IN VITRO INDUCTION OF THYMUS-DERIVED CELL- MEDIATED CYTOTOXICITY TO TRINITROPHENOL-

MODIFIED SYNGENEIC LYMPHOCYTE SURFACE PROTEINS

Gene M. Shearer

Immunology Branch National Cancer Institute

Bethesda, Maryland 20014

Introduction

Thymus-derived lymphocytes appear to play an active role in many aspects of immunity. These include: (1 ) helper cell l-’ and suppressor or regulator cell 5-7 functions for modulation of antibody production; (2) delayed hyper- sensitivity; (3) graft-versus-host (GvH) reactions; (4) mixed lymphocyte reactions; lo- and ( 5 ) cell-mediated cytotoxicity.12-20 In the mouse, the evidence that the induction and effector phases of specific cell-mediated cyto- toxicity involve thymusderived cells has been provided by studies demonstrating that both of these phases are sensitive to anti-8 antiserum and comple- merit I?. 17-20 and that in vitro induction of cell-mediated cytotoxicity can be generated by thymus-derived thoracic duct lymphocytes and by cortisone- resistant thymocytes.15q If;

Murine cell mediated cytotoxic reactions have been demonstrated against alloantigens, usually involving differences at the major histocompatibility com- plex between responding lymphocytes and stimulating cells.*2-20 Cytotoxic reactions against either modified or unmodified “self-antigens” have been more difficult to demonstrate. Mouse spleen cells cultured in vitro with syngeneic fibroblasts were capable of syngeneic GvH reactions when injected into syn- geneic neonates,?’ and mixed lymphocyte reactivity between syngeneic cells of certain mouse strains have been demonstrated.11. 22 A few examples of cell- mediated cytotoxicity directed against unmodified =- ? ’ and modified ?5 syn- geneic tumor cell antigens have been reported. The present study shows that trinitrophenol (TNP) modification of mouse spleen cells resulted in the forma- tion of new antigenic regions on the modified cells. In vitro culture of unmodi- fied, syngeneic, thymus-derived lymphocytes with TNP-modified spleen cells resulted in the generation of cytotoxic effector cells that were not TNP-specific but were directed primarily against modified cell-surface proteins. The speci- ficity observed between TNP-modified “immunogen” and target cells suggested that the proteins modified by TNP were controlled by genes within distinct regions of the major (H-2) histocompatibility complex.

Materials and Methods

Spleen cells from normal, young, adult mice were cultured in vitro with unmodified or TNP-modified syngeneic or congenic spleen cells by a modifica- tion 26 of the Mishell-Dutton technique.?‘ Five days later, the effector cells

47

48 AM& New York Academy of Sciences

generated in culture were harvested and incubated with Wr-labeled unmodified or TNP-modified syngeneic or congenic spleen target cells. The percentage of specific lysis was determined after a four-hour incubation of effector and target cells by the method described by Canty and Wunderlich.2s Spontaneous release of W r by spleen cell targets in the presence of unsensitized spleen cells was 15-30%. Nevertheless, significant specific lysis was obtained after subtraction of the relatively high spontaneous W r release characteristic of spleen cell targets. These results have been verified using TNP-modified tumor target cells (which give lower spontaneous lysis, 5-15% ) with the appropriate H-2 types.

Spleen and tumor cells were modified with TNP by incubation of the cells with 10 mM 2,4,6-trinitrobenzene sulfonic acid at pH 7.3 for 10 minutes at 37" C. The cells were then washed twice in Hank's balanced salt solution containing 10% fetal bovine serum.

Results and Discussion

The cytotoxic results obtained using unmodified and TNP-modified syn- geneic as well as congenic target cells are summarized in TABLE 1 . Trinitro- phenol modification of the target cells was necessary but not sufficient to obtain lysis of target cells. Unmodified BIO.A spleen targets were not significantly lysed, whereas 28.6% lysis was obtained when the same target cells were modified with TNP (first two lines of TABLE 1). In order to determine whether the cytotoxicity was specific for the TNP moiety, BIO.A effector spleen cells that had been sensitized in vitro with TNP-modified syngeneic spleen cells were assayed with modified spleen targets from the B10.D2, B1O.BR and C57BL/ 10 congenic lines. These mouse strains differ at distinct regions of the major histocompatibility complex (MHC) as discussed elsewhere.** Designations of the K, Ir, Ss-SIP,. and D regions of the MHC for the four strains studied are

TABLE 1 I n Vitro INDUCTION OF CYTOTOXICITY OF B1O.A SPLEEN CELLS

TO TNP-MODIFIED SYNGENEIC SPLEEN CELLS ASSAYED WITH UNMODIFIED AND TNP-MODIFIED SYNGENEIC AND CONGENIC SPLEEN TARGET CELLS *

'Target Cells

% Specific Lysis & S.E.

MHC 'r

K Ir Ss-Slp D

BIO.A 2.7 f 2.6 B 1O.A-TNP 28.6 f 3.3 B10.D2 4.1 f 2.5

BI0.A-TNP 23.2 f 1 . 1 B 1 O.BR-TN P 14.1 f 2.1 B1O.A-TNP 37.3 & 1.6 BIO-TNP 6.9 & 1 . 1

* Effector:target cell ratio=8: 1. t MHC: Major histocompatibility complex.

B1O.DZ-TNP 6.3 2 2.3

K K K K D D D D K K K K K K B B

D D D D D D D D D D K K D D B B

Shearer: Thymus-derived Cell-mediated Cytotoxicity 49

TABLE 2 SUMMARY OF EXPERIMENTS INDICATING THAT THE PHENOMENON

IS DUE TO T-CELL-MEDIATED CYTOTOXICITY

96 Specific Lysis f S.E.

Parameter Tested in the Induction or Effector Phase

Positive Experimental Control

1. lymphocyte-dependent antibody is not detected in the culture media 0 37.3 f 1.6

2. TNP-lysine does not block the effector phase 20.0 f 2.2 23.2 f 1.1 3. spleen cells from althymic nude donors do not

4. effector cells are sensitive to rabbit anti-mouse

5. effector cells can be generated by cortisone-

generate effector cells -0.9 & 0.4 13.8 f 2.1

brain serum -0.2 f 1.0 17.0 f 1.5

resistant thymocytes 10.0 & 1.1 10.4 & 2.2

shown on the right side of the table. Specific lysis did not occur when either modified B10.D2 or C57BL/ 10 spleen cells were used as targets (compare lines 2 and 7 with lines 4 and 8, respectively). Significant cytotoxicity (14.1 f 2.1%) was obtained, however, when TNP-modified B1 O.BR spleen cells targets were used, although it was not as great as in the case of target cells syngeneic with the TNP-modified “immunogen” and responding cell population (compare line 5 with 6). Thus, the cytotoxicity was not specific for TNP exclusively, but was primarily directed against modified syngeneic cell-surface proteins, which may or may not have included the TNP moiety as an integral part of the recognition unit. Were TNP the only requirement for recognition and cytolysis, B10.D2 (B10 is an exceptional strain as shown below) modified spleen cells should have been as effective targets as modified B1O.A spleen cells when B 10.A responding cells were sensitized against TNP-modified syngeneic spleen cells. Since these congenic mouse strains differ only at the major histocom- patibility complex (H-2) , the results suggest that the cellular proteins modified by TNP so as to be immunogenic to syngeneic lymphocytes were controlled by genes within distinct regions of the MHC. One or more of these genes would appear to be located within the K, Ir portion of the MHC, since some cross- reactivity was observed between TNP-modified BIO.A and BlO.BR spleen cells when cells for these two strains were used as “immunogen” and targets, respec- tively.

A number of criteria were used to demonstrate that the phenomenon was attributed to thymus-derived cell mediated cytotoxicity against modified lympho- cyte proteins and that it was not due to lymphocyte-dependent antibody.‘e These include (see TABLE 2) : ( 1 ) lack of detectable lymphocyte-dependent antibody in the culture media; ( 2 ) failure of TNP-lysine to block the effector phase of cytolysis under conditions identical to those that block lysis due to lymphocyte-dependent antibody; ( 3) failure of spleen cells from athymic nude donors to generate effector cells; Ifi ( 4 ) sensitivity of effector cells to rabbit anti-mouse brain serum and complement; 18. 29, 30 and ( 5 ) generation of effector cells in vilro using cortisone-resistant thymocytes.l5* Thus, both the effector and induction phases of cytotoxicity to TNP-modified syngeneic BIO.A spleen

50 Annals New York Academy of Sciences

cells are thymus-dependent, and the response can be generated exclusively from a population of cortisone-resistant thymocytes.

The ability of various congenic and noncongenic inbred mouse strains to generate in vitro cell-mediated cytotoxicity to TNP-modified syngeneic spleen cells when assayed with modified syngeneic targets was also controlled by genes within the major histocompatibility complex (TABLE 3). Of the C57BL/ 10 congenic strains tested, BlO.A, B10.D2, and B1O.BR were “responders,” whereas C57BL/ 10 was a “nonresponder.” The (B10 X BlO.A)F, hybrid was a high responder, suggesting that the gene( s) controlling this phenomenon is domi- nant.31

The SJL/J and DBA/ 1 inbred strains were also found to be responder and nonresponders, respectively. However, in contrast to the (B10 X BlO.A)F, mice, (SJL X DBA/ 1)F, mice were nonresponders. The apparent recessive nature of responsiveness in this case is similar to the “recessive” immune re-

TABLE 3 STRAIN DISTRIBUTION OF INDUCTION OF CELL-MEDIATED CYTOTOXICITY

TO TNP-MODIFIED SYNCENEIC SPLEEN CELLS In Vitro *

Mouse Strain % Specific

Lysis & S.E. H-2 Type BIO.A 37.3 +- 1.6 a B10 1.8 +- 1.8 b (B10 X B1O.A)R 32.9 & 1.4 a/b B 10.D2 34.4 2 1.0 d B 1 O.BR 37.1 f 2.7 k SJL/J 34.5 +- 1.7 S DBA/ 1 6.0 +- 2.5 9 (DBA/l X SJL/J)K 3.9 * s /q

NZB 2.6 d NZW 0.0 f Z

* Effector:target cell ratio=l:l.

sponse gene controlling antibody production to TNP on a mouse serum albumin carrier.32 This raised the possibility that a portion of the specificity of cytolysis in the SJL/J strain was directed against TNP exclusively. In fact, some cyto- toxic cross-reactivity was observed when the effector cells from cultures in which SJL/ J spleen cells were sensitized with TNP-modified syngeneic spleen cells were assayed with H-2-unrelated modified target cells (data not shown).

Both young adult NZB and NZW mice were nonresponders to modified syngeneic spleen cells. Older animals of these strains that exhibit autoimmune disorders 33 have not yet been tested.

The cellular and molecular bases for unresponsiveness in the C57BL/ 10 and DBA/l strains are currently under investigation. Since the effector cells are of thymic origin, one might speculate that the phenomenon is controlled by a classical immune response (Ir) gene involving a defect in the responding population of thymus-derived cells.31 Unlike most other known Ir genes, however, a cellular component of this model is contributed by the “immunogen”

Shearer: Thymus-derived Cell-mediated Cytotoxicity 51

itself. Therefore, a defect could reside in the H-Zlinked control of the syn- thesis of a cell-surface protein on C57BL/10 spleen cells, which cannot be conformationally altered by TNP so as to be immunogenic in the B10 congenic strains. Were this to be the site of the defect (not necessarily the only defect), (B10 X BlO.A)F, responder cells should respond to TNP-modified F, or BIO.A spleen cells, but not to TNP-modified B10 cells. Furthermore, the use of modified BlO spleen cells as targets should fail to detect F, cytotoxic effector cells even when immunized against modified F, or BIO.A spleen cells. Both of these predictions were verified (data not shown). Thus, at least one genetic defect involves the failure of B10 cells to be modified in such a way as to be immunogenic to responding cells.

It remains to be established whether the H-Zlinked genetic parameters associated with this phenomenon map in the same region of the MHC as classical Ir genes,34- 30 and whether this model serves any significant role in natural immunobiology. The possibility is raised that these lymphocyte surface proteins modified by TNP and controlled by genes within the MHC represent the so-called Ir gene products.

Acknowledgments

I wish to gratefully acknowledge the skilled technical assistance of Carol Garbarino. I thank Dr. Pierre Henkart for testing culture media for lymphocyte- dependent antibody, Drs. Barry S. Handwerger and Ronald S. Schwartz for providing rabbit anti-mouse brain serum, and Marilyn Schoenfelder for typing the manuscript.

[NOTE ADDED IN PROOF: More recent studies indicate that C57BI/lO spleen cells can be modified by TNP so as to be immunogenic to syngeneic-responding lympho- cytes. Studies are in progress to elucidate possible differences in the immunogenicity of modified B10 spleen cells as a function of the degree of TNP-lation.]

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