634
Eur. J. Immunol. 1978.8: 634-640
D.J. Schendel, R. Wank and B. Dupont
11 Morris, R.J., Letarte-Muirhead,M. and Williams, A.F., Eur. J. Immunol. 1975.5: 282. 12 Snary, D., Goodfellow, P., Hayman, M.J., Bodmer, W.F. and Crumpton, M.J., Nature 1974. 2 4 7 457. 13 Snary, D., Goodfellow, P., Bodmer, W.F. and Crumpton, M.J., Nature 1975. 258: 240. 14 Wickner, W., Proc. Nut. Acad. Sci. US 1976. 73: 1159. 15 Racker, E., Chien, T.F. and Kandrach, A., FEES Lett. 1975. 57: 14. 16 Bangham, A.D., de Gier, J. and Greville, G.D., Chem. Phys. Lipids 1967. I : 225. 17 Tyrrel, D.A., Heath, T.D., Colley, C.M. and Ryman, B.E., Biochim. Biophys. Acta 1976. 457: 259.
Dolores J. Schendel. R. Wank and 6. Dupont Sloan-Kettering Institute for Cancer Research, New York
18 Weissmann, G., Bloomgarden, D., Kaplan, R., Cohen, C., Hoffstein, S., Collins, T., Gotlieb, A. and Nagle, D., Proc. Nut. Acad. Sci. US 1975. 72: 88. 19 Cullen, S.E., Freed, J.H. and Nathenson, S.G., Transplant. Rev. 1976. 30: 236. 20 McKenzie, I.F.C., Clarke, A. and Parish, C.R., J. Exp. Med. 1977. 145: 1039. 21 Haughton, G., Transplantation 1966. 4: 238. 22 Sessa, G. and Weissmann, G., J. Biol. Chem. 1970.245: 3295. 23 Jilka, R.L. and Martonosi, A.N., J. Biol. Chem. 1975. 250: 7511. 24 Holloway, P.W. and Katz, J.T., J. Biol. Chem. 1975. 250: 9002. 25 Zborowski, J., Roerdink, F. and Scherphof, G., Biochim. Biophys. Acta 1977. 4 9 7 183.
Cell-mediated lympholysis: examination of H LA genetic fine structure and complementation using cytotoxic Iy mphocytes* Human cytotoxic lymphocytes (CTL), sensitized in vitro in one-way mixed lymphocyte cultures between t w o unrelated individuals, have been used t o study target cell determinants. One pair of CTL shows strong cell-mediated lympholysis responses against the specific target cells as well as against thirdparty target cells obtained from unrelated individuals. Testing of sixty target cells shows a significant association between the two CTL with a correlation coefficient of 0.67 in the panel. The target determinant recognized by the CTL is not an HLA-A,B,C or D specificity, as defined by standard serological and cellular reagents. Segregation studies in a large family reveal that the new specificity defined by the CTL is controlled by HLA, and its recognition may be restricted by the HLA complex.
1 Introduction The immune response t o foreign alloantigens i n many species is primarily directed against antigens of a major histocompatibility complex (MHC) (reviewed in [ 11). Activation of the allograft response by t h e MHC has been extensively investigated using the in vitro techniques of mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML). When lymphocytes from A are sensitized t o x-irradiated inactivated lymphocytes from B (B,) ( i . e . AB,), cells of A recognize foreign alloantigens of B and respond with proliferation which can be measured in MLC. Th e A responding cells also develop specific cytotoxic activity which can be measured in CML [ 2-51.
[I 20571
* This work was in part supported by NIH Grants CA-17404, CA22507, CA-19267, CA-08748, andEY01616. Correspondence: Dolores J. Schendel, Sloan-KetteringInstitute for Cancer Research, 1275 York Avenue, New York, NY 10021, USA Abbreviations: C M L Cell-mediated lympholysis Con A: Concanavalin A CTL: Cytotoxic lymphocytes LU: Lytic unit MR: Maximum release MRBC Mouse erythrocytes PHA: Phythohemagglutinin RCR: Relative cytotoxic response SR: Spontaneous release S R B C Sheep erythrocytes X x-irradiated
Different antigens of the MHC function in the stimulation of proliferation, induction of cytotoxic lymphocytes (CTL) and recognition of target cells. In this respect, striking similarities are observed between antigens of the HLA complex in man and of the H-2 complex in mouse. HLA-D-controlled determinant s and H-21-cont rolle d determinant s stimulate strong proliferation and enhance the development of CTL [6- lo]. Determinants controlled by HLA-A,B and C and H-2K and H-2D function in the sensitization of CTL and serve as target antigens [6,9-171. In the mouse, it has been observed that l a antigens are recognized as target determinants by CTL [ 17191, and additional determinants of t h e H-2 complex also seem t o be involved in CML [20-221. Recognition of HLAD determinants has not been observed in human CML [ 13, 14, 23, 241, but recent studies of our own and others [23331 indicate that other determinants in man, also controlled by the HLA complex, function in CML. These determinants are not currently identified with serological reagents which define the HLA-A,B and C products, but they can be identified in CML. In the present study, human CTL have been used t o identify new HLA-associated determinants and t o study their specificity and expression on panels of target cells. These CTL further characterize the genetic fine structure of the HLA complex and its role in the control of t h e in vitro allograft response.
Eur. J. Immunol. 1978.8: 634-640
Analysis of the HLA region with cytotoxic lymphocytes
635
2 Materials and methods
2.4 CMLassay
2.1 CTL
CTL were suspended at a concentration of 5 x l o 6 - 1 x 10' cells/ml, and t w o serial dilutions were made from this suspension. Triplicate 0.1 ml aliquots were incubated with 0.1 ml aliquots of target cells ( 5 x 1O4 - 1 x 1O5 cells/ml) in round-bottom microtiter plates (Cooke Co., Alexandria, VA). After 4 h, supernatant samples were harvested and counted in a gamma counter (Nuclear Chicago, Hartsdale, NY). Spontaneous release (SR) was determined b y incubating target cells in medium alone. Comparison of this value with that obtained when target cells were exposed t o an activated CTL (i.e. AB,/A*) showed a mean CML value of 2.5 3.3 % in 2 2 experiments. Maximum release (MR) was determined from target cells exposed t o a 5 % detergent solution (Triton X-100, New England Nuclear). Values for the mean MR cpm and % SR (SR/MR x 100) for 1 x lo4 cells are given in the table legends.
After density gradient separation (Lymphoprep, Accurate Chemical Co., Hicksville, NY) from whole blood, lymphocytes were diluted in complete culture medium, consisting of RPMI 1640 supplemented with L-glutamine ( 2 mM), penicillin (1 00 units/ml), streptomycin (1 00 pg/ml) and 15 % pooled human serum. Ten x l o 6 responding cells were coculture flasks (Corning, NY, No. 25 100) containing 2 0 ml of complete medium. Stimulating cells were inactivated 20 ml of complete medium. Stimulating cells were inactivated with 2000 rads of x-irradiation. The flasks were incubated at 37.5 "C standing upright in a humidified incubator with 5 % C02. After six days, the cytotoxic cells were used directly in the CML assay or cryopreserved f o r future use in medium containing 50 % human serum and 1 0 % dimethylsulfoxide (Sigma Co., St. Louis, MO) [34].
*
2.5 Calculation of data
2.2 Target cells Target cells were used either without mitogen stimulation or cultured with phytohemagglutinin (PHA-m, 1 :1 0 0 dilution of the stock source, 0528-57; Difco Co., Detroit, MI) o r concanavalin A (Con A, 10 pg/ml, 3351-56; Difco). Cryopreserved samples were thawed 48-72 h before t h e CML assay, and 5 x 106- 1 0 x 106 cells were incubated with mitogen in flasks containing 1 0 ml of complete medium. Nonstimulated cryopreserved target cells were thawed 18-24 h before use, and fresh cells were cultured f o r 6 days in complete medium without mitogen. On the day of the CML assay, t h e cell suspensions were transferred t o plastic test tubes (Falcon 2001; Becton Dickinson and Co., Cockeysville, MD) and centrifuged at 150 x g f o r 8 min. The cell pellets were resuspended in 0.2 ml of the supernatant medium. Samples were labeled with 250 pCi of NazCrs104 (spec. act. 200500 mCi/mg Cr; New England Nuclear, Boston, MA) for 1-2 h at 37.5 OC in a humidified incubator with 5 % C02. Labeled cells were washed twice with 1 5 % serum medium, counted and suspended at a concentration of 5 x lo4 - 1 x i051mi.
A percent cytotoxicity was calculated by the formula: %CML =
Experimental release - SR x 100. MR - SR
To pool data from several experiments, a normalized cytotoxicity value was used [34]. A percent relative cytotoxic response (RCR) based on the specific response for a given CTL was determined as follows: % RCR =
% CML of experimental target X* -x 100, % CML of specific target B*
where a CTL of A.B specificity (A responding cells sensitized to Bx stimulating cells) was tested for lytic activity directed against the experimental target X* and expressed as a percentage of t h e lytic activity directed against the specific target B*, which defines the reference value for 100 5%.
3 Results 3.1 Identification of CTL detecting new target specificities
2.3 Subpopulations of target cells Subpopulations of peripheral blood lymphocytes were prepared as previously described [35-371. Briefly, T lymphocytes, forming rosettes with neuraminidase-treated sheep erythrocytes (SRBC), were separated from nonrosetting cells on a Lymphoprep gradient. The nonrosetting fraction was then exposed t o mouse erythrocytes (MRBC) and the rosettes, representing a B subset [ 3 7 ] , were separated from the nonrosetting fraction on a second Lymphoprep gradient. Attached erythrocytes were lysed with ammonium chloride (0.8 %), and the washed lymphocytes were incubated for four days in culture medium containing no mitogen before use as CML targets. This separation procedure did not provide pure T and B cell subpopulations but rather provided enriched T and B fractions. Rosetting of the interface fractions after the separation showed < 10 % contamination with SRBC and MRBC rosetting cells, respectively.
CTL were made by mutual in uitro sensitization of peripheral blood lymphocytes from three unrelated individuals. The HLA-A,B and C antigens differing between the responding and stimulating cells of t h e CTL are listed in Table 1. Responses mediated by these CTL reveal several unusual reactions if the HLA-A and B antigens are presumed t o be the predominant CML sensitizing and target antigens. CTL A . l causes the expected specific lysis of target 1*, yet shows an unexpected equal lysis of target 2* (Table 2). Similarly, CTL A.2 specifically lyses 2* and 1 * in a quantitatively identical manner. Since CTL A.l and A.2 have been stimulated by different antigens of the HLA-A and B series, they must be sensitized t o and recognize other determinants shared by cells 1 and 2. In contrast, when the reciprocal CML responses are measured between cells 1 and 2, using CTL 1.2 and 2.1, the specific cytotoxic responses are much weaker. Of particular interest is t h e reaction of CTL 1.2 against target 2*. In this combination, HLA-B 12, which has been claimed t o be one of t h e
636
Eur. J. Immunol. 1978.8: 634-640
D.J. Schendel, R. Wank and B. Dupont
strongest CML antigens of the HLA-B series [ 151, should serve as a sensitizing and target antigen, yet despite the difference for HLA-B12 between the responding and stimulating cells in CTL 1.2 the cytotoxic response is low. Presumably this low response is observed because 1 and 2 share the CML target specificity which is &cognized by CTL A.l and A.2. This hypothesis is strengthened by target cell inhibition studies showing that cells 1 and 2 can equally inhibit the specific responses of A.l and A.2 (Schendel, unpublished observations). Table 1. Sensitizing antigens for cytotoxic lymphocytes CTLa) Responder A. 1 A.2 1.2 2.1 1.A 2.A
Stimulatorb)
HLA antigen difference&
1, 2, 2, 1,
A3,Bw35,Bw37,Cw4 B12,CwS B12,CwS A3,Bw35,Bw37,Cw4 B8.Bw40 B8,Bw40
A A 1 2 1 2
AX AX
a) The known HLA antigens for individuals A, 1 and 2 are A:(A2,
/A~,BW~~); B8/Bw40); ~ : ( A ~ , B w ~ ~ , C W ~ , D W ~2:(A2,B12,Cw5). b) Stimulating cells are x-irradiated with 2000 R. c) Antigens expressed by stimulating cells which are not expressed
by responding cells. Table 2. Quantitative CML study with six CTLa)
3.2 Evaluation of the role of HLA-C antigens In evaluating what could be servirrg as t h e unknown target determinant recognized by CTL A.l and A.2, the HLA-C antigens were first considered. Cytotoxicity by CTL A.l and A.2, as well as by the autologous sensitized CTL A.A, was measured on a panel of target cells obtained from unrelated individuals with defined H L A C specificities. The CML reactivity did not reveal any shared HLA-C antigen which could serve as the CML specificity. Selected cells from this panel are shown in Table 3 , including cells that express one or more of the five HLAC-controlled antigens which are currently identified serologically (Cwl -Cw5). No single one of these HLA-C specificities can explain the positive reactions of CTL A. 1 and A.2. Furthermore, because target cells E* and F* have both H L A C specificities defined, the reactions cannot be ascribed t o an unknown HLA-C specificity. Table 3. Analysis of HLA-Cilirected cytotoxicitya) CTL HLAC A* 1* 2* B*bf C* D* E* F* antigen cw4 cw5 cw5 cw4 cw2 Cwl cw4 - - differences cw3 cw5 cw5 A.A A.l A.2
0.5 1.8 1.9 1.5 1.4 Cw4I- 0.4 52.3 45.2 4.1 5.3 CW5/- - 1.4 56.3 46.1 11.0 11.0
0.3 5.4 7.6
0.6 0.4 31.4 35.9 37.9 36.8
a) Data are expressed as 9% CML; MR=2023-9890 cpm; SR < 23 %.
Target cells (*) are stimulated with PHA-m. CTL
Target
A.1
1* 2* 1* 2* 2* 1* A* A*
A.2 1.2 2.1 l.A 2.A
20lb)
40:l
60:l
r2C)
LUd)
36.4 32.5 38.2 37.3 9.0 10.9 9.9 10.1
50.5 41.4 55.4 51.6 12.6 17.5 18.7 14.8
64.5 50.2 72.6 66.0 16.1 24.1 27.6 19.4
0.91 0.92 0.91 0.94 0.96 0.99 1.00 0.81
1.1 1.1 1.1 1.0 13.8 8.0 6.5 10.6
a) Data are expressed as % CML: MR=1024-1321 cpm; SR < 21 %. Target cells (*) are stimulated with PHA-m. b) Ratio of CTL to target cell. c) Coefficient of determination for linear CML activity. required to give 30 % CML. d) Lytic unit = number of CTL x Linear regression analyses show that the responses of all t h e CTL fit straight lines when the effector-to-target cell ratio is varied. Extrapolation from these lines enables determination of the number of lymphocytes of each CTL required t o cause 30 % CML, which defines a lytic unit (LU). According t o this definition, a larger LU size reflects a weaker response since more CTL are required t o cause a 30 % CML response. Comparison of the LU for the CTL is shown in Table 2. The LU is identical for CTL A.l and A.2 against target 1* but is eightfold more for CTL 2.1 against 1*. At least twelve times more of CTL 1.2 is required t o give 30 % CML of 2* than is required by either CTL A.l or A.2.
b) The HLA-A and B antigens for targets B* -F* are as follows:
B*(Al,A2,B7,B12); C*(Ag,AlO,B12,B14);D*(A2,All,B27); E*(A2,B12,B27); Fr(A2,B12,Bw35). 3.3 Evaluation of the role of HLA-D antigens The role of HLA-Dcontrolled antigens was also evaluated on the target panel, and n o known shared HLA-D determinant could explain all the CML reactions of CTL A.l and A.2. A second apprQach used t o evaluate the role of HLA-D, as well as that of B cell (DRw) alloantigens [38], was comparison of subpopulations of enriched T and B lymphocytes as target cells. If the new target specificity is a n antigen of the HLA-D series o r a B cell alloantigen shared by cells 1 and 2, which has not yet been identified, then CML against target subpopulations should reveal a high level of lysis of the B-enriched target population and loss of CML reactivity against the Tenriched target population. Peripheral blood lymphocytes from individual 1 were separated into fractions of enriched T and B cells and a remaining fraction (N) after removal of the T and B fractions. Equal levels of cytotoxicity are observed (Table 4) when the CML responses of CTL A.l and A.2 are measured on the three subpopulations of 1* target cells. Therefore, the HLA-D o r B cell alloantigens do not explain the CML specificity. The level of lysis in repeated testings was very high on all target cell subpopulations, so responses t o any one subpopulation are not explained by contamination with cells of another subpopulation. 3.4 Analysis of cytotoxicity of a target cell panel
Cells from 1 and 2 were also sensitized t o lymphocytes from individual A (CTL l.A and 2.A). These t w o CTL react similarly with A* target cells, and they can also lyse unrelated target cells sharing n o known HLA-A,B or C antigens with A*.
The cytotoxic responses of CTL A.1 and A.2 were tested simultaneously on a panel of sixty target cells. Data were pooled from four experiments for this analysis using the
Eur. .IImmunol. . 1978.8: 634-640
Analysis of the HLA region with cytotoxic lymphocytes
637
Table 4. Comparison of target cell subpopulationsa) 0
n L
A*-@)
Target cells 2*-U l*-U * 1*-TC)
I*.@
1*-Ne)
A.A
-0.3 1.0 1.0
35 1.9 6.5 63.6 72.6 74.4 54.5 59.5 74.2
8.7 75.3 69.6
9.3 73.1 69.4
A.l A3
0
3
80
a) Data are expressed as % CML; MR=2290-25 000 cpm; SR < 18 %. All target cells (*) are tested without mitogen stimulation. b) Unseparated (U) peripheral blood lymphocytes. c) Enriched T cells forming SRBC rosettes. d) Enriched B cells forming MRBC rosettes. e) Remaining cells after removd of T and B fractions.
0
f
c
70-
0
al
60-
Lz
0
r2=0.9 b = 1.03
10
20 30 40 50 60 70 80 90 100 110 120 1%
-1
The second unusual characteristic noted in this study is t h e quantitative relationship between the activities of t h e t w o CTL in their recognition of different target cells. (Fig. 1). The % RCR of an individual target cell with CTL A.l is plotted against its % RCR with CTL A.2. Althoughpositive responses range from 11 -130 % RCR, most target cells are lysed t o t h e same degree by both CTL A.l and A.2. For example, the target cell indicated by the * shows a RCR of 75 % with CTL A.l and a RCR of 76 %with CTL A.2. Linear regression analysis fits t h e CML responses of CTL A.l and A.2 t o a straight line with a slope of 1.03 (r2=0.9), and repeated testings show t h e quantitative distinction of individual cells t o be very consistent.
Figure 1. Quantitative recognition of panel target cells with CTL A . l and A.2. The % RCR by CTL A.1 of an individual target cell is plotted against the % RCR with CTL A.2. There is a strong linear relationsh (r2=0.9) fitting a line with a slope (b) of 1.03.
target cells recognized by CTL A.l and A.2. Most of the cells of the target panel were studied in t h e VII. International Histocompatibility Workshop and are well characterized for their IILA-A,B,C, D and DRw (Ia-like) alloantigens [39]. This enabled comparison of the CML specificity with 1 8 HLA-A antigens, 20 HLA-B antigens, w4 and w6, 5 HLA-C antigens, 1 0 HLA-D specificities, and 7 DRw specificities defined by clusters of B cell sera. Table 6 summarizes information from these analyses. Table 6. Associations of CTL specificitiesand HLA antigens CTL
A.l
CMLresponsewith CTL A.2
+
-
24 4
A.2
-
5
21
a) Cytotoxicity was measured on 60 target cells of unrelated individuals. A RCR < 10 % was considered negative, and a RCR > 10 %was considered positive. The association between CTL A.l and A.2 is highly significant (x2=26.6; p < 0.001), and their
correlation coefficient in this panel is r=0.67.
3.5 Association analyses with HLA antigens Two by t w o comparisons were made between different HLA antigens and the CML specificity t o determine whether particular HLA specificities are frequently expressed o n those
Serologically defined HLA antigen Bw35 cw4 B12 CW5
with CTL A.1
+
%
Relative Cytotoxic Response with C T L A.1
Table 5 . Two by two association analysis of the CML responses of CTL A . l and A.2a)
CML response
N.60
0
relative cytotoxic response (% RCR, see Sect. 2.5). The range of the responses of CTL A.l against 1* was 52-88 % CML, and of CTL A.2 against 2* was 46-72 % CML. RCR of < 10 % was considered negative. Based o n the specific responses, this reflects an actual % CML of 4.6-8.8 %. Two findings characterize this analysis. First, CTL A.l and A.2 distinguish similar groups of positive and negative cells. Table 5 shows t h e two by t w o comparison analysis indicating a highly significant association in the responses 9f CTL A.l and A.2. It should be emphasized that the basis for this association cannot be explained by recognition of any shared HLA-A,B,C or D antigen, at least as they are currently defined.
0
0
A.l
+
A.2
B12+Bw35 B12 cw5 Bw35 cw4 B12+Bw35 B12 Bw35 Cw4 cw5 B12+Bw35
12 12 10 6 19 13 I 10 11 20 10 9 9 6 16
14 14 16 20 I 16 22 19 18 9 13 14 14 11 7
I 2 5 1
6 2 0 3 3 5 5 4 5 1 9
27 24 23 25 22 23 23 22 20 20 26 27 24 28 22
11.1 I .9 1.9 2.6 12.5 1.3 4.5 2.6 2.9 11.1 3.7 3.6 2.1 3.9 7.2
0.001 0.005 0.166 0.104 0.000 0.006 0.034 0.108 0.090 0.001 0.055 0.05 7 0.146 0.049 0.007
a) The first sign indicates the response with the given CTL ("+"= RCR > 10 %; "-" = RCR < 10 %), and the second sign indicates
"+" or "-"serological typing for the given HLA antigen. The 2x2 analysis was also done defining RCR values of < 5 % and < 15 % a s negative; this did not show a different association or a better correlation between the CML specificity and a serological specificity. b) x2 value for the positive association. c) Significance level of the association.
Eur. J. Immunol. 1978.8: 634-640
D.J. Schendel, R. Wank and B. Dupont
638
Recognition by CTL A.l shows associations t o HLA-Bw35 and HLA-Cw4, both of which are expressed by the stimulating cells of this CTL (see Table 1). CTL A.2 shows associations t o HLA-B12 and Cw5, also expressed by stimulating cell 2. No significant positive associations were found for any HLA-A antigens, w4, w6, HLA-D antigens or DRw specificities. Analyses with both CTL A.l and A.2 (i.e. target cells must be positive o r negative with both CTL) show a decrease in the associations with HLA-B12, Bw35, Cw4 and Cw5. It was noted that the discordant reactions in the 2 x 2 tables were mainly positive with the CTL and negative for t h e serological specificity, indicating that t h e serological specificities might be included in the CML specificity. Therefore, additional analyses were done combining all cells positive for either B12 or Bw35 t o determine whether the combined antigens would show a stronger correlation with the CML specificity, and t o see whether the number of CML-positive but serologically negative (+-) reactions would decrease. The value of the association with CTL A . l , A.2 and A.l + A.2 does in fact increase with the B12 + Bw35 combination. However, discordant reactions are still observed, and they now show an equal distribution in the (+-) and (-+) categories. The finding that some target cells expressing neither of these antigens are in the high positive group (RCR 60 %), and others carrying these antigens are in the negative group (RCR < 10 %),emphasizes that t h e CML determinant is in fact distinct from these serologically defined HLA antigensHowever, t h e positive associations between t h e CML antigen and HLA-B antigens d o indicate that t h e target specificity is controlled by t h e HLA complex.
x2
>
3.6 HLA segregation analysis Additional evidence for the control of the CML specificity b y the HLA region was obtained in a family segregation study. Individual Ka (indicated by the * in Fig. 1) was used as a sensitizing cell t o generate a third CTL (A.3) and tested simultaneously with A.l and A.2 against target cells from her family. (Table 7). Three of t h e family target cells were strongly lysed by CTL A.l, A.2 and A.3. These three target cells are from HLA-identical siblings sharing t h e “b” and “c” haplotypes. Quantitatively, t h e three CTL react similarly with each of t h e HLA-identical target cells. The recognition of these three target cells demonstrates that HLA is determining the CML specificity. However, the failure t o find equally strong positive responses against either of the parental target cells was unexpected. Because of the association between the CML specificity and HLA-Bw35 in the panel one might have expected strong lysis of t h e cells of the mother (M) who carries the Bw35 antigen on her “c” haplotype. T o rule out a technical failure t o detect this response, two separate blood samples were used t o prepare target cells. From both of these samples targets stimulated with PHA o r Con A and nonstimulated cells were compared, and in each case the CML reaction was less than 1 3 %. Only a low level of CML (7-10 7%) is detected against the cells of the father. If t h e reaction t o Ka, T i and J e is directed against determinants controlled only by the “b” haplotype, one would have expected t o see strong positive lysis not only of the cells of the father but also of t h e sibling Da. Thus, HLA appears t o govern the CML specificity detected by the three
Table 7. HLA segregation study in family Le
Targets
Sex
F* M* Ka*(3) Ti* Je *
Da* Vi* Jo*
Ma* Ja*
d
9 9
d 9
d 9 3
Y
9
HLA~) haplotypes
alb cld
blc bl c blc bld ald ald a1d ald
CTLb) A.l
A.2
A.3
7.3 1.9 41.3 54.1 34.3 1.o 0.6 0.9 0.0 0.0
6.8 0.1 52.5 62.2 35.4 1.7 1.9
9.8 8.1 54.1 61.8 35.9c) 4.0 3.5 1.8 0. I 2.1
1 .I
0.0 0.0
a) The HLA-A,B and C antigens for the different haplotypes are a(A2,B27,Cwl), b(A2,B13), c(Aw24,Bw35,Cw4)and d(A3,B7). b) Data are expressed as % CML; MR-1860-3717 cpm; SR < 20 %. Targets (*) are stimulated with PHA-m. c) These target cells are obtained from an individual undergoing long-term kidney dialysis which may account for the lower level of CML. CTL, but the reaction is not uniquely associated with determinants controlled by a single HLA haplotype. Complementation between the “b” and “c” haplotypes must occur for CML recognition. 4 Discussion
Initial studies on t h e genetic control of CML indicated that the serologically defined HLA-A and B antigens are the primary CML target determinants. The determinants identified by CTL A.l and A.2 are distinct from known HLA-A,B,C and D specificities but are associated with t h e HLA complex. Earlier quantitative studies on the CML response t o different HLA antigens claimed that HLA-B 12 is particularly strong, yet the finding that a distinct, but HLA-B12-associated, determinant can be recognized by CTL A.l and A.2 raises the question of whether such HLA-B antigens act as markers for other target determinants. Comparison of the lytic activities of CTL A.l and A.2 on targets 1* and 2*, which shows the strong response t o the new determinant, and the lytic activities of CTL 1.2 and 2.1, which shows t h e weak responses between cells sharing this specificity but differing for HLAA,B and C locus antigens, requires reconsideration as t o what determinants are primarily recognized b y human CTL. Although recognition of individual antigens of the HLA-A,B,C and D series does not explain the CML specificity, an association is seen with some HLA-B locus antigens in the panel. Several possibilities can explain this association. First, the target determinant may be a specificity shared by some HLAB antigens which is detected by CTL but has not been identified serologically. Models of this type have been reported for H-2 antigens in which cells from mice carrying mutations in the H-2 complex can stimulate strong CML responses, but are poor in stimulating antibody production t o t h e mutant specificity [20, 221. Second, t h e CML target molecule might be preferentially associated in the membrane with particular HLA-B antigens. Interactions between such associated molecules could lead t o CML recognition. Many examples of interacting specificities have been reported in the mouse for recognition of minor histocompatibility antigens, viral antigens and chemical modifications of cell surface molecules (reviewed in
Eur. J. Immunol. 1978.8: 634-640 [40]). In one important respect these results differ from t h e H-2 models in that identity between t h e responding cell and target cell for HLA-A,B or C antigens (in anology with H-2K and H-2D) is not required for CML recognition. Third, t h e CML target specificity may be completely distinct from HLA-B antigens, but if the genes controlling these antigens are in linkage disequilibrium in the population, the antigens would frequently occur simultaneously o n t h e cell surface. Such linkage disequilibria are already known for t h e genes controlling HLA-Bw35 and Cw4 and HLA-B12 and Cw5 [41]. Clear evidence which would distinguish between distinct molecular cytotoxic and serological specificities can be obtained by separation of the determinants through genetic recombination of their controlling genes o r by their biochemical separation.
.
The ability t o study the target cell panel with t w o different, but highly correlated CTL, not only enables evaluation of responses t o cells sharing HLA-A,B,C or D antigens with the CTL-stimulating cells, but reveals parallel patterns in t h e level of lysis of individual cells. This variation could reflect recognition of several specificities by CTL A.l and A.2 with those cells in the lower category expressing only some of the specificities. If this is t h e case, these new specificities must be frequently shared by unrelated individuals because CTL A.l and A.2 can be used t o select individuals t o sensitize new CTL (A.3), and these CTL behave similarly t o CTL A.l and A.2 not only in family studies but also in panel studies. O n t h e other hand, the cells in the high category (>5 0 %) may express a common CML determinant while those in t h e intermediate category (10-50 %) may express cross-reactive determinants. T o examine this possibility, association analyses t o HLA antigens were done comparing t w o categories: < 10 % and 2 10 % RCR, and three categories: < 10 %, 10 % - 50 % and > 50 % RCR. Associations t o HLA-B locus antigens did not show any distinctions between t h e single positive group (> 10 % and) intermediate ( 10 % - 5 0 %) and strong (> 5 0 %) positive groups. Therefore, if recognition of a cross-reactive specificity accounts for the intermediate responses, the crossreacting specificities show identical HLA-B antigen associations. The variation may also be explained by complementation effects similar t o those observed in the family Le. If CML recognition in the unrelated panel is dependent upon interactions, then different components may lead t o more o r less efficient complementation. The complementation patterns observed in the family Le reveal that the interacting factors which lead t o CML recognition are determined by HLA. This complementation could either occur at t h e genetic level o r at t h e cell surface. F o r example, t h e target specificity may be determined by a single HLA haplotype, but other genetic factors may interact with this specificity and allow CML recognition. Recognition of target determinants may be restricted by HLA, similar t o the pattern reported for recognition of H-Y [42] and other non-HLA-A,B,C and D antigens in man [32, 43, 441. Our results cannot be explained by H-Y since target cells of both sexes are recognized by the CTL as seen in Table 7. In one of these studies [32], a strikingly similar pattern was seen in CML analysis of a family carrying t h e HLA-B12 and Bw35 antigens. Such a restriction could be due t o recognition of a hybrid antigen formed through genetic interaction of the “b” and “c” haplotypes, or dual recognition of t w o specificities, each of which is determined by a single haplotype in this
Analysis of the HLA region with cytotoxic lymphocytes
639
family. Again, it should be stressed that this HLA “restriction” does not require HLA-A,B or C identity between CTL and the target cell. Alternatively, expression of target specificities may not be equal o r even codominant on the surface of the cell. Thus, homozygosity for t h e same determinant or heterozygosity for t w o determinants, each of which is recognized by the three CTL, could account for strong lysis of the HLA-identical cells. In t h e parental cells, a lower antigen density or expression of the antigen on only a portion of the lymphocytes would lead t o low lysis. Distinction of these models is n o t yet possible, but studies underway t o examine CML responses between Le family members, and by CTL sensitized t o other individuals of t h e panel, may provide new information about t h e basis of the complementation. CTL provide a useful immunogenetic reagent for studying the genetic fine structure of the HLA complex. The CTL described in these studies identify new HLA-associated cytotoxic determinants which may eventually prove t d be controlled by distinct genes of the HLA complex. The strong response directed against these determinants indicates that they may be of significant importance in the histocompatibility response. The pattern of complementation leading t o CML recognition raises new questions regarding the role of HLA haplotypes in cytotoxicity, and leads us t o consider interactions in the control of t h e allograft response. The authors wish to thank Mss. Melvyn Blanco and Melna Hall for excellent technical assistance, Ms. Loretta Hall for help in the preparation of this manuscript. Dr. David Braun, Jr., for performing the statistical analysis, and Ka Le and her family for participating in these studies.
Received March 1, 1978; in revised form June 5, 1978.
5 References 1 Gotze, D. (Ed.), The Major Histocompatibility System in Man and Animals, Springer Verlag , Berlin 1977. 2 Lightbody, J., Bernoco, D., Miggiano, V. and Ceppellini, R., G. Batt. Virol. Immunol. 1971. 64: 243. 3 Solliday, S. and Bach, F., Science 1970.170: 1406. 4 Hayry, P. and Defendi, A. Science 1970. 168: 133. 5 Hodes, R. and Svedmyr, E., Transplantation 1970. 9: 470. 6 Eijsvoogel, V., du Bois, M., Melief, C., de Groot-Kooy, M., de Koning. C. van Rood, J., van Leeuwen, A.. d u Toit, E. and Schellekens, P., in Dausset, J. and Colornbiani,J. (Eds.), Histocompatibility Testing 1972, Munksgaard, Copenhagen 1 9 7 3 , ~501. . 7 Bonnard, G., Lemos, L. and Chapuis, M., S a n d . J. Immunol. 1974. 3: 97. 8 Long, M. Handwerger, B., Amos B. and Yunis, E., J. Immunol. 1976. I 1 7: 2092. 9 Alter, B., Schendel, D.J., Bach, M., Bach, F . , Klein, J. and Stimpfling, J . , J . Exp-Med. 1973.137: 1303. 10 Schendel, D.J. Alter, B. and Bach, F., Transplant. Proc. 1973. 5: 1651. 11 Miggiano, V., Bernoco, D., Lightbody, J., Trinchieri, G. and Ceppellini, R., Transplant. Proc. 1972. 4 : 231. 12 Bonnard, G., Chapuis, M., Glauser, A., Mempel, W., Baumann, P., Grosse-Wilde, H. and Albert,E., nansplant. Proc. 1973. 5: 1679. 13 Eijsvoogel, V., d u Bois, R., Melief, C., Zeijlemaker, W., RaatKooning, L. and de Groot-Kooy, L., Transplant. Proc. 1973.5: 1301. 14 Trinchieri, G., Bernoco, D., Curtoni, S. Miggiano, V. and Ceppellini, R., in Dausset, J. and Colombiani, J. (Eds.), Histocompatibility Testing 1972, Munksgaard, Copenhagen 1973, p. 509.
640
Eur. J. Immunol. 1978.8: 640-645
J. H. Russell, A. H. Hale, D. Inbar and H. N. Eisen
15 Kristensen, T., Grunnet, N. and Kissmeyer-Nielsen, F., Tissue Antigens 1975.6: 221. 16 Grunnet, N., Kristensen, T. and Kissmeyer-Nielsen, F., Tissue Antigens 1976. 7: 301. 17 Nabholz, M., Vives, J., Young, H.M., Meo, T., Miggiano, V., Rijnbeek, A. and Shreffler, D. C., Eur. J. Immunol. 1974.4: 378. 18 Schendel, D.J. and Bach, F . , J . Exp. Med. 1974.140: 1534. 19 Schendel, D.J. and Bach, F., Eur. J. Immunol. 1975. 5: 880. 20 Widmer, M., Alter, B., Bach, F. and Bach, M., Nature-New Biol. 1973.242: 239. 21 Phillips, S . Carpenter, C. arid Strom, T., 7kansplant. Proc. 1973. 5: 1669. 22 Nabholz, M., Young, H.M., Meo, T., Miggiano, V., Rijnbeek, A. and Shremer, D.C., Immunogenetics 1975.5: 469. 23 Long, M. Handwerger, B. and Yunis, E., in Kissmeyer-Nielsen, F. (Ed.), Histocompatibility Testing I 9 75, Munksgaard, Copenhagen 1975, p. 849. 24 Mawas, C., Dominique, C. and Sasportes, M., in Kissmeyer-Nielsen, F., (Ed.), Histocompatibility Testing 1975, Munksgaard, Copenhagen 1975, p. 855. 25 Kristensen, T., Grunnet, N. and Kissmeyer-Nielsen, F., Tissue Antigens 1974. 4 : 378. 26 Mawas, C., Christen, Y., Legrand, L., Sasportes, M. and Dausset, J., Transplantation 1974.18: 256. 27 Schapira, M. and Jeannet, M., Tissue Antigens 1974. 4 : 178. 28 Willumsen, J. and Heron, I., Tissue Antigens 1974.4: 172.
Histocompatibility Testing 1 9 75, Munksgaard, Copenhagen 1975, p. 835. 31 Sondel, P. and Bach, F., J. Exp. Med. 1975.142: 1339. 32 Goulmy, E., Termijtelen, A., Bradley, B. and van Rood, J., Tissue Antigens 1976.8: 317. 33 Schendel, D.J., Wank, R., Hansen, J.A. and Dupont, B., Transplant. Proc. 1977. 9 : 1777. 34 Schendel, D.J., Wank, R., Hall, M. and Dupont, B., TissueAntigens, in press. 35 Weiner, M., Bianco, C. and Nussenzweig, V., Blood 1973.42: 939. 36 Gallie, U. and Schlesinger, M., J. of Immunol. 1974.112: 1628. 37 Gupta, S., Good, R. and Siegel, F., Clin. Exp. Immunol. 1976. 25: 319. 38 Bodmer, W., Batchelor, R., Morris, P., Festenstein, H. and Bodmer, J., (Eds.) Histocompatibility Testing I 9 77, Munksgaard, Copenhagen, in press. 39 Dupont, B., Yunis, E., Duquesnoy, R., Pollack, M., Noreen, H., Hansen, J., Reinsmoen, N., Annen, K., Greenberg, L., Lee, T., Whitsett, C., Antonelli, P. and Braun, D., in Bodmer, W., Batchelor, R., Morris, P., Festenstein, H. and Bodmer, J. (Eds.), Histocompatibility Testing I 9 77, Munksgaard, Copenhagen, in press. 40 Doherty, P., Blanden, R. and Zinkernagel, R., 7kansplant. Rev. 1976.29: 89. 41 Mayr, W., in Kissmeyer-Nielsen, F. (Ed.), Histocompatibility Testing 1975, Munksgaard, Copenhagen 1975, p. 330. 42 Goulmy, E., Termijtelen, A., Bradley, B. and van Rood, J., Nature 1977.266: 544.
29 Goulmy, E., Termijtelen, A., Keuning, J. and van Rood, J., in Kissmeyer-Nielsen, F. (Ed.), Histocompatibility Testing 1975, Munksgaard, Copenhagen 1975, p. 845.
43 McMichael, A., Ting, A., Zweerink, H. and Askonas, B., Nature 1977.270: 524.
30 Kristensen, T. and Grunnet, M., in Kissmeyer-Nielsen, F. (Ed.),
44 Dickmeiss, E., Socber, B. and Svejgaard, A., Nature 1977.270:526.
J. H. Russelo, A. H. Hale', D. Inbar' and
H. N. Eisen Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge
Loss of reactivity of a BALB/c myeloma tumor with allogeneic and syngeneic cytotoxic T lymphocytes* I t was previously observed that MOPC-315EL, a subline o f t h e BALB/c m y e l o m a t u m o r MOPC-315, varies in its ability to interact w i t h primary anti-H-2d c y t o t o x i c thymus-derived l y m p h o c y t e s ( C T L ) while remaining invariant in its expression of cell surface antigens recognized b y anti-H-2d sera. This paper demonstrates (a) t h a t secondary anti-H-2d C T L also fail to recognize t h e late t u m o r cells, a n d (b) t h a t t w o o t h e r C T L systems (anti-minor histocompatibility antigens a n d anti-2,4,6trinitrophenyl), which require recognition of both H-2 p r o d u c t s a n d o t h e r surface antigens, also fail to react w i t h t h e late t u m o r cells. The defect in the late t u m o r cells w a s evident w h e n they w e r e used as targets, inhibitors, a n d stimulators of C T L activity [I 21261
1 Introduction
*
This work was supported in part by a research grant (CA-15472) and and a center grant to the Massachusetts Institute of Technology, Center for Cancer Research (CA-14051) from the National Cancer Institute, Department of Health, Education and Welfare and by additional aid from the James and Lynelle Holden Fund. o Helen Hay Whitney Postdoctoral Fellow + National Cancer Institute Postdoctoral Fellow (5f32CA05685) Present address: Ames Yissum, Ltd., Har Hotzvin, Sanhedria Murchevet, Jerusalem, Israel Correspondence: Herman N. Eisen, E l 7-128, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Abbreviations: Con A: Concanavalin A C T L Cytotoxic thymusderived lymphocyte(s) ip: Intraperitoneal minor: Minor histocompatibility antigens MLR: One-way mixed lymphocyte reaction PBS: Phosphate-buffered saline (0.015 M NaCl/O.Ol M potassium phosphate, pH 7.4) Tnp: 2,4,6-Trinitrophenyl E :T ratio: Effector : target cell ratio
In a previous c o m m u n i c a t i o n w e reported t h a t MOPC-315EL, a subline o f MOPC-315 ( t h e BALB/c m y e l o m a t u m o r that produces a 2,4dinitrophenyl-binding IgA myeloma protein), undergoes late in e a c h t r a n s p l a n t a t i o n cycle in BALB/c mice a reversible loss in ability to react w i t h allogeneic c y t o t o x i c thymus-derived l y m p h o c y t e s (CTL) [ 11. A t t h e s a m e t i m e , the t u m o r cells retained undiminished on their surface t h e serologically recognized p r o d u c t s o f t h e H-2d haplotype. T h e C T L with which t h e late t u m o r cells lost reactivity had been elicited b y stimulating B10 spleen cells w i t h B10.D2 cells o r BALB.B spleen cells w i t h BALB/c cells. Hence, the CTL (H-2b haplot y p e ) w e r e specific f o r H-2d, t h e h a p l o t y p e o f t h e BALB/c t u m o r . T h e lack o f reactivity o f l a t e t u m o r cells with these C T L was evident w h e n t h e t u m o r cells were used as labeled
Eur. J. Immunol. 1978.8: 634-640
D.J. Schendel, R. Wank and B. Dupont
11 Morris, R.J., Letarte-Muirhead,M. and Williams, A.F., Eur. J. Immunol. 1975.5: 282. 12 Snary, D., Goodfellow, P., Hayman, M.J., Bodmer, W.F. and Crumpton, M.J., Nature 1974. 2 4 7 457. 13 Snary, D., Goodfellow, P., Bodmer, W.F. and Crumpton, M.J., Nature 1975. 258: 240. 14 Wickner, W., Proc. Nut. Acad. Sci. US 1976. 73: 1159. 15 Racker, E., Chien, T.F. and Kandrach, A., FEES Lett. 1975. 57: 14. 16 Bangham, A.D., de Gier, J. and Greville, G.D., Chem. Phys. Lipids 1967. I : 225. 17 Tyrrel, D.A., Heath, T.D., Colley, C.M. and Ryman, B.E., Biochim. Biophys. Acta 1976. 457: 259.
Dolores J. Schendel. R. Wank and 6. Dupont Sloan-Kettering Institute for Cancer Research, New York
18 Weissmann, G., Bloomgarden, D., Kaplan, R., Cohen, C., Hoffstein, S., Collins, T., Gotlieb, A. and Nagle, D., Proc. Nut. Acad. Sci. US 1975. 72: 88. 19 Cullen, S.E., Freed, J.H. and Nathenson, S.G., Transplant. Rev. 1976. 30: 236. 20 McKenzie, I.F.C., Clarke, A. and Parish, C.R., J. Exp. Med. 1977. 145: 1039. 21 Haughton, G., Transplantation 1966. 4: 238. 22 Sessa, G. and Weissmann, G., J. Biol. Chem. 1970.245: 3295. 23 Jilka, R.L. and Martonosi, A.N., J. Biol. Chem. 1975. 250: 7511. 24 Holloway, P.W. and Katz, J.T., J. Biol. Chem. 1975. 250: 9002. 25 Zborowski, J., Roerdink, F. and Scherphof, G., Biochim. Biophys. Acta 1977. 4 9 7 183.
Cell-mediated lympholysis: examination of H LA genetic fine structure and complementation using cytotoxic Iy mphocytes* Human cytotoxic lymphocytes (CTL), sensitized in vitro in one-way mixed lymphocyte cultures between t w o unrelated individuals, have been used t o study target cell determinants. One pair of CTL shows strong cell-mediated lympholysis responses against the specific target cells as well as against thirdparty target cells obtained from unrelated individuals. Testing of sixty target cells shows a significant association between the two CTL with a correlation coefficient of 0.67 in the panel. The target determinant recognized by the CTL is not an HLA-A,B,C or D specificity, as defined by standard serological and cellular reagents. Segregation studies in a large family reveal that the new specificity defined by the CTL is controlled by HLA, and its recognition may be restricted by the HLA complex.
1 Introduction The immune response t o foreign alloantigens i n many species is primarily directed against antigens of a major histocompatibility complex (MHC) (reviewed in [ 11). Activation of the allograft response by t h e MHC has been extensively investigated using the in vitro techniques of mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML). When lymphocytes from A are sensitized t o x-irradiated inactivated lymphocytes from B (B,) ( i . e . AB,), cells of A recognize foreign alloantigens of B and respond with proliferation which can be measured in MLC. Th e A responding cells also develop specific cytotoxic activity which can be measured in CML [ 2-51.
[I 20571
* This work was in part supported by NIH Grants CA-17404, CA22507, CA-19267, CA-08748, andEY01616. Correspondence: Dolores J. Schendel, Sloan-KetteringInstitute for Cancer Research, 1275 York Avenue, New York, NY 10021, USA Abbreviations: C M L Cell-mediated lympholysis Con A: Concanavalin A CTL: Cytotoxic lymphocytes LU: Lytic unit MR: Maximum release MRBC Mouse erythrocytes PHA: Phythohemagglutinin RCR: Relative cytotoxic response SR: Spontaneous release S R B C Sheep erythrocytes X x-irradiated
Different antigens of the MHC function in the stimulation of proliferation, induction of cytotoxic lymphocytes (CTL) and recognition of target cells. In this respect, striking similarities are observed between antigens of the HLA complex in man and of the H-2 complex in mouse. HLA-D-controlled determinant s and H-21-cont rolle d determinant s stimulate strong proliferation and enhance the development of CTL [6- lo]. Determinants controlled by HLA-A,B and C and H-2K and H-2D function in the sensitization of CTL and serve as target antigens [6,9-171. In the mouse, it has been observed that l a antigens are recognized as target determinants by CTL [ 17191, and additional determinants of t h e H-2 complex also seem t o be involved in CML [20-221. Recognition of HLAD determinants has not been observed in human CML [ 13, 14, 23, 241, but recent studies of our own and others [23331 indicate that other determinants in man, also controlled by the HLA complex, function in CML. These determinants are not currently identified with serological reagents which define the HLA-A,B and C products, but they can be identified in CML. In the present study, human CTL have been used t o identify new HLA-associated determinants and t o study their specificity and expression on panels of target cells. These CTL further characterize the genetic fine structure of the HLA complex and its role in the control of t h e in vitro allograft response.
Eur. J. Immunol. 1978.8: 634-640
Analysis of the HLA region with cytotoxic lymphocytes
635
2 Materials and methods
2.4 CMLassay
2.1 CTL
CTL were suspended at a concentration of 5 x l o 6 - 1 x 10' cells/ml, and t w o serial dilutions were made from this suspension. Triplicate 0.1 ml aliquots were incubated with 0.1 ml aliquots of target cells ( 5 x 1O4 - 1 x 1O5 cells/ml) in round-bottom microtiter plates (Cooke Co., Alexandria, VA). After 4 h, supernatant samples were harvested and counted in a gamma counter (Nuclear Chicago, Hartsdale, NY). Spontaneous release (SR) was determined b y incubating target cells in medium alone. Comparison of this value with that obtained when target cells were exposed t o an activated CTL (i.e. AB,/A*) showed a mean CML value of 2.5 3.3 % in 2 2 experiments. Maximum release (MR) was determined from target cells exposed t o a 5 % detergent solution (Triton X-100, New England Nuclear). Values for the mean MR cpm and % SR (SR/MR x 100) for 1 x lo4 cells are given in the table legends.
After density gradient separation (Lymphoprep, Accurate Chemical Co., Hicksville, NY) from whole blood, lymphocytes were diluted in complete culture medium, consisting of RPMI 1640 supplemented with L-glutamine ( 2 mM), penicillin (1 00 units/ml), streptomycin (1 00 pg/ml) and 15 % pooled human serum. Ten x l o 6 responding cells were coculture flasks (Corning, NY, No. 25 100) containing 2 0 ml of complete medium. Stimulating cells were inactivated 20 ml of complete medium. Stimulating cells were inactivated with 2000 rads of x-irradiation. The flasks were incubated at 37.5 "C standing upright in a humidified incubator with 5 % C02. After six days, the cytotoxic cells were used directly in the CML assay or cryopreserved f o r future use in medium containing 50 % human serum and 1 0 % dimethylsulfoxide (Sigma Co., St. Louis, MO) [34].
*
2.5 Calculation of data
2.2 Target cells Target cells were used either without mitogen stimulation or cultured with phytohemagglutinin (PHA-m, 1 :1 0 0 dilution of the stock source, 0528-57; Difco Co., Detroit, MI) o r concanavalin A (Con A, 10 pg/ml, 3351-56; Difco). Cryopreserved samples were thawed 48-72 h before t h e CML assay, and 5 x 106- 1 0 x 106 cells were incubated with mitogen in flasks containing 1 0 ml of complete medium. Nonstimulated cryopreserved target cells were thawed 18-24 h before use, and fresh cells were cultured f o r 6 days in complete medium without mitogen. On the day of the CML assay, t h e cell suspensions were transferred t o plastic test tubes (Falcon 2001; Becton Dickinson and Co., Cockeysville, MD) and centrifuged at 150 x g f o r 8 min. The cell pellets were resuspended in 0.2 ml of the supernatant medium. Samples were labeled with 250 pCi of NazCrs104 (spec. act. 200500 mCi/mg Cr; New England Nuclear, Boston, MA) for 1-2 h at 37.5 OC in a humidified incubator with 5 % C02. Labeled cells were washed twice with 1 5 % serum medium, counted and suspended at a concentration of 5 x lo4 - 1 x i051mi.
A percent cytotoxicity was calculated by the formula: %CML =
Experimental release - SR x 100. MR - SR
To pool data from several experiments, a normalized cytotoxicity value was used [34]. A percent relative cytotoxic response (RCR) based on the specific response for a given CTL was determined as follows: % RCR =
% CML of experimental target X* -x 100, % CML of specific target B*
where a CTL of A.B specificity (A responding cells sensitized to Bx stimulating cells) was tested for lytic activity directed against the experimental target X* and expressed as a percentage of t h e lytic activity directed against the specific target B*, which defines the reference value for 100 5%.
3 Results 3.1 Identification of CTL detecting new target specificities
2.3 Subpopulations of target cells Subpopulations of peripheral blood lymphocytes were prepared as previously described [35-371. Briefly, T lymphocytes, forming rosettes with neuraminidase-treated sheep erythrocytes (SRBC), were separated from nonrosetting cells on a Lymphoprep gradient. The nonrosetting fraction was then exposed t o mouse erythrocytes (MRBC) and the rosettes, representing a B subset [ 3 7 ] , were separated from the nonrosetting fraction on a second Lymphoprep gradient. Attached erythrocytes were lysed with ammonium chloride (0.8 %), and the washed lymphocytes were incubated for four days in culture medium containing no mitogen before use as CML targets. This separation procedure did not provide pure T and B cell subpopulations but rather provided enriched T and B fractions. Rosetting of the interface fractions after the separation showed < 10 % contamination with SRBC and MRBC rosetting cells, respectively.
CTL were made by mutual in uitro sensitization of peripheral blood lymphocytes from three unrelated individuals. The HLA-A,B and C antigens differing between the responding and stimulating cells of t h e CTL are listed in Table 1. Responses mediated by these CTL reveal several unusual reactions if the HLA-A and B antigens are presumed t o be the predominant CML sensitizing and target antigens. CTL A . l causes the expected specific lysis of target 1*, yet shows an unexpected equal lysis of target 2* (Table 2). Similarly, CTL A.2 specifically lyses 2* and 1 * in a quantitatively identical manner. Since CTL A.l and A.2 have been stimulated by different antigens of the HLA-A and B series, they must be sensitized t o and recognize other determinants shared by cells 1 and 2. In contrast, when the reciprocal CML responses are measured between cells 1 and 2, using CTL 1.2 and 2.1, the specific cytotoxic responses are much weaker. Of particular interest is t h e reaction of CTL 1.2 against target 2*. In this combination, HLA-B 12, which has been claimed t o be one of t h e
636
Eur. J. Immunol. 1978.8: 634-640
D.J. Schendel, R. Wank and B. Dupont
strongest CML antigens of the HLA-B series [ 151, should serve as a sensitizing and target antigen, yet despite the difference for HLA-B12 between the responding and stimulating cells in CTL 1.2 the cytotoxic response is low. Presumably this low response is observed because 1 and 2 share the CML target specificity which is &cognized by CTL A.l and A.2. This hypothesis is strengthened by target cell inhibition studies showing that cells 1 and 2 can equally inhibit the specific responses of A.l and A.2 (Schendel, unpublished observations). Table 1. Sensitizing antigens for cytotoxic lymphocytes CTLa) Responder A. 1 A.2 1.2 2.1 1.A 2.A
Stimulatorb)
HLA antigen difference&
1, 2, 2, 1,
A3,Bw35,Bw37,Cw4 B12,CwS B12,CwS A3,Bw35,Bw37,Cw4 B8.Bw40 B8,Bw40
A A 1 2 1 2
AX AX
a) The known HLA antigens for individuals A, 1 and 2 are A:(A2,
/A~,BW~~); B8/Bw40); ~ : ( A ~ , B w ~ ~ , C W ~ , D W ~2:(A2,B12,Cw5). b) Stimulating cells are x-irradiated with 2000 R. c) Antigens expressed by stimulating cells which are not expressed
by responding cells. Table 2. Quantitative CML study with six CTLa)
3.2 Evaluation of the role of HLA-C antigens In evaluating what could be servirrg as t h e unknown target determinant recognized by CTL A.l and A.2, the HLA-C antigens were first considered. Cytotoxicity by CTL A.l and A.2, as well as by the autologous sensitized CTL A.A, was measured on a panel of target cells obtained from unrelated individuals with defined H L A C specificities. The CML reactivity did not reveal any shared HLA-C antigen which could serve as the CML specificity. Selected cells from this panel are shown in Table 3 , including cells that express one or more of the five HLAC-controlled antigens which are currently identified serologically (Cwl -Cw5). No single one of these HLA-C specificities can explain the positive reactions of CTL A. 1 and A.2. Furthermore, because target cells E* and F* have both H L A C specificities defined, the reactions cannot be ascribed t o an unknown HLA-C specificity. Table 3. Analysis of HLA-Cilirected cytotoxicitya) CTL HLAC A* 1* 2* B*bf C* D* E* F* antigen cw4 cw5 cw5 cw4 cw2 Cwl cw4 - - differences cw3 cw5 cw5 A.A A.l A.2
0.5 1.8 1.9 1.5 1.4 Cw4I- 0.4 52.3 45.2 4.1 5.3 CW5/- - 1.4 56.3 46.1 11.0 11.0
0.3 5.4 7.6
0.6 0.4 31.4 35.9 37.9 36.8
a) Data are expressed as 9% CML; MR=2023-9890 cpm; SR < 23 %.
Target cells (*) are stimulated with PHA-m. CTL
Target
A.1
1* 2* 1* 2* 2* 1* A* A*
A.2 1.2 2.1 l.A 2.A
20lb)
40:l
60:l
r2C)
LUd)
36.4 32.5 38.2 37.3 9.0 10.9 9.9 10.1
50.5 41.4 55.4 51.6 12.6 17.5 18.7 14.8
64.5 50.2 72.6 66.0 16.1 24.1 27.6 19.4
0.91 0.92 0.91 0.94 0.96 0.99 1.00 0.81
1.1 1.1 1.1 1.0 13.8 8.0 6.5 10.6
a) Data are expressed as % CML: MR=1024-1321 cpm; SR < 21 %. Target cells (*) are stimulated with PHA-m. b) Ratio of CTL to target cell. c) Coefficient of determination for linear CML activity. required to give 30 % CML. d) Lytic unit = number of CTL x Linear regression analyses show that the responses of all t h e CTL fit straight lines when the effector-to-target cell ratio is varied. Extrapolation from these lines enables determination of the number of lymphocytes of each CTL required t o cause 30 % CML, which defines a lytic unit (LU). According t o this definition, a larger LU size reflects a weaker response since more CTL are required t o cause a 30 % CML response. Comparison of the LU for the CTL is shown in Table 2. The LU is identical for CTL A.l and A.2 against target 1* but is eightfold more for CTL 2.1 against 1*. At least twelve times more of CTL 1.2 is required t o give 30 % CML of 2* than is required by either CTL A.l or A.2.
b) The HLA-A and B antigens for targets B* -F* are as follows:
B*(Al,A2,B7,B12); C*(Ag,AlO,B12,B14);D*(A2,All,B27); E*(A2,B12,B27); Fr(A2,B12,Bw35). 3.3 Evaluation of the role of HLA-D antigens The role of HLA-Dcontrolled antigens was also evaluated on the target panel, and n o known shared HLA-D determinant could explain all the CML reactions of CTL A.l and A.2. A second apprQach used t o evaluate the role of HLA-D, as well as that of B cell (DRw) alloantigens [38], was comparison of subpopulations of enriched T and B lymphocytes as target cells. If the new target specificity is a n antigen of the HLA-D series o r a B cell alloantigen shared by cells 1 and 2, which has not yet been identified, then CML against target subpopulations should reveal a high level of lysis of the B-enriched target population and loss of CML reactivity against the Tenriched target population. Peripheral blood lymphocytes from individual 1 were separated into fractions of enriched T and B cells and a remaining fraction (N) after removal of the T and B fractions. Equal levels of cytotoxicity are observed (Table 4) when the CML responses of CTL A.l and A.2 are measured on the three subpopulations of 1* target cells. Therefore, the HLA-D o r B cell alloantigens do not explain the CML specificity. The level of lysis in repeated testings was very high on all target cell subpopulations, so responses t o any one subpopulation are not explained by contamination with cells of another subpopulation. 3.4 Analysis of cytotoxicity of a target cell panel
Cells from 1 and 2 were also sensitized t o lymphocytes from individual A (CTL l.A and 2.A). These t w o CTL react similarly with A* target cells, and they can also lyse unrelated target cells sharing n o known HLA-A,B or C antigens with A*.
The cytotoxic responses of CTL A.1 and A.2 were tested simultaneously on a panel of sixty target cells. Data were pooled from four experiments for this analysis using the
Eur. .IImmunol. . 1978.8: 634-640
Analysis of the HLA region with cytotoxic lymphocytes
637
Table 4. Comparison of target cell subpopulationsa) 0
n L
A*-@)
Target cells 2*-U l*-U * 1*-TC)
I*.@
1*-Ne)
A.A
-0.3 1.0 1.0
35 1.9 6.5 63.6 72.6 74.4 54.5 59.5 74.2
8.7 75.3 69.6
9.3 73.1 69.4
A.l A3
0
3
80
a) Data are expressed as % CML; MR=2290-25 000 cpm; SR < 18 %. All target cells (*) are tested without mitogen stimulation. b) Unseparated (U) peripheral blood lymphocytes. c) Enriched T cells forming SRBC rosettes. d) Enriched B cells forming MRBC rosettes. e) Remaining cells after removd of T and B fractions.
0
f
c
70-
0
al
60-
Lz
0
r2=0.9 b = 1.03
10
20 30 40 50 60 70 80 90 100 110 120 1%
-1
The second unusual characteristic noted in this study is t h e quantitative relationship between the activities of t h e t w o CTL in their recognition of different target cells. (Fig. 1). The % RCR of an individual target cell with CTL A.l is plotted against its % RCR with CTL A.2. Althoughpositive responses range from 11 -130 % RCR, most target cells are lysed t o t h e same degree by both CTL A.l and A.2. For example, the target cell indicated by the * shows a RCR of 75 % with CTL A.l and a RCR of 76 %with CTL A.2. Linear regression analysis fits t h e CML responses of CTL A.l and A.2 t o a straight line with a slope of 1.03 (r2=0.9), and repeated testings show t h e quantitative distinction of individual cells t o be very consistent.
Figure 1. Quantitative recognition of panel target cells with CTL A . l and A.2. The % RCR by CTL A.1 of an individual target cell is plotted against the % RCR with CTL A.2. There is a strong linear relationsh (r2=0.9) fitting a line with a slope (b) of 1.03.
target cells recognized by CTL A.l and A.2. Most of the cells of the target panel were studied in t h e VII. International Histocompatibility Workshop and are well characterized for their IILA-A,B,C, D and DRw (Ia-like) alloantigens [39]. This enabled comparison of the CML specificity with 1 8 HLA-A antigens, 20 HLA-B antigens, w4 and w6, 5 HLA-C antigens, 1 0 HLA-D specificities, and 7 DRw specificities defined by clusters of B cell sera. Table 6 summarizes information from these analyses. Table 6. Associations of CTL specificitiesand HLA antigens CTL
A.l
CMLresponsewith CTL A.2
+
-
24 4
A.2
-
5
21
a) Cytotoxicity was measured on 60 target cells of unrelated individuals. A RCR < 10 % was considered negative, and a RCR > 10 %was considered positive. The association between CTL A.l and A.2 is highly significant (x2=26.6; p < 0.001), and their
correlation coefficient in this panel is r=0.67.
3.5 Association analyses with HLA antigens Two by t w o comparisons were made between different HLA antigens and the CML specificity t o determine whether particular HLA specificities are frequently expressed o n those
Serologically defined HLA antigen Bw35 cw4 B12 CW5
with CTL A.1
+
%
Relative Cytotoxic Response with C T L A.1
Table 5 . Two by two association analysis of the CML responses of CTL A . l and A.2a)
CML response
N.60
0
relative cytotoxic response (% RCR, see Sect. 2.5). The range of the responses of CTL A.l against 1* was 52-88 % CML, and of CTL A.2 against 2* was 46-72 % CML. RCR of < 10 % was considered negative. Based o n the specific responses, this reflects an actual % CML of 4.6-8.8 %. Two findings characterize this analysis. First, CTL A.l and A.2 distinguish similar groups of positive and negative cells. Table 5 shows t h e two by t w o comparison analysis indicating a highly significant association in the responses 9f CTL A.l and A.2. It should be emphasized that the basis for this association cannot be explained by recognition of any shared HLA-A,B,C or D antigen, at least as they are currently defined.
0
0
A.l
+
A.2
B12+Bw35 B12 cw5 Bw35 cw4 B12+Bw35 B12 Bw35 Cw4 cw5 B12+Bw35
12 12 10 6 19 13 I 10 11 20 10 9 9 6 16
14 14 16 20 I 16 22 19 18 9 13 14 14 11 7
I 2 5 1
6 2 0 3 3 5 5 4 5 1 9
27 24 23 25 22 23 23 22 20 20 26 27 24 28 22
11.1 I .9 1.9 2.6 12.5 1.3 4.5 2.6 2.9 11.1 3.7 3.6 2.1 3.9 7.2
0.001 0.005 0.166 0.104 0.000 0.006 0.034 0.108 0.090 0.001 0.055 0.05 7 0.146 0.049 0.007
a) The first sign indicates the response with the given CTL ("+"= RCR > 10 %; "-" = RCR < 10 %), and the second sign indicates
"+" or "-"serological typing for the given HLA antigen. The 2x2 analysis was also done defining RCR values of < 5 % and < 15 % a s negative; this did not show a different association or a better correlation between the CML specificity and a serological specificity. b) x2 value for the positive association. c) Significance level of the association.
Eur. J. Immunol. 1978.8: 634-640
D.J. Schendel, R. Wank and B. Dupont
638
Recognition by CTL A.l shows associations t o HLA-Bw35 and HLA-Cw4, both of which are expressed by the stimulating cells of this CTL (see Table 1). CTL A.2 shows associations t o HLA-B12 and Cw5, also expressed by stimulating cell 2. No significant positive associations were found for any HLA-A antigens, w4, w6, HLA-D antigens or DRw specificities. Analyses with both CTL A.l and A.2 (i.e. target cells must be positive o r negative with both CTL) show a decrease in the associations with HLA-B12, Bw35, Cw4 and Cw5. It was noted that the discordant reactions in the 2 x 2 tables were mainly positive with the CTL and negative for t h e serological specificity, indicating that t h e serological specificities might be included in the CML specificity. Therefore, additional analyses were done combining all cells positive for either B12 or Bw35 t o determine whether the combined antigens would show a stronger correlation with the CML specificity, and t o see whether the number of CML-positive but serologically negative (+-) reactions would decrease. The value of the association with CTL A . l , A.2 and A.l + A.2 does in fact increase with the B12 + Bw35 combination. However, discordant reactions are still observed, and they now show an equal distribution in the (+-) and (-+) categories. The finding that some target cells expressing neither of these antigens are in the high positive group (RCR 60 %), and others carrying these antigens are in the negative group (RCR < 10 %),emphasizes that t h e CML determinant is in fact distinct from these serologically defined HLA antigensHowever, t h e positive associations between t h e CML antigen and HLA-B antigens d o indicate that t h e target specificity is controlled by t h e HLA complex.
x2
>
3.6 HLA segregation analysis Additional evidence for the control of the CML specificity b y the HLA region was obtained in a family segregation study. Individual Ka (indicated by the * in Fig. 1) was used as a sensitizing cell t o generate a third CTL (A.3) and tested simultaneously with A.l and A.2 against target cells from her family. (Table 7). Three of t h e family target cells were strongly lysed by CTL A.l, A.2 and A.3. These three target cells are from HLA-identical siblings sharing t h e “b” and “c” haplotypes. Quantitatively, t h e three CTL react similarly with each of t h e HLA-identical target cells. The recognition of these three target cells demonstrates that HLA is determining the CML specificity. However, the failure t o find equally strong positive responses against either of the parental target cells was unexpected. Because of the association between the CML specificity and HLA-Bw35 in the panel one might have expected strong lysis of t h e cells of the mother (M) who carries the Bw35 antigen on her “c” haplotype. T o rule out a technical failure t o detect this response, two separate blood samples were used t o prepare target cells. From both of these samples targets stimulated with PHA o r Con A and nonstimulated cells were compared, and in each case the CML reaction was less than 1 3 %. Only a low level of CML (7-10 7%) is detected against the cells of the father. If t h e reaction t o Ka, T i and J e is directed against determinants controlled only by the “b” haplotype, one would have expected t o see strong positive lysis not only of the cells of the father but also of t h e sibling Da. Thus, HLA appears t o govern the CML specificity detected by the three
Table 7. HLA segregation study in family Le
Targets
Sex
F* M* Ka*(3) Ti* Je *
Da* Vi* Jo*
Ma* Ja*
d
9 9
d 9
d 9 3
Y
9
HLA~) haplotypes
alb cld
blc bl c blc bld ald ald a1d ald
CTLb) A.l
A.2
A.3
7.3 1.9 41.3 54.1 34.3 1.o 0.6 0.9 0.0 0.0
6.8 0.1 52.5 62.2 35.4 1.7 1.9
9.8 8.1 54.1 61.8 35.9c) 4.0 3.5 1.8 0. I 2.1
1 .I
0.0 0.0
a) The HLA-A,B and C antigens for the different haplotypes are a(A2,B27,Cwl), b(A2,B13), c(Aw24,Bw35,Cw4)and d(A3,B7). b) Data are expressed as % CML; MR-1860-3717 cpm; SR < 20 %. Targets (*) are stimulated with PHA-m. c) These target cells are obtained from an individual undergoing long-term kidney dialysis which may account for the lower level of CML. CTL, but the reaction is not uniquely associated with determinants controlled by a single HLA haplotype. Complementation between the “b” and “c” haplotypes must occur for CML recognition. 4 Discussion
Initial studies on t h e genetic control of CML indicated that the serologically defined HLA-A and B antigens are the primary CML target determinants. The determinants identified by CTL A.l and A.2 are distinct from known HLA-A,B,C and D specificities but are associated with t h e HLA complex. Earlier quantitative studies on the CML response t o different HLA antigens claimed that HLA-B 12 is particularly strong, yet the finding that a distinct, but HLA-B12-associated, determinant can be recognized by CTL A.l and A.2 raises the question of whether such HLA-B antigens act as markers for other target determinants. Comparison of the lytic activities of CTL A.l and A.2 on targets 1* and 2*, which shows the strong response t o the new determinant, and the lytic activities of CTL 1.2 and 2.1, which shows t h e weak responses between cells sharing this specificity but differing for HLAA,B and C locus antigens, requires reconsideration as t o what determinants are primarily recognized b y human CTL. Although recognition of individual antigens of the HLA-A,B,C and D series does not explain the CML specificity, an association is seen with some HLA-B locus antigens in the panel. Several possibilities can explain this association. First, the target determinant may be a specificity shared by some HLAB antigens which is detected by CTL but has not been identified serologically. Models of this type have been reported for H-2 antigens in which cells from mice carrying mutations in the H-2 complex can stimulate strong CML responses, but are poor in stimulating antibody production t o t h e mutant specificity [20, 221. Second, t h e CML target molecule might be preferentially associated in the membrane with particular HLA-B antigens. Interactions between such associated molecules could lead t o CML recognition. Many examples of interacting specificities have been reported in the mouse for recognition of minor histocompatibility antigens, viral antigens and chemical modifications of cell surface molecules (reviewed in
Eur. J. Immunol. 1978.8: 634-640 [40]). In one important respect these results differ from t h e H-2 models in that identity between t h e responding cell and target cell for HLA-A,B or C antigens (in anology with H-2K and H-2D) is not required for CML recognition. Third, t h e CML target specificity may be completely distinct from HLA-B antigens, but if the genes controlling these antigens are in linkage disequilibrium in the population, the antigens would frequently occur simultaneously o n t h e cell surface. Such linkage disequilibria are already known for t h e genes controlling HLA-Bw35 and Cw4 and HLA-B12 and Cw5 [41]. Clear evidence which would distinguish between distinct molecular cytotoxic and serological specificities can be obtained by separation of the determinants through genetic recombination of their controlling genes o r by their biochemical separation.
.
The ability t o study the target cell panel with t w o different, but highly correlated CTL, not only enables evaluation of responses t o cells sharing HLA-A,B,C or D antigens with the CTL-stimulating cells, but reveals parallel patterns in t h e level of lysis of individual cells. This variation could reflect recognition of several specificities by CTL A.l and A.2 with those cells in the lower category expressing only some of the specificities. If this is t h e case, these new specificities must be frequently shared by unrelated individuals because CTL A.l and A.2 can be used t o select individuals t o sensitize new CTL (A.3), and these CTL behave similarly t o CTL A.l and A.2 not only in family studies but also in panel studies. O n t h e other hand, the cells in the high category (>5 0 %) may express a common CML determinant while those in t h e intermediate category (10-50 %) may express cross-reactive determinants. T o examine this possibility, association analyses t o HLA antigens were done comparing t w o categories: < 10 % and 2 10 % RCR, and three categories: < 10 %, 10 % - 50 % and > 50 % RCR. Associations t o HLA-B locus antigens did not show any distinctions between t h e single positive group (> 10 % and) intermediate ( 10 % - 5 0 %) and strong (> 5 0 %) positive groups. Therefore, if recognition of a cross-reactive specificity accounts for the intermediate responses, the crossreacting specificities show identical HLA-B antigen associations. The variation may also be explained by complementation effects similar t o those observed in the family Le. If CML recognition in the unrelated panel is dependent upon interactions, then different components may lead t o more o r less efficient complementation. The complementation patterns observed in the family Le reveal that the interacting factors which lead t o CML recognition are determined by HLA. This complementation could either occur at t h e genetic level o r at t h e cell surface. F o r example, t h e target specificity may be determined by a single HLA haplotype, but other genetic factors may interact with this specificity and allow CML recognition. Recognition of target determinants may be restricted by HLA, similar t o the pattern reported for recognition of H-Y [42] and other non-HLA-A,B,C and D antigens in man [32, 43, 441. Our results cannot be explained by H-Y since target cells of both sexes are recognized by the CTL as seen in Table 7. In one of these studies [32], a strikingly similar pattern was seen in CML analysis of a family carrying t h e HLA-B12 and Bw35 antigens. Such a restriction could be due t o recognition of a hybrid antigen formed through genetic interaction of the “b” and “c” haplotypes, or dual recognition of t w o specificities, each of which is determined by a single haplotype in this
Analysis of the HLA region with cytotoxic lymphocytes
639
family. Again, it should be stressed that this HLA “restriction” does not require HLA-A,B or C identity between CTL and the target cell. Alternatively, expression of target specificities may not be equal o r even codominant on the surface of the cell. Thus, homozygosity for t h e same determinant or heterozygosity for t w o determinants, each of which is recognized by the three CTL, could account for strong lysis of the HLA-identical cells. In t h e parental cells, a lower antigen density or expression of the antigen on only a portion of the lymphocytes would lead t o low lysis. Distinction of these models is n o t yet possible, but studies underway t o examine CML responses between Le family members, and by CTL sensitized t o other individuals of t h e panel, may provide new information about t h e basis of the complementation. CTL provide a useful immunogenetic reagent for studying the genetic fine structure of the HLA complex. The CTL described in these studies identify new HLA-associated cytotoxic determinants which may eventually prove t d be controlled by distinct genes of the HLA complex. The strong response directed against these determinants indicates that they may be of significant importance in the histocompatibility response. The pattern of complementation leading t o CML recognition raises new questions regarding the role of HLA haplotypes in cytotoxicity, and leads us t o consider interactions in the control of t h e allograft response. The authors wish to thank Mss. Melvyn Blanco and Melna Hall for excellent technical assistance, Ms. Loretta Hall for help in the preparation of this manuscript. Dr. David Braun, Jr., for performing the statistical analysis, and Ka Le and her family for participating in these studies.
Received March 1, 1978; in revised form June 5, 1978.
5 References 1 Gotze, D. (Ed.), The Major Histocompatibility System in Man and Animals, Springer Verlag , Berlin 1977. 2 Lightbody, J., Bernoco, D., Miggiano, V. and Ceppellini, R., G. Batt. Virol. Immunol. 1971. 64: 243. 3 Solliday, S. and Bach, F., Science 1970.170: 1406. 4 Hayry, P. and Defendi, A. Science 1970. 168: 133. 5 Hodes, R. and Svedmyr, E., Transplantation 1970. 9: 470. 6 Eijsvoogel, V., du Bois, M., Melief, C., de Groot-Kooy, M., de Koning. C. van Rood, J., van Leeuwen, A.. d u Toit, E. and Schellekens, P., in Dausset, J. and Colornbiani,J. (Eds.), Histocompatibility Testing 1972, Munksgaard, Copenhagen 1 9 7 3 , ~501. . 7 Bonnard, G., Lemos, L. and Chapuis, M., S a n d . J. Immunol. 1974. 3: 97. 8 Long, M. Handwerger, B., Amos B. and Yunis, E., J. Immunol. 1976. I 1 7: 2092. 9 Alter, B., Schendel, D.J., Bach, M., Bach, F . , Klein, J. and Stimpfling, J . , J . Exp-Med. 1973.137: 1303. 10 Schendel, D.J. Alter, B. and Bach, F., Transplant. Proc. 1973. 5: 1651. 11 Miggiano, V., Bernoco, D., Lightbody, J., Trinchieri, G. and Ceppellini, R., Transplant. Proc. 1972. 4 : 231. 12 Bonnard, G., Chapuis, M., Glauser, A., Mempel, W., Baumann, P., Grosse-Wilde, H. and Albert,E., nansplant. Proc. 1973. 5: 1679. 13 Eijsvoogel, V., d u Bois, R., Melief, C., Zeijlemaker, W., RaatKooning, L. and de Groot-Kooy, L., Transplant. Proc. 1973.5: 1301. 14 Trinchieri, G., Bernoco, D., Curtoni, S. Miggiano, V. and Ceppellini, R., in Dausset, J. and Colombiani, J. (Eds.), Histocompatibility Testing 1972, Munksgaard, Copenhagen 1973, p. 509.
640
Eur. J. Immunol. 1978.8: 640-645
J. H. Russell, A. H. Hale, D. Inbar and H. N. Eisen
15 Kristensen, T., Grunnet, N. and Kissmeyer-Nielsen, F., Tissue Antigens 1975.6: 221. 16 Grunnet, N., Kristensen, T. and Kissmeyer-Nielsen, F., Tissue Antigens 1976. 7: 301. 17 Nabholz, M., Vives, J., Young, H.M., Meo, T., Miggiano, V., Rijnbeek, A. and Shreffler, D. C., Eur. J. Immunol. 1974.4: 378. 18 Schendel, D.J. and Bach, F . , J . Exp. Med. 1974.140: 1534. 19 Schendel, D.J. and Bach, F., Eur. J. Immunol. 1975. 5: 880. 20 Widmer, M., Alter, B., Bach, F. and Bach, M., Nature-New Biol. 1973.242: 239. 21 Phillips, S . Carpenter, C. arid Strom, T., 7kansplant. Proc. 1973. 5: 1669. 22 Nabholz, M., Young, H.M., Meo, T., Miggiano, V., Rijnbeek, A. and Shremer, D.C., Immunogenetics 1975.5: 469. 23 Long, M. Handwerger, B. and Yunis, E., in Kissmeyer-Nielsen, F. (Ed.), Histocompatibility Testing I 9 75, Munksgaard, Copenhagen 1975, p. 849. 24 Mawas, C., Dominique, C. and Sasportes, M., in Kissmeyer-Nielsen, F., (Ed.), Histocompatibility Testing 1975, Munksgaard, Copenhagen 1975, p. 855. 25 Kristensen, T., Grunnet, N. and Kissmeyer-Nielsen, F., Tissue Antigens 1974. 4 : 378. 26 Mawas, C., Christen, Y., Legrand, L., Sasportes, M. and Dausset, J., Transplantation 1974.18: 256. 27 Schapira, M. and Jeannet, M., Tissue Antigens 1974. 4 : 178. 28 Willumsen, J. and Heron, I., Tissue Antigens 1974.4: 172.
Histocompatibility Testing 1 9 75, Munksgaard, Copenhagen 1975, p. 835. 31 Sondel, P. and Bach, F., J. Exp. Med. 1975.142: 1339. 32 Goulmy, E., Termijtelen, A., Bradley, B. and van Rood, J., Tissue Antigens 1976.8: 317. 33 Schendel, D.J., Wank, R., Hansen, J.A. and Dupont, B., Transplant. Proc. 1977. 9 : 1777. 34 Schendel, D.J., Wank, R., Hall, M. and Dupont, B., TissueAntigens, in press. 35 Weiner, M., Bianco, C. and Nussenzweig, V., Blood 1973.42: 939. 36 Gallie, U. and Schlesinger, M., J. of Immunol. 1974.112: 1628. 37 Gupta, S., Good, R. and Siegel, F., Clin. Exp. Immunol. 1976. 25: 319. 38 Bodmer, W., Batchelor, R., Morris, P., Festenstein, H. and Bodmer, J., (Eds.) Histocompatibility Testing I 9 77, Munksgaard, Copenhagen, in press. 39 Dupont, B., Yunis, E., Duquesnoy, R., Pollack, M., Noreen, H., Hansen, J., Reinsmoen, N., Annen, K., Greenberg, L., Lee, T., Whitsett, C., Antonelli, P. and Braun, D., in Bodmer, W., Batchelor, R., Morris, P., Festenstein, H. and Bodmer, J. (Eds.), Histocompatibility Testing I 9 77, Munksgaard, Copenhagen, in press. 40 Doherty, P., Blanden, R. and Zinkernagel, R., 7kansplant. Rev. 1976.29: 89. 41 Mayr, W., in Kissmeyer-Nielsen, F. (Ed.), Histocompatibility Testing 1975, Munksgaard, Copenhagen 1975, p. 330. 42 Goulmy, E., Termijtelen, A., Bradley, B. and van Rood, J., Nature 1977.266: 544.
29 Goulmy, E., Termijtelen, A., Keuning, J. and van Rood, J., in Kissmeyer-Nielsen, F. (Ed.), Histocompatibility Testing 1975, Munksgaard, Copenhagen 1975, p. 845.
43 McMichael, A., Ting, A., Zweerink, H. and Askonas, B., Nature 1977.270: 524.
30 Kristensen, T. and Grunnet, M., in Kissmeyer-Nielsen, F. (Ed.),
44 Dickmeiss, E., Socber, B. and Svejgaard, A., Nature 1977.270:526.
J. H. Russelo, A. H. Hale', D. Inbar' and
H. N. Eisen Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge
Loss of reactivity of a BALB/c myeloma tumor with allogeneic and syngeneic cytotoxic T lymphocytes* I t was previously observed that MOPC-315EL, a subline o f t h e BALB/c m y e l o m a t u m o r MOPC-315, varies in its ability to interact w i t h primary anti-H-2d c y t o t o x i c thymus-derived l y m p h o c y t e s ( C T L ) while remaining invariant in its expression of cell surface antigens recognized b y anti-H-2d sera. This paper demonstrates (a) t h a t secondary anti-H-2d C T L also fail to recognize t h e late t u m o r cells, a n d (b) t h a t t w o o t h e r C T L systems (anti-minor histocompatibility antigens a n d anti-2,4,6trinitrophenyl), which require recognition of both H-2 p r o d u c t s a n d o t h e r surface antigens, also fail to react w i t h t h e late t u m o r cells. The defect in the late t u m o r cells w a s evident w h e n they w e r e used as targets, inhibitors, a n d stimulators of C T L activity [I 21261
1 Introduction
*
This work was supported in part by a research grant (CA-15472) and and a center grant to the Massachusetts Institute of Technology, Center for Cancer Research (CA-14051) from the National Cancer Institute, Department of Health, Education and Welfare and by additional aid from the James and Lynelle Holden Fund. o Helen Hay Whitney Postdoctoral Fellow + National Cancer Institute Postdoctoral Fellow (5f32CA05685) Present address: Ames Yissum, Ltd., Har Hotzvin, Sanhedria Murchevet, Jerusalem, Israel Correspondence: Herman N. Eisen, E l 7-128, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Abbreviations: Con A: Concanavalin A C T L Cytotoxic thymusderived lymphocyte(s) ip: Intraperitoneal minor: Minor histocompatibility antigens MLR: One-way mixed lymphocyte reaction PBS: Phosphate-buffered saline (0.015 M NaCl/O.Ol M potassium phosphate, pH 7.4) Tnp: 2,4,6-Trinitrophenyl E :T ratio: Effector : target cell ratio
In a previous c o m m u n i c a t i o n w e reported t h a t MOPC-315EL, a subline o f MOPC-315 ( t h e BALB/c m y e l o m a t u m o r that produces a 2,4dinitrophenyl-binding IgA myeloma protein), undergoes late in e a c h t r a n s p l a n t a t i o n cycle in BALB/c mice a reversible loss in ability to react w i t h allogeneic c y t o t o x i c thymus-derived l y m p h o c y t e s (CTL) [ 11. A t t h e s a m e t i m e , the t u m o r cells retained undiminished on their surface t h e serologically recognized p r o d u c t s o f t h e H-2d haplotype. T h e C T L with which t h e late t u m o r cells lost reactivity had been elicited b y stimulating B10 spleen cells w i t h B10.D2 cells o r BALB.B spleen cells w i t h BALB/c cells. Hence, the CTL (H-2b haplot y p e ) w e r e specific f o r H-2d, t h e h a p l o t y p e o f t h e BALB/c t u m o r . T h e lack o f reactivity o f l a t e t u m o r cells with these C T L was evident w h e n t h e t u m o r cells were used as labeled
