML) development, and that full CML development requires the continuous presence of KAF thereafter.
INTRODUCTION In vitro development of murine killer T cells with specificity for cellular alloantigens requires cooperation between at least two types of cells: helper T lymphocytes and pre-killer T lymphocytes (1, 2). Furthermore, full differentiation of the latter into cytotoxic effector cells requires two signals : an antigen borne on foreign cells, most likely an H-2K or H-2D gene product (3, 4), and a humoral assisting factor produced by helper T cells upon contact with other foreign cellular antigens, most likely I region associated gene products (2-4). When added at the initiation of in vitro sensitization, in the absence of either helper T cells (5)) or antigens which stimulate helper T cells (6, 7), the assisting factor facilitates the development of killer T cells, provided an allogeneic H-2K or H-2D antigen is also present. We have shown that a soluble factor is required to generate cytotoxicity in mixed lymphocyte cultures (MLCs) in which allogeneic thymocytes are used as stimulators (7). Presumably, since allogeneic thymocytes display few helper T cell stimulating antigens (i.e., Ia antigens) (8)) generation of cytotoxic T cells cannot occur unless an exogenously produced helper T cell signal is provided. The helper cell signal, killer assisting factor (KAF), can be recovered as spleen86 0008-8749/78/0371-0086$02.00/O 6 1978 by Acadkmic P;ess,Inc.
Copyright All rights
of reproduction
in
my
farm
reserved.
KAF
IN
CELL-MEDIATED
LYMPHOLYSIS
87
stimulated MLC supernatant fluids (S-7). We have also shown that KAF is produced by, and acts on, T lymphocytes in promoting the generation of cytotoxic T cells (7). Further, in the presence of allogeneic thymus cells, KAF assists in the generation of cytotoxic T cells which are specific only for the histocompatibility antigens used as stimulators in the MLC. Since KAF-like supernatant fluids can also assist pre-killer T cells which are not histocompatible with the KAF-producing cells (9)) the KAF moiety itself probably lacks strain specificity. Using the assay we have recently described (7), we investigated the requirements for KAF in the in vitro generation of cytotoxic T cells. We found that to generate these cells, the pre-killer T cell must be presented with both KAF and alloantigen very early in the sensitization phase. Apparently CML development does not begin until both KAF and alloantigen are presented together. Furthermore, generation of maximum CML requires continuous KAF presence. MATERIALS
AND
METHODS
Mice. C57BL/6 (H-2b) and CBA/J (H-2k) (Jackson Laboratory, Bar Harbor, Maine), CBA/H-T6 mice (H-2k) ( courtesy of Dr. Bryan Helyer, Metropolitan General Hospital, Cleveland, Ohio), and (CBA/J x C57BL/6) F1 mice, bred in our laboratory were used for these experiments. These mice were of both sexes and were 8 to 12 weeks of age. Cell-mediated cytotoxicity assay. Splenic responder cells, obtained from C57BL/6 mice, were co-cultured with splenic or thymic stimulator cells taken from CBA/ H-T6 mice. Cells from CBA/H-T6 mice intended as stimulators were irradiated with 2000R at lOOR/min (Philips RT 100 X-ray apparatus). After washing, responder and stimulator cells ( lo7 of each) were mixed in 2.5 cm2 tissue culture flasks (Falcon plastics No. 3013) in 10 ml of RPM1 1640 medium supplemented with 15 mM Hepes buffer, 10% fetal calf serum, 1% penicillin-streptomycin (Grand Island Biological Company, Grand Island, N.Y.) and 5 x 1O-5 2-mercaptoethanol (2ME). Some pre-effector cells were cultured in the absence of stimulator cells to produce an unsensitized control population. All cultures were incubated for 5 days at 37°C in 5% COz in air. On the fifth day of incubation, the remaining effector cells were washed, resuspended in fresh media, and co-incubated at ratios of 2: 1 and 4: 1 with lo5 CBA/J target spleen cells. CBA/J mice were used as target cell donors since they display the same MHC target antigens as CBA/H-T6 mice, which were in limited supply. Target cells were pre-cultured for 2 to 3 days in the presence of Concanavalin A (Con A) (1 N Con A/lo6 cells) and then labeled with radioactive sodium chromate (New England Nuclear). For labeling, 20 x lo6 mitogen stimulated target cells were incubated with NaZ5Cr04 (100 &i/30 x lo6 cells) for 90 min in 1.0 cc of complete medium and then washed two times prior to use. Triplicate assays prepared in microtiter plates were centrifuged at 5Og for 10 min, and incubated for 3 hr at 37°C in 5% CO2 in air. Thereafter, 0.1 cc of centrifuged (3009) supernatant fluid from each well was assayed in a gamma counter for released 51Cr. Cytotoxicity was calculated as : y. specific lysis =
CPM (sensitized) CPM (maximum release)
’ loo
Maximum release of 51Cr was obtained by freezing and thawing similar aliquots of lo5 labeled target cells three times. Spontaneous release from target cells was de-
88
OROSZ
AND
FINKE
termined in wells without effector cells. 51Chromium release achieved by nonsensitized effector cells was always less than spontaneous release. In all of these experiments, maximum release ranged from 6 x 103 to 12 x 103 CPM, and spantaneous release averaged 20% of maximum release. Except where indicated, data shown represent observations made in three or more experiments. Production of KAF. C57BL/6 spleen cells (20 x 106) were co-cultured with an equal number of CBA/H-T6 or CBA/J spleen cells that had been x-irradiated with 2000R. The cells were suspended in a final volume of 6.0 cc of complete RPM1 1640 medium containing 5 x 10V5 M 2ME. After 2 days of incubation at 37°C in 5% CO2 in air, the culture supernate was removed, centrifuged at 300g for 10 min, and filtered through 0.45 p Millipore filters. Such supernates were used fresh and after storage at - 79°C. Storage at this temperature, and thawing, produced no noticeable reduction in KAF activity. In all experiments, KAF was added so that it constituted 50% of the final culture volume. Several different lots of KAF were used during the course of these investigations ; some were more active than others. Removal of KAF front cultures. In certain experiments, KAF activity was removed from mixed lymphocyte cultures by carefully withdrawing all but the final 0.5 cc of culture fluid from above the settled, undisturbed cell layer. The volume withdrawn was replaced by gentle addition of fresh media with minimal disturbance of the cells. To control for the residual KAF in these cultures, only 0.5 cc of KAF (diluted 1: 1 with media) was included in other cultures containing thymus cells as stimulators. This small quantity (2.57 o vol/vol) never facilitated significant increases in cytotoxicity over background levels. To control for any effect of disturbance due to replacement of media on the sensitization process, the supernatant was withdrawn .and then returned in some control cultures. These did not differ from unmanipulated cultures in the degree of cytotoxicity generated. RESULTS Splenic T cells can be stimulated by allogeneic thymic cells to become killer cells, provided an assisting T cell humoral factor is present (7). As shown in Table 1, when C57BL/6 splenic effector cells were co-cultured in vitro with irradiated CBA/H-T6 splenic stimulator cells, considerable cytotoxicity against CBA/J targets (51.1%) was generated by the fifth day of culture. If, in place of splenic stimulator cells, thymic stimulator cells were used, only minimal levels of cytotoxicity were induced within this period (10.0%). High levels of cytotoxicity in cultures stimulated by thymic cells were readily obtained, however, when exogenous KAF was added at the initiation of the sensitization period (55.5%). Addition of KAF to cultures of pre-effector cells in the absence of stimulator cells did not result in significant cytotoxicity (2.4%). Similarly, when both KAF and stimulator cells from pre-effector cell cultures were withheld throughout the five day period of incubation, cytotoxic cells were not generated. Hence, two signals are required to produce killer cells, one soluble (KAF), and the other a cellular alloantigen that can be presented by the use of thymic cells as stimulators. To identify the period of time during which KAF has its influence on pre-killer cells, KAF was added at various times after the initiation of cultures stimulated with irradiated thymic cells. When added at time 0, a full cytotoxic response, as compared with cytotoxicity generated in spleen-stimulated MLCs, was measurable
KAF
IN
CELL-MEDIATED
of WI-Mediated
Lympholysis
TABLE Generation
Sensitization
1 Requires
80th Alloantigen Effector
phase
C57BL/6 splenic responder cells co-cultured with irradiated CBA/H-T6 stimulator cells from :
KAFa added to cultures at time 0
Spleen” Thymus Thymus No stimulatorsd
+ -
80
LYMI’HOLYStS
and KAl< _.-__--
_.._
phase
$& specific lysisb on Day 5 at effector : (CBA/J) target cell ratios of -.. 2:l
4:l
34.0 f 8.5 3.5 f 3.2 39.7 f 15.1 0.0
51.1 i 12.4 10.0 f 6.1 55.5 i 16.1 0.0
-
-
a For method of production and concentration of KAF used, see Materials and Methods. b y0 Specific lysis ($&SL) given as the mean of 10 experiments &SD. c In two of the above experiments, KAF was added to spleen-stimulated ML&. The average cytotoxicity subsequently observed was 57.4$$ SL at the 2: 1, and 75.1% SL at the 4:l effector/ target ratios. The average oJ,SL generated in spleen-stimulated MLCs without exogenous KAF in these two experiments was 34.6(r, at 2: 1 and 57.8% at 4: 1 effecter/target ratios. d In one of the above experiments, KAF was added to responder cell cultures in the absence of allogeneic stimulators. Subsequent cytotoxicity was 2.4y0 SL at 4: 1 effecter/target ratio.
5 days later (see Fig. 1). If addition of KAF was delayed by 1 day, cytotoxic cells were still generated, albeit at somewhat reduced levels. Allowing 48 or more hours to elapse before addition of KAF reduced killing capacity to background levels. Since irradiated cells die rapidly in culture, results obtained in these experiments might reflect, in part, a progressive loss of stimulating alloantigens. To control for this, parallel experiments were run using nonirradiated (C57BL/6 X CBA/J)F, spleen or thymus cells as stimulators. Viability studies showed that these cells survive longer in culture than irradiated splenic stimulators (data not shown). In all cases where this was done, little or no anti-parental cytotoxicity could be detected (data not shown), and results were virtually identical to data obtained with irradiated cells as stimulators (see Fig. 1). Thus, in the 5 day MLC, cytotoxicity may still be generated when the KAF signal is received 24 hr after exposure to antigen, but optimal induction of killer cells is seen when KAF is added at the time of initiation of culture. Postponing the KAF signal might simply delay the peak cytotoxic response, of Therefore, KAF was added to thymus-stimulated MLCs either at the initiation culture or after the first 3 days of incubation. Residual viable cells were then assayed for cytotoxic capacity both on Day 5 and Day 8 after initiation of cultures. As shown in Table 2, when added at time 0, KAF induced a good cytotoxic response by Day 5 (45.5%). If KAF addition was delayed by 3 days, a poor CML response, relative to background, was observed on Day 5 (19.0%), but by Day 8 cytotoxicity had continued to develop to a much higher level ( 39.6y0 ) Thus, development of optimal CML activity was delayed when there was a delay in the addition of KAF. AS seen also in Table 2, when KAF was added to thymus-stimulated MLCs on Day 0, cytotoxicity continued to develop beyond Day 5 (45.5%) to an even higher level on day 8 (60.0%). In other similar experiments, it has been repeatedly observed that thymus-stimulated MLCs supplemented with KAF develop a plateau
OROSZ
I 0
24
AND
FISKE
I 46
I 72
96
I NO KAF ADDED
HOUR OF KAF ADDITION AFTER INITIATION OF CULTURE
FIG. 1. Effect on CML of late addition of KAF to thymus-stimulated ML&. In these representative experiments C57BL/6 spleen cells were co-cultured with irradiated CBA/H-T6 thymus cells (0) or nonirradiated (57BL/6 X CBA/J) F1 thymus cells (0). After the designated intervals, KAF was added to the respective cultures as 50% of the culture media. On the 5th day of culture, the surviving cells were utilized as effecters at 2 : 1 (- - - -) or 4 : 1 () ratios against labeled CBA/J target cells. For comparison: MLCs stimulated by irradiated CBA/HT6 spleen cells developed 52.1% specific lysis at the 4: 1 effecter/target ratio; MLCs stimulated by nonirradiated (57BL/6 X CBA/J)K spleen cells developed 49.2% specific lysis at the 4: 1 effecter/target ratio.
of maximal cytotoxic response which usually begins about the fifth day of culture and may last for several days thereafter (see also Table 3). A similar but reduced pattern of CML development was observed in cultures where no exogenous KAF was added. In these cultures, cytotoxicity is probably a result of a weak endogenous production, and slow accumulation of KAF activity, as discussed below. Having determined when pre-effector cells, in the presence of alloantigen, must be presented with KAF, we next examined when pre-effector cells in the presence of KAF must be exposed to the alloantigen. C57BL/6 spleen cells were cultured TABLE
2
Kinetics of CML Development Following Delayed KAF Addition to Thymus-Stimulated $!$Specific lysis
Day of KAF addition relative to initiation of culture’
0 3 None added
MLCs
Day 5
Day 8
2:l
4:l
2:l
4:l
24.4 7.8 7.7
45.5 19.0 15.2
45.1 24.8 17.4
60.0 39.6 30.7
(1In this representative of four experiments, C57BL/6 spleen cells were co-cultured with irradiated CBA/H-T6 thymus cells, and exogenous KAF (50% vol/vol) was added either on Day 0 or Day 3. On Day 5 and Day 8 surviving cells were used as effecters against labeled CBA/J target cells at effecter/target ratios of 2: 1 and 4: 1.
KAF
IN
CELL-MEDIATED
91
LYMPHOLYSTS
TABLE
3
Kinetics of CML Development Following Delayed Addition of Alloantigen to KAF-Supplemented MLCs Effector phase
Sensitization phase Elements included in culture with CS?%L/6 effector cells on Day 0
1. CBA/H-T6 + KAFb 2. KAFb 3. 4. 5. None 6. 7. 8. CBA/H-T6 9. CBA/H-T6
Elements added after initiation of culture
thymusa None CBA/H-T6 thymus”
CBA/H-T6 thymusa + KAFd spleen” thymusa
None None
To Specific lysis
Day of addition
Day 7
Day 5
-
2:l
4:l
49.5 21.2 13.1 7.9 24.4
38.7 34.7 N.D. 5.6 24.4
45.1 43.7 9.0 10.8 35.2
5.0 -0.4 51.4 14.1
8.9 3.0 20.0 16.2
9.4 4.5 33.8 24.0
2:l
4:i
1 2 3 1
37.4 13.0 8.6 4.4 13.5
2 3 -
-0.2 -0.5 38.0 9.0
-
-
a X-irradiated with 2000R. b KAF added as SOY0vol/vol of culture media.
in SO’% KAF and, at specified times, irradiated CBA/H-T6 thymic cells were added. On the fifth and seventh day of incubation, surviving cells were used as effecters against labeled CBA/J target cells. As may be seen in Table 3, maximal specific lysis was obtained when allogeneic stimulator thymic cells were introduced into culture ‘on Day 0 (49.5%). Thereafter, cytotoxicity, when observed on Day 5, declined in proportion to the length of the interval between initiation of culture and the addition of alloantigen, in spite of the presence of KAF from the outset. Thus, to generate cytotoxic T cells, in the S-day MLC, alloantigen, as well as KAF, must be presented to the pre-killer cells very early in the sensitization phase for the maximal lytic response to occur. The reduction in CML activity observed after late addition of alloantigen might reflect a delay in CML development. Indeed, when addition of thymus cells, but not KAF, was delayed by 24 hr, development of optimum cytotoxicity was also delayed (Table 3, line 2). This again suggests that KAF and alloantigen must be present together before cytotoxic cells can be generated against alloantigens. If, however, 48 or more hours were allowed to elapse before alloantigen was added to the cultures, the shift in CML development was no longer observed (Table 3, lines 3 and 4). This was not due to a progressive loss of KAF activity, since an identical pattern of response was observed if the delayed alloantigen was accompanied by addition of fresh KAF (Table 3, lines 5-7). It appears that responder cells cultured 48 hr or more in the absence of alloantigen lose most of their ability to respond to alloantigen. As seen earlier (Table Z), cytotoxic capacity in thymus-stimulated MLCs supplemented on Day 0 with KAF was not significantly reduced by Day 7, relative to Day 5 (Table 3, line 1). This is in marked contrast to spleen-stimulated MLCs, where there was a prominent reduction in cytotoxicity between Day 5 and Day 7
92
OROSZ
AND
FINKE
TABLE Effect on CML
of KAL; Removal
Total time (days) KAF remained in culturea
1 2 3 4 5b No KAF
4 from Thymus-Stimulated
MLC’s
y. Specific lysis at effecter/target ratios of 2:i
4:l
0.3 15.7 21.7 46.9 67.7 -1.7
-0.3 25.9 32.0 64.7 78.7 -3.0
n In this representative of four experiments, cultures of C57BL/6 spleen cells, irradiated CBA/ H-T6 thymic cells, and KAF (500/, vol/vol) were initiated. At the indicated times thereafter, KAF was removed and replaced by normal media. On Day 5, surviving cells were used in 2: 1 and 4: 1 ratios as effecters against labeled CBA/J target cells. b As a control for the technique of KAF removal (See Methods and Materials), in one culture after 48 hr of incubation the media was removed and returned to the same culture. Subsequent cytotoxicity generated in this culture was 79.5% SL at 4: 1 effecter/target ratios. For comparison, C57BL/6 spleen cells, sensitized toward irradiated CBA/H spleen cells, yielded 31.7% specific lysis at 4: 1 effecter/target ratios.
(Table 3, line 8). The reasons for the discrepancy between the two systems are under investigation. Finally, to determine how long KAF must be present in culture to be most effective, thymic-stimulated cultures were initiated, each containing KAF from the outset. At various times thereafter, the KAF-containing supernatant fluid was carefully removed and replaced with fresh medium, as described in Materials and Methods. Under these conditions, KAF was unable to promote cytotoxicity if left in culture for only 24 hr (- 0.3%, see Table 4). However, significant killing was detected when KAF remained in culture for 48 hr (25.9%). Thereafter, the longer KAF remained in culture, the greater was the observed percent specific lysis. Indeed, maximal cytotoxicity was observed only when KAF was present constantly during the 5 day incubation period (78.7%). This pattern of response was observed in several other experiments where less potent lots of KAF induced levels of CML more comparable to those observed in the spleen-stimulated controls. Hence, no importance is attached to the fact that, in this experiment, levels of CML observed after only 3 days of exposure to KAF and alloantigen equaled levels generated in the spleen-stimulated control MLCs. Taken together, these data suggest that the level of cytotoxic ability attained in thymus-stimulated MLCs is proportional to the duration of contact with KAF (see also Table 2). DISCUSSION In an earlier paper (7), we showed that the T cell factor(s), KAF, elevates the low levels of CML generated in thymus-stimulated MLCs to the significantly higher levels observed in spleen-stimulated MLCs. We now show that CML development does not begin until KAF and alloantigen are present together in cultures with pie-killer cells, and that maximal CML development requires the continuous presence of KAF thereafter.
KAF
IN
CELL-MEDIATED
LYMPHOLYSIS
93
During these studies, we consistently observed weak CML development ill thymus-stimulated MLCs, even without the addition of exogenous KAF. We attribute this to low level endogenous production of KAF caused by a small population of cells in the thymus bearing lymphocyte-defined (LD) antigens, and thus capable of stimulating helper cells. Indeed, a small subpopulation of thymus cells appears to display Ia (LD) antigens (10). Subsequent studies have shown that heat-treated allogeneic thymus cells do not stimulate even weak KAF production by normal splenocytes in MLCs, nor do they induce the generation of any detectable CML unless exogenous KAF is included in the culture media (Scott and Finke, manuscript in preparation). Heat treatment of stimulator cells has been shown to functionally inactivate lymphocyte-defined (LD) , but not serologically defined (SD), lymphocyte surface determinants (11). These facts, in conjunction with the observation that removal of KAF within the first 24 hr of the MLC abrogates development of CML (Table 4), indicate that the helper T cell signal, KAF, is prohably essential for generation of cytotoxic lymphocytes and does not simply amplify an otherwise weak response by antigen-stimulated pre-killer T cells. All data presented in this paper have been derived from experiments in which the assay cultures used to determine KAF activity utilize strain combinations which are homologous with the combinations used to produce the KAF-containing supernatants. In other experiments, we have observed that like spleen-stimulated MLCs, cytotoxic activity generated in thymus-stimulated MLCs supplemented with KAF is restricted to cells of the haplotype experienced by the responder cells during sensitization (7). However, KAF activity is not specific with respect to the haplotype of the responder cell it can assist. Furthermore, KAF can assist in the development of cytotoxicity against haplotypes unrelated to those used to stimulate its production (Orosz and Finke, manuscript in preparation). Similar observations have been made about the factor(s) investigated by Plate (9) and Altman and Cohen (1.2). KAF, however, is not the only requisite signal for pre-killer T cell differentiation, as evidenced by the fact that KAF is incapable of inducing cytotoxicity in MLC responder populations unless allogeneic cells are included (9, Table 1) . (This also suggests that KAF activity probably is not attributable just to solubilized alloantigen or cell membrane fragments carried over from KAF-producing MLCs.) Further, when either signal is delayed (alloantigen for up to 24 hr, KAF for up to 72 hr) there is a shift in CML generation kinetics. Bach has also reported a similar shift when either the cell-bound SD or LD stimulus is withheld from MLCs for 24 hr (13). This suggests that (a) alloantigen and KAF must be experienced together before primary killer T cells can begin to develop and (b) the development of primary CML activity takes a minimum of 3-4 days after both signals have been received. We observed that, in contrast to thymus-stimulated MLCs where addition of KAF was delayed, no significant cytotoxicity could be generated in MLCs where alloantigen was withheld for 48 or more hours, even if the day of assay was likewise delayed (Table 3). Apparently, pre-effector cells cultured for 48 hr in the absence of stimulating alloantigens appear to lose their ability to generate CML activity. Hodes and Hathcock have described a suppressor T cell which develops under these same conditions (14). If, however, alloantigen in the form of thymocytes was added within 24 hr of culture initiation, responder cells retain their ability to develop CML activity (Table 3).
94
OROSZ
AND
FINKE
In the course of these experiments, we also observed that in thymus-stimulated MLCs supplemented with KAF, cytotoxicity observed on Day 7 or 8 was essentially undiminished from that seen on Day 5 (Tables 2 and 3). This is in contrast to spleen-stimulated MLCs, in which marked reduction in cytotoxicity was observed by this time (Table 3). These observations suggest that thymus-stimulated MLCs might lack a regulatory mechanism, which is operative in spleen-stimulated MLCs. Such a possibility is the subject of our continuing investigations. Another feature of KAF activity is that net cytotoxicity developed during 5 days of culture appears to be dependent on duration of pre-effector cell contact with KAF, while in the presence of alloantigen (Table 4). This suggests that prekiller cells need multiple or constant input from KAF, and that without it, CML development terminates. Another possibility is that more than one signal is present in the KAF preparation: one acting early in the development process, another somewhat later. In this case, early removal of KAF would eliminate a terminal signal, resulting in decreased net cytotoxicity. Altman and Cohen have shown that 48 hr of contact with mixed lymphocyte culture supernatant fluid is sufficient to induce maximal cytotoxicity by fibroblast-stimulated pre-killer cells ( 15). We have observed that 48 hr of contact with KAF, does, indeed, result in significant cytotoxicity, but that maximal cytotoxicity results only when KAF is present for the full 5 day sensitization period (Table 4). As has been shown elsewhere, the effector populations in these two systems may differ, at least on the basis of hydrocortisone sensitivity (16), and disparity in results may simply reflect this difference. Of interest, however, is the fact that, in both systems, only 24 hr of contact with soluble factor is insufficient for induction of cytotoxicity. The reciprocal experiments, based on removal of stimulator cells from pre-killer/KAF cultures at intervals after culture initiation, would have been advantageous, but are not technically feasible in our assay system. If, as has been suggested, development of cytotoxic cells in 5 day MLCs is controlled by (a) absolute concentration of KAF (5), (b) time of first exposure to both KAF and alloantigen and/or (c) duration of contact with KAF, one would predict that even spleen-stimulated MLCs should exhibit enhanced cytotoxicity if cultured with exogenous KAF. Indeed, enhancement of CML under these conditions has been observed (Table 1, footnote c). We consider this as supportive evidence for the foregoing observations. In a more general context, the mechanism of in vitro sensitization for cellmediated lympholysis is somewhat similar to that for in vitro antibody production in that both depend on a signal from a soluble factor. Nonetheless, several differences between these two immunological systems exist at the level of the helper factors. For example, allogeneic effect factor (AEF), the humoral signal active in production of IgG, must be presented to B cells no sooner than 48 hr after the presentation of antigen to achieve a full primary antibody response (17). In contrast, KAF, the humoral signal active in generating CML, must be present initially in the cultures ; a delay of 48 hr does not permit development of cytotoxicity by the fifth day of culture (Fig. 1) . Secondly, a delay beyond 48 hr of culture in the addition of AEF to B cells and antigen does not postpone maximal plaque-forming responses (Yen, personal communication) whereas delay in KAF addition up to 72 hr to T cells and antigens simply retards development of peak CML activity by a similar length of time (Table 2). Third, the B cell need be in contact with AEF only briefly (1 hr) ; AEF may then be removed from culture, with little or no
KAF
IN
CELL-MEDIATED
LYMPHOLYSIS
9.5
reduction iu full antibody response (18). On the other hand, T cells need prolonged contact with KAF and removal of KAF from such culture reduces further CML generation (Table 4). These observations indicate that certain differences exists between AEF and KAF activities. Both activities, however, can be recovered from the same mixed lymphocyte culture supernates. Whether KAF and AEF are distinct molecules with separate functions or one molecule with multiple functions is not known. Furthermore, like AEF, KAF activity may even be mediated by several molecules. Resolution of these possibilites awaits molecular characterization of KAF activities, as has been done for AEF (19). ACKNOWLEDGMENTS This work was supported in part by Grant Number 12468, awarded to J. R. Battisto by the National Institute of Allergy and Infectious Diseases and the American Cancer Society. We would like to thank Ms. M. Wright for typing the manuscript.
REFERENCES 1. Wagner, H., I. Exp. Med. 138, 1379, 1973. 2. Cantor, H., and Boyse, E., J. Exp. Med. 141, 1390, 1975. 3. Bach, F., J. Allergy Cl&. Immunol. 57, 9.5, 1976. 4. Schreffler, D., and David, C., Adv. Immunol. 20, 125, 1975. 5. Plate, J., Nature 260, 329, 1976. 6. Altman, A., and Cohen, I., Eur. J. Immunol. 5, 437, 1975. 7. Finke, J., Orosz, C., and Battisto, J., Nature 267, 353, 1977. 8. Niederhuber, J., and Frelinger, J., Transplant. Rev. 30, 101, 1976. 9. Plate, J., Cell. Immzcnol. 32, 183, 1977. 10. Frelinger, J., Niederhuber, J., David, C., and Shreffler, D., J. Exp. Med. 140, 1273, 1974. 11. Schendel, D., and Bach, F., J. Exp. Med. 140, 1534, 1974. 12. Altman, A., and Cohen, I., Eur. J. Immunol. 5, 437, 1975. 13. Bach, F., Bach, M., and Sondel, P., Nature 259, 273, 1976. 14. Hodes, R., and Hathcock, K., J. Immunol. 116, 167, 1976. 15. Altman, A., and Cohen, I., Adv. Exp. Med. Biol. 66, 289, 1976. 16. Altman, A., and Cohen, I., J. Exp. Med. 142, 790, 1975. 17. Schimpl, A., and Wecker, E., J. Exp. Med. 137, 547, 1973. 18. Yen, B., and Battisto, J., J. Immunol. (in press). 19. Watson, J., J. Immunol. 111, 1301, 1973.
37, 86-95 (1978)
IMMUNOLOGY
Influence
of Killer
Assisting
Factor
of Cytotoxic
T Cells
CHARLES G.OROSZANDJAMES Dept.
(KAF) on Generation
H. FINKED
of Biology and Health Sciences, Cleveland State University, Cleveland, Ohio 44115 and 1 Department of Immunology, Research Division, The Cleveland Clinic Fozlndation, Cleveland, Ohio 44106 Received
October 11.1977
Generation of killer T cells in murine mixed lymphocyte culture required that prekiller cells be presented with both a killer assisting factor(s) (KAF) and certain cellular alloantigens (presumably H-2K or H-2D) early in the sensitization process. Late addition of either KAF or alloantigen-bearing cells to pre-killer cell cultures decreased the levels of cytotoxicity seen by the fifth day of incubation. In both cases this decrease reflected a delay in the development of cytotoxicity. Periodic removal of KAF from mixed lymphocyte cultures showed that maximum cytotoxicity was obtained when KAF was present along with the alloantigen for the full S-day sensitization period. However, a significant cytotoxicity could be detected when contact with KAF, in the presence of alloantigen, was allowed for as few as 48 hr of culture. These observations suggest that both alloantigen and KAF must be experienced together to initiate cell mediated lympholysis (CML) development, and that full CML development requires the continuous presence of KAF thereafter.
INTRODUCTION In vitro development of murine killer T cells with specificity for cellular alloantigens requires cooperation between at least two types of cells: helper T lymphocytes and pre-killer T lymphocytes (1, 2). Furthermore, full differentiation of the latter into cytotoxic effector cells requires two signals : an antigen borne on foreign cells, most likely an H-2K or H-2D gene product (3, 4), and a humoral assisting factor produced by helper T cells upon contact with other foreign cellular antigens, most likely I region associated gene products (2-4). When added at the initiation of in vitro sensitization, in the absence of either helper T cells (5)) or antigens which stimulate helper T cells (6, 7), the assisting factor facilitates the development of killer T cells, provided an allogeneic H-2K or H-2D antigen is also present. We have shown that a soluble factor is required to generate cytotoxicity in mixed lymphocyte cultures (MLCs) in which allogeneic thymocytes are used as stimulators (7). Presumably, since allogeneic thymocytes display few helper T cell stimulating antigens (i.e., Ia antigens) (8)) generation of cytotoxic T cells cannot occur unless an exogenously produced helper T cell signal is provided. The helper cell signal, killer assisting factor (KAF), can be recovered as spleen86 0008-8749/78/0371-0086$02.00/O 6 1978 by Acadkmic P;ess,Inc.
Copyright All rights
of reproduction
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stimulated MLC supernatant fluids (S-7). We have also shown that KAF is produced by, and acts on, T lymphocytes in promoting the generation of cytotoxic T cells (7). Further, in the presence of allogeneic thymus cells, KAF assists in the generation of cytotoxic T cells which are specific only for the histocompatibility antigens used as stimulators in the MLC. Since KAF-like supernatant fluids can also assist pre-killer T cells which are not histocompatible with the KAF-producing cells (9)) the KAF moiety itself probably lacks strain specificity. Using the assay we have recently described (7), we investigated the requirements for KAF in the in vitro generation of cytotoxic T cells. We found that to generate these cells, the pre-killer T cell must be presented with both KAF and alloantigen very early in the sensitization phase. Apparently CML development does not begin until both KAF and alloantigen are presented together. Furthermore, generation of maximum CML requires continuous KAF presence. MATERIALS
AND
METHODS
Mice. C57BL/6 (H-2b) and CBA/J (H-2k) (Jackson Laboratory, Bar Harbor, Maine), CBA/H-T6 mice (H-2k) ( courtesy of Dr. Bryan Helyer, Metropolitan General Hospital, Cleveland, Ohio), and (CBA/J x C57BL/6) F1 mice, bred in our laboratory were used for these experiments. These mice were of both sexes and were 8 to 12 weeks of age. Cell-mediated cytotoxicity assay. Splenic responder cells, obtained from C57BL/6 mice, were co-cultured with splenic or thymic stimulator cells taken from CBA/ H-T6 mice. Cells from CBA/H-T6 mice intended as stimulators were irradiated with 2000R at lOOR/min (Philips RT 100 X-ray apparatus). After washing, responder and stimulator cells ( lo7 of each) were mixed in 2.5 cm2 tissue culture flasks (Falcon plastics No. 3013) in 10 ml of RPM1 1640 medium supplemented with 15 mM Hepes buffer, 10% fetal calf serum, 1% penicillin-streptomycin (Grand Island Biological Company, Grand Island, N.Y.) and 5 x 1O-5 2-mercaptoethanol (2ME). Some pre-effector cells were cultured in the absence of stimulator cells to produce an unsensitized control population. All cultures were incubated for 5 days at 37°C in 5% COz in air. On the fifth day of incubation, the remaining effector cells were washed, resuspended in fresh media, and co-incubated at ratios of 2: 1 and 4: 1 with lo5 CBA/J target spleen cells. CBA/J mice were used as target cell donors since they display the same MHC target antigens as CBA/H-T6 mice, which were in limited supply. Target cells were pre-cultured for 2 to 3 days in the presence of Concanavalin A (Con A) (1 N Con A/lo6 cells) and then labeled with radioactive sodium chromate (New England Nuclear). For labeling, 20 x lo6 mitogen stimulated target cells were incubated with NaZ5Cr04 (100 &i/30 x lo6 cells) for 90 min in 1.0 cc of complete medium and then washed two times prior to use. Triplicate assays prepared in microtiter plates were centrifuged at 5Og for 10 min, and incubated for 3 hr at 37°C in 5% CO2 in air. Thereafter, 0.1 cc of centrifuged (3009) supernatant fluid from each well was assayed in a gamma counter for released 51Cr. Cytotoxicity was calculated as : y. specific lysis =
CPM (sensitized) CPM (maximum release)
’ loo
Maximum release of 51Cr was obtained by freezing and thawing similar aliquots of lo5 labeled target cells three times. Spontaneous release from target cells was de-
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termined in wells without effector cells. 51Chromium release achieved by nonsensitized effector cells was always less than spontaneous release. In all of these experiments, maximum release ranged from 6 x 103 to 12 x 103 CPM, and spantaneous release averaged 20% of maximum release. Except where indicated, data shown represent observations made in three or more experiments. Production of KAF. C57BL/6 spleen cells (20 x 106) were co-cultured with an equal number of CBA/H-T6 or CBA/J spleen cells that had been x-irradiated with 2000R. The cells were suspended in a final volume of 6.0 cc of complete RPM1 1640 medium containing 5 x 10V5 M 2ME. After 2 days of incubation at 37°C in 5% CO2 in air, the culture supernate was removed, centrifuged at 300g for 10 min, and filtered through 0.45 p Millipore filters. Such supernates were used fresh and after storage at - 79°C. Storage at this temperature, and thawing, produced no noticeable reduction in KAF activity. In all experiments, KAF was added so that it constituted 50% of the final culture volume. Several different lots of KAF were used during the course of these investigations ; some were more active than others. Removal of KAF front cultures. In certain experiments, KAF activity was removed from mixed lymphocyte cultures by carefully withdrawing all but the final 0.5 cc of culture fluid from above the settled, undisturbed cell layer. The volume withdrawn was replaced by gentle addition of fresh media with minimal disturbance of the cells. To control for the residual KAF in these cultures, only 0.5 cc of KAF (diluted 1: 1 with media) was included in other cultures containing thymus cells as stimulators. This small quantity (2.57 o vol/vol) never facilitated significant increases in cytotoxicity over background levels. To control for any effect of disturbance due to replacement of media on the sensitization process, the supernatant was withdrawn .and then returned in some control cultures. These did not differ from unmanipulated cultures in the degree of cytotoxicity generated. RESULTS Splenic T cells can be stimulated by allogeneic thymic cells to become killer cells, provided an assisting T cell humoral factor is present (7). As shown in Table 1, when C57BL/6 splenic effector cells were co-cultured in vitro with irradiated CBA/H-T6 splenic stimulator cells, considerable cytotoxicity against CBA/J targets (51.1%) was generated by the fifth day of culture. If, in place of splenic stimulator cells, thymic stimulator cells were used, only minimal levels of cytotoxicity were induced within this period (10.0%). High levels of cytotoxicity in cultures stimulated by thymic cells were readily obtained, however, when exogenous KAF was added at the initiation of the sensitization period (55.5%). Addition of KAF to cultures of pre-effector cells in the absence of stimulator cells did not result in significant cytotoxicity (2.4%). Similarly, when both KAF and stimulator cells from pre-effector cell cultures were withheld throughout the five day period of incubation, cytotoxic cells were not generated. Hence, two signals are required to produce killer cells, one soluble (KAF), and the other a cellular alloantigen that can be presented by the use of thymic cells as stimulators. To identify the period of time during which KAF has its influence on pre-killer cells, KAF was added at various times after the initiation of cultures stimulated with irradiated thymic cells. When added at time 0, a full cytotoxic response, as compared with cytotoxicity generated in spleen-stimulated MLCs, was measurable
KAF
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of WI-Mediated
Lympholysis
TABLE Generation
Sensitization
1 Requires
80th Alloantigen Effector
phase
C57BL/6 splenic responder cells co-cultured with irradiated CBA/H-T6 stimulator cells from :
KAFa added to cultures at time 0
Spleen” Thymus Thymus No stimulatorsd
+ -
80
LYMI’HOLYStS
and KAl< _.-__--
_.._
phase
$& specific lysisb on Day 5 at effector : (CBA/J) target cell ratios of -.. 2:l
4:l
34.0 f 8.5 3.5 f 3.2 39.7 f 15.1 0.0
51.1 i 12.4 10.0 f 6.1 55.5 i 16.1 0.0
-
-
a For method of production and concentration of KAF used, see Materials and Methods. b y0 Specific lysis ($&SL) given as the mean of 10 experiments &SD. c In two of the above experiments, KAF was added to spleen-stimulated ML&. The average cytotoxicity subsequently observed was 57.4$$ SL at the 2: 1, and 75.1% SL at the 4:l effector/ target ratios. The average oJ,SL generated in spleen-stimulated MLCs without exogenous KAF in these two experiments was 34.6(r, at 2: 1 and 57.8% at 4: 1 effecter/target ratios. d In one of the above experiments, KAF was added to responder cell cultures in the absence of allogeneic stimulators. Subsequent cytotoxicity was 2.4y0 SL at 4: 1 effecter/target ratio.
5 days later (see Fig. 1). If addition of KAF was delayed by 1 day, cytotoxic cells were still generated, albeit at somewhat reduced levels. Allowing 48 or more hours to elapse before addition of KAF reduced killing capacity to background levels. Since irradiated cells die rapidly in culture, results obtained in these experiments might reflect, in part, a progressive loss of stimulating alloantigens. To control for this, parallel experiments were run using nonirradiated (C57BL/6 X CBA/J)F, spleen or thymus cells as stimulators. Viability studies showed that these cells survive longer in culture than irradiated splenic stimulators (data not shown). In all cases where this was done, little or no anti-parental cytotoxicity could be detected (data not shown), and results were virtually identical to data obtained with irradiated cells as stimulators (see Fig. 1). Thus, in the 5 day MLC, cytotoxicity may still be generated when the KAF signal is received 24 hr after exposure to antigen, but optimal induction of killer cells is seen when KAF is added at the time of initiation of culture. Postponing the KAF signal might simply delay the peak cytotoxic response, of Therefore, KAF was added to thymus-stimulated MLCs either at the initiation culture or after the first 3 days of incubation. Residual viable cells were then assayed for cytotoxic capacity both on Day 5 and Day 8 after initiation of cultures. As shown in Table 2, when added at time 0, KAF induced a good cytotoxic response by Day 5 (45.5%). If KAF addition was delayed by 3 days, a poor CML response, relative to background, was observed on Day 5 (19.0%), but by Day 8 cytotoxicity had continued to develop to a much higher level ( 39.6y0 ) Thus, development of optimal CML activity was delayed when there was a delay in the addition of KAF. AS seen also in Table 2, when KAF was added to thymus-stimulated MLCs on Day 0, cytotoxicity continued to develop beyond Day 5 (45.5%) to an even higher level on day 8 (60.0%). In other similar experiments, it has been repeatedly observed that thymus-stimulated MLCs supplemented with KAF develop a plateau
OROSZ
I 0
24
AND
FISKE
I 46
I 72
96
I NO KAF ADDED
HOUR OF KAF ADDITION AFTER INITIATION OF CULTURE
FIG. 1. Effect on CML of late addition of KAF to thymus-stimulated ML&. In these representative experiments C57BL/6 spleen cells were co-cultured with irradiated CBA/H-T6 thymus cells (0) or nonirradiated (57BL/6 X CBA/J) F1 thymus cells (0). After the designated intervals, KAF was added to the respective cultures as 50% of the culture media. On the 5th day of culture, the surviving cells were utilized as effecters at 2 : 1 (- - - -) or 4 : 1 () ratios against labeled CBA/J target cells. For comparison: MLCs stimulated by irradiated CBA/HT6 spleen cells developed 52.1% specific lysis at the 4: 1 effecter/target ratio; MLCs stimulated by nonirradiated (57BL/6 X CBA/J)K spleen cells developed 49.2% specific lysis at the 4: 1 effecter/target ratio.
of maximal cytotoxic response which usually begins about the fifth day of culture and may last for several days thereafter (see also Table 3). A similar but reduced pattern of CML development was observed in cultures where no exogenous KAF was added. In these cultures, cytotoxicity is probably a result of a weak endogenous production, and slow accumulation of KAF activity, as discussed below. Having determined when pre-effector cells, in the presence of alloantigen, must be presented with KAF, we next examined when pre-effector cells in the presence of KAF must be exposed to the alloantigen. C57BL/6 spleen cells were cultured TABLE
2
Kinetics of CML Development Following Delayed KAF Addition to Thymus-Stimulated $!$Specific lysis
Day of KAF addition relative to initiation of culture’
0 3 None added
MLCs
Day 5
Day 8
2:l
4:l
2:l
4:l
24.4 7.8 7.7
45.5 19.0 15.2
45.1 24.8 17.4
60.0 39.6 30.7
(1In this representative of four experiments, C57BL/6 spleen cells were co-cultured with irradiated CBA/H-T6 thymus cells, and exogenous KAF (50% vol/vol) was added either on Day 0 or Day 3. On Day 5 and Day 8 surviving cells were used as effecters against labeled CBA/J target cells at effecter/target ratios of 2: 1 and 4: 1.
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LYMPHOLYSTS
TABLE
3
Kinetics of CML Development Following Delayed Addition of Alloantigen to KAF-Supplemented MLCs Effector phase
Sensitization phase Elements included in culture with CS?%L/6 effector cells on Day 0
1. CBA/H-T6 + KAFb 2. KAFb 3. 4. 5. None 6. 7. 8. CBA/H-T6 9. CBA/H-T6
Elements added after initiation of culture
thymusa None CBA/H-T6 thymus”
CBA/H-T6 thymusa + KAFd spleen” thymusa
None None
To Specific lysis
Day of addition
Day 7
Day 5
-
2:l
4:l
49.5 21.2 13.1 7.9 24.4
38.7 34.7 N.D. 5.6 24.4
45.1 43.7 9.0 10.8 35.2
5.0 -0.4 51.4 14.1
8.9 3.0 20.0 16.2
9.4 4.5 33.8 24.0
2:l
4:i
1 2 3 1
37.4 13.0 8.6 4.4 13.5
2 3 -
-0.2 -0.5 38.0 9.0
-
-
a X-irradiated with 2000R. b KAF added as SOY0vol/vol of culture media.
in SO’% KAF and, at specified times, irradiated CBA/H-T6 thymic cells were added. On the fifth and seventh day of incubation, surviving cells were used as effecters against labeled CBA/J target cells. As may be seen in Table 3, maximal specific lysis was obtained when allogeneic stimulator thymic cells were introduced into culture ‘on Day 0 (49.5%). Thereafter, cytotoxicity, when observed on Day 5, declined in proportion to the length of the interval between initiation of culture and the addition of alloantigen, in spite of the presence of KAF from the outset. Thus, to generate cytotoxic T cells, in the S-day MLC, alloantigen, as well as KAF, must be presented to the pre-killer cells very early in the sensitization phase for the maximal lytic response to occur. The reduction in CML activity observed after late addition of alloantigen might reflect a delay in CML development. Indeed, when addition of thymus cells, but not KAF, was delayed by 24 hr, development of optimum cytotoxicity was also delayed (Table 3, line 2). This again suggests that KAF and alloantigen must be present together before cytotoxic cells can be generated against alloantigens. If, however, 48 or more hours were allowed to elapse before alloantigen was added to the cultures, the shift in CML development was no longer observed (Table 3, lines 3 and 4). This was not due to a progressive loss of KAF activity, since an identical pattern of response was observed if the delayed alloantigen was accompanied by addition of fresh KAF (Table 3, lines 5-7). It appears that responder cells cultured 48 hr or more in the absence of alloantigen lose most of their ability to respond to alloantigen. As seen earlier (Table Z), cytotoxic capacity in thymus-stimulated MLCs supplemented on Day 0 with KAF was not significantly reduced by Day 7, relative to Day 5 (Table 3, line 1). This is in marked contrast to spleen-stimulated MLCs, where there was a prominent reduction in cytotoxicity between Day 5 and Day 7
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TABLE Effect on CML
of KAL; Removal
Total time (days) KAF remained in culturea
1 2 3 4 5b No KAF
4 from Thymus-Stimulated
MLC’s
y. Specific lysis at effecter/target ratios of 2:i
4:l
0.3 15.7 21.7 46.9 67.7 -1.7
-0.3 25.9 32.0 64.7 78.7 -3.0
n In this representative of four experiments, cultures of C57BL/6 spleen cells, irradiated CBA/ H-T6 thymic cells, and KAF (500/, vol/vol) were initiated. At the indicated times thereafter, KAF was removed and replaced by normal media. On Day 5, surviving cells were used in 2: 1 and 4: 1 ratios as effecters against labeled CBA/J target cells. b As a control for the technique of KAF removal (See Methods and Materials), in one culture after 48 hr of incubation the media was removed and returned to the same culture. Subsequent cytotoxicity generated in this culture was 79.5% SL at 4: 1 effecter/target ratios. For comparison, C57BL/6 spleen cells, sensitized toward irradiated CBA/H spleen cells, yielded 31.7% specific lysis at 4: 1 effecter/target ratios.
(Table 3, line 8). The reasons for the discrepancy between the two systems are under investigation. Finally, to determine how long KAF must be present in culture to be most effective, thymic-stimulated cultures were initiated, each containing KAF from the outset. At various times thereafter, the KAF-containing supernatant fluid was carefully removed and replaced with fresh medium, as described in Materials and Methods. Under these conditions, KAF was unable to promote cytotoxicity if left in culture for only 24 hr (- 0.3%, see Table 4). However, significant killing was detected when KAF remained in culture for 48 hr (25.9%). Thereafter, the longer KAF remained in culture, the greater was the observed percent specific lysis. Indeed, maximal cytotoxicity was observed only when KAF was present constantly during the 5 day incubation period (78.7%). This pattern of response was observed in several other experiments where less potent lots of KAF induced levels of CML more comparable to those observed in the spleen-stimulated controls. Hence, no importance is attached to the fact that, in this experiment, levels of CML observed after only 3 days of exposure to KAF and alloantigen equaled levels generated in the spleen-stimulated control MLCs. Taken together, these data suggest that the level of cytotoxic ability attained in thymus-stimulated MLCs is proportional to the duration of contact with KAF (see also Table 2). DISCUSSION In an earlier paper (7), we showed that the T cell factor(s), KAF, elevates the low levels of CML generated in thymus-stimulated MLCs to the significantly higher levels observed in spleen-stimulated MLCs. We now show that CML development does not begin until KAF and alloantigen are present together in cultures with pie-killer cells, and that maximal CML development requires the continuous presence of KAF thereafter.
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During these studies, we consistently observed weak CML development ill thymus-stimulated MLCs, even without the addition of exogenous KAF. We attribute this to low level endogenous production of KAF caused by a small population of cells in the thymus bearing lymphocyte-defined (LD) antigens, and thus capable of stimulating helper cells. Indeed, a small subpopulation of thymus cells appears to display Ia (LD) antigens (10). Subsequent studies have shown that heat-treated allogeneic thymus cells do not stimulate even weak KAF production by normal splenocytes in MLCs, nor do they induce the generation of any detectable CML unless exogenous KAF is included in the culture media (Scott and Finke, manuscript in preparation). Heat treatment of stimulator cells has been shown to functionally inactivate lymphocyte-defined (LD) , but not serologically defined (SD), lymphocyte surface determinants (11). These facts, in conjunction with the observation that removal of KAF within the first 24 hr of the MLC abrogates development of CML (Table 4), indicate that the helper T cell signal, KAF, is prohably essential for generation of cytotoxic lymphocytes and does not simply amplify an otherwise weak response by antigen-stimulated pre-killer T cells. All data presented in this paper have been derived from experiments in which the assay cultures used to determine KAF activity utilize strain combinations which are homologous with the combinations used to produce the KAF-containing supernatants. In other experiments, we have observed that like spleen-stimulated MLCs, cytotoxic activity generated in thymus-stimulated MLCs supplemented with KAF is restricted to cells of the haplotype experienced by the responder cells during sensitization (7). However, KAF activity is not specific with respect to the haplotype of the responder cell it can assist. Furthermore, KAF can assist in the development of cytotoxicity against haplotypes unrelated to those used to stimulate its production (Orosz and Finke, manuscript in preparation). Similar observations have been made about the factor(s) investigated by Plate (9) and Altman and Cohen (1.2). KAF, however, is not the only requisite signal for pre-killer T cell differentiation, as evidenced by the fact that KAF is incapable of inducing cytotoxicity in MLC responder populations unless allogeneic cells are included (9, Table 1) . (This also suggests that KAF activity probably is not attributable just to solubilized alloantigen or cell membrane fragments carried over from KAF-producing MLCs.) Further, when either signal is delayed (alloantigen for up to 24 hr, KAF for up to 72 hr) there is a shift in CML generation kinetics. Bach has also reported a similar shift when either the cell-bound SD or LD stimulus is withheld from MLCs for 24 hr (13). This suggests that (a) alloantigen and KAF must be experienced together before primary killer T cells can begin to develop and (b) the development of primary CML activity takes a minimum of 3-4 days after both signals have been received. We observed that, in contrast to thymus-stimulated MLCs where addition of KAF was delayed, no significant cytotoxicity could be generated in MLCs where alloantigen was withheld for 48 or more hours, even if the day of assay was likewise delayed (Table 3). Apparently, pre-effector cells cultured for 48 hr in the absence of stimulating alloantigens appear to lose their ability to generate CML activity. Hodes and Hathcock have described a suppressor T cell which develops under these same conditions (14). If, however, alloantigen in the form of thymocytes was added within 24 hr of culture initiation, responder cells retain their ability to develop CML activity (Table 3).
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In the course of these experiments, we also observed that in thymus-stimulated MLCs supplemented with KAF, cytotoxicity observed on Day 7 or 8 was essentially undiminished from that seen on Day 5 (Tables 2 and 3). This is in contrast to spleen-stimulated MLCs, in which marked reduction in cytotoxicity was observed by this time (Table 3). These observations suggest that thymus-stimulated MLCs might lack a regulatory mechanism, which is operative in spleen-stimulated MLCs. Such a possibility is the subject of our continuing investigations. Another feature of KAF activity is that net cytotoxicity developed during 5 days of culture appears to be dependent on duration of pre-effector cell contact with KAF, while in the presence of alloantigen (Table 4). This suggests that prekiller cells need multiple or constant input from KAF, and that without it, CML development terminates. Another possibility is that more than one signal is present in the KAF preparation: one acting early in the development process, another somewhat later. In this case, early removal of KAF would eliminate a terminal signal, resulting in decreased net cytotoxicity. Altman and Cohen have shown that 48 hr of contact with mixed lymphocyte culture supernatant fluid is sufficient to induce maximal cytotoxicity by fibroblast-stimulated pre-killer cells ( 15). We have observed that 48 hr of contact with KAF, does, indeed, result in significant cytotoxicity, but that maximal cytotoxicity results only when KAF is present for the full 5 day sensitization period (Table 4). As has been shown elsewhere, the effector populations in these two systems may differ, at least on the basis of hydrocortisone sensitivity (16), and disparity in results may simply reflect this difference. Of interest, however, is the fact that, in both systems, only 24 hr of contact with soluble factor is insufficient for induction of cytotoxicity. The reciprocal experiments, based on removal of stimulator cells from pre-killer/KAF cultures at intervals after culture initiation, would have been advantageous, but are not technically feasible in our assay system. If, as has been suggested, development of cytotoxic cells in 5 day MLCs is controlled by (a) absolute concentration of KAF (5), (b) time of first exposure to both KAF and alloantigen and/or (c) duration of contact with KAF, one would predict that even spleen-stimulated MLCs should exhibit enhanced cytotoxicity if cultured with exogenous KAF. Indeed, enhancement of CML under these conditions has been observed (Table 1, footnote c). We consider this as supportive evidence for the foregoing observations. In a more general context, the mechanism of in vitro sensitization for cellmediated lympholysis is somewhat similar to that for in vitro antibody production in that both depend on a signal from a soluble factor. Nonetheless, several differences between these two immunological systems exist at the level of the helper factors. For example, allogeneic effect factor (AEF), the humoral signal active in production of IgG, must be presented to B cells no sooner than 48 hr after the presentation of antigen to achieve a full primary antibody response (17). In contrast, KAF, the humoral signal active in generating CML, must be present initially in the cultures ; a delay of 48 hr does not permit development of cytotoxicity by the fifth day of culture (Fig. 1) . Secondly, a delay beyond 48 hr of culture in the addition of AEF to B cells and antigen does not postpone maximal plaque-forming responses (Yen, personal communication) whereas delay in KAF addition up to 72 hr to T cells and antigens simply retards development of peak CML activity by a similar length of time (Table 2). Third, the B cell need be in contact with AEF only briefly (1 hr) ; AEF may then be removed from culture, with little or no
KAF
IN
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LYMPHOLYSIS
9.5
reduction iu full antibody response (18). On the other hand, T cells need prolonged contact with KAF and removal of KAF from such culture reduces further CML generation (Table 4). These observations indicate that certain differences exists between AEF and KAF activities. Both activities, however, can be recovered from the same mixed lymphocyte culture supernates. Whether KAF and AEF are distinct molecules with separate functions or one molecule with multiple functions is not known. Furthermore, like AEF, KAF activity may even be mediated by several molecules. Resolution of these possibilites awaits molecular characterization of KAF activities, as has been done for AEF (19). ACKNOWLEDGMENTS This work was supported in part by Grant Number 12468, awarded to J. R. Battisto by the National Institute of Allergy and Infectious Diseases and the American Cancer Society. We would like to thank Ms. M. Wright for typing the manuscript.
REFERENCES 1. Wagner, H., I. Exp. Med. 138, 1379, 1973. 2. Cantor, H., and Boyse, E., J. Exp. Med. 141, 1390, 1975. 3. Bach, F., J. Allergy Cl&. Immunol. 57, 9.5, 1976. 4. Schreffler, D., and David, C., Adv. Immunol. 20, 125, 1975. 5. Plate, J., Nature 260, 329, 1976. 6. Altman, A., and Cohen, I., Eur. J. Immunol. 5, 437, 1975. 7. Finke, J., Orosz, C., and Battisto, J., Nature 267, 353, 1977. 8. Niederhuber, J., and Frelinger, J., Transplant. Rev. 30, 101, 1976. 9. Plate, J., Cell. Immzcnol. 32, 183, 1977. 10. Frelinger, J., Niederhuber, J., David, C., and Shreffler, D., J. Exp. Med. 140, 1273, 1974. 11. Schendel, D., and Bach, F., J. Exp. Med. 140, 1534, 1974. 12. Altman, A., and Cohen, I., Eur. J. Immunol. 5, 437, 1975. 13. Bach, F., Bach, M., and Sondel, P., Nature 259, 273, 1976. 14. Hodes, R., and Hathcock, K., J. Immunol. 116, 167, 1976. 15. Altman, A., and Cohen, I., Adv. Exp. Med. Biol. 66, 289, 1976. 16. Altman, A., and Cohen, I., J. Exp. Med. 142, 790, 1975. 17. Schimpl, A., and Wecker, E., J. Exp. Med. 137, 547, 1973. 18. Yen, B., and Battisto, J., J. Immunol. (in press). 19. Watson, J., J. Immunol. 111, 1301, 1973.
