Implications of TL Phenotype Changes in an H-2-Loss Variant of a Transplanted H-2b/H-2a Leukemia*

Transcription

1 Proceedings of the National Academy of Sciences Vol. 65, No. 4, pp , April 1970 Implications of TL Phenotype Changes in an H-2-Loss Variant of a Transplanted H-2b/H-2a Leukemia* Edward A. Boyse, Elisabeth Stockert, Chika A. Iritani, and Lloyd J. Old DIVISION OF IMMUNOLOGY, SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH, NEW YORK, NEW YORK Communicated by L. C. Dunn, December 19, 1969 Abstract. An H-2-loss variant line, lacking H-2a antigens, was obtained from an H-2b/H-2a (C57BL X A)Fj transplanted leukemia by immunoselection. The TL phenotype of the unselected line was TL.1,2,3,4 and that of the variant TL.1,2,4, which is the phenotype of TL+ leukemias of C57BL. This finding, and the observed quantitative alterations of antigens TL.1,2 and 4, indicate that the Tla (Thymus leukemia antigen) locus was functionally deleted together with the H-2 locus in the process of variant formation, despite absence of selection against cells carrying TL antigens. Representation of H-2 antigens of the remaining haplotype, H-2b, was quantitatively unchanged. The Thymus leukemia antigen (Tla) locus specifies cell surface alloantigens (TL antigens) that in normal mice are expressed only on thymocytes, the relevant Tla genes being apparently repressed in cells of all other types (see review').-mice of several strains (TL- strains) do not express any of the known TL antigens on their thymocytes. But TL antigens may be. expressed on leukemia cells of these TL- strains, from which it is inferred that they carry the respective structural genes. In other words, in TL- mice certain Tla genes fail to undergo derepression in the course of thymocyte differentiation, as they do in TL + strains, although derepression may occur in the event of leukemogenesis. As evidence that TL antigens are truly unexpressed in TL- strains, such mice form the respective antibodies when appropriately immunized, whereas TL + mice do not. Genetic derepression of Tla genes consequent on malignancy indicates that this region of chromosome IX may in some way be intimately involved in a type of leukemogenesis in the mouse. These and other unusual features of the TL system such as antigenic modulation (see below) point to the need for further approaches to help substantiate our present view of the Tla locus. One of these approaches, not so far explored, is the use of "parental variants" selected from TL + leukemias of mice heterozygous for H-2. Such variants can be derived from transplanted leukemias induced in hybrid mice of crosses between two inbred strains differing at H-2. Passage of the hybrid leukemia in either parent line may result in immunoselection of a variant clone lacking the set of H-2 alloantigens (haplotype) of the other parent line, and therefore selectively histocompatible with the first parent line. Klein and his colleagues,2 on the basis of a penetrating and extensive analysis, favored a genetic 933

2 934 GENETICS: BOYSE ET AL. PROC. N. A. S. rather than an epigenetic explanation of variant formation, especially on the grounds that the H-2 losses were generally irreversible. A study of partial H-2- loss variants, obtained by immunoselection against only some antigens of a haplotype, revealed that loss of certain D-end antigens always entailed loss of K-end antigens (Fig. 1), but not vice versa. This polarity of the losses suggested somatic crossing over or terminal chromosomal deletion as mechanisms of variant formation, assuming the centromere to lie on l the D side of H-2. According to more T recent reports, however, the centromere 3 of chromosome IX lies on the K side of : H-2 (Fig. 1) ;3 4 this is a useful paradox and an added stimulus to find out what tf may happen to other group IX loci that may be implicated in parental variant formation. K end In the past, the only usable group IX D end markers on the variants have been the H-2 H-2 antigens themselves, for no other C57 has genes for -Tla -A strain has genes for group IX gene had a cellular phenotype specificities 1, 2 specificities 1, 2 and and 4; expressed 3; expressed in thymocytes recognizable in malignant cells. With only in leukemia cells and leukemia cells the description of the Tia locus, a second FIG. 1-Linkage group IX. Approximately set of markers (the TL antigens) became to scale. Not complete. available. Tia is linked to the D end of H-2 (Fig. 1). This association evidently is not fortuitous, for D-end H-2 antigens and TL antigens occupy adjacent sites on the thymocyte surface.5 The variant described in this report was derived from a TL+ leukemia which we induced by X irradiation in a (C57 X A)F1 hybrid mouse6 (C57 = C57BL/6), and is now in the ascites form. Table 1 and Figure 1 are guides to the TL phenotypes involved. To circumvent the difficulty that the parent strains differ at many loci other than H-2, a variant was selected by passage in (C57 X A.SW)F1 recipients; because A.SW (H-28) is congenic with strain A, the only known histoincompatability with respect of the transplanted leukemia is thus reduced to the H-2a haplotype of the A strain. After several consecutive passages in (C57 X A.SW)- TABLE 1. TL phenotypes. TL phenotype of TL phenotype of thymocytes TL + leukemia cells* C57 (H-2b) Negative 1,2,4 A (H-2a) 1,2,3 (full quantity) 1,2,3 (C57 X A)F1 1,2,3 (half quantity) 1,2,3,4 * Antigens represented in this column but not in the preceding column owe their appearance to genetic derepression accompanying leukemogenesis. Note that TL.3 is a marker for chromosome IX from A, and TL.4 is a marker for chromosome IX from C57.

3 VoL. 65, 1970 GENETICS: BOYSE ET AL. 935 F1 mice, either untreated or presensitized with A strain tissue, a variant line of cells emerged that was freely transplantable in hosts of this genotype, indicating that antigens of the H-2a haplotype had been lost. This acquired histocompatibility was specific, for the variant was rejected normally in recipients of several other H-2 types; thus it was not a variant of the "nonspecific" type in which there is a general increase in homotransplantability. The variant was continued in passage in (C57 X A.SW)F1 hosts while repeated determinations of its content of antigens were performed by a sensitive and accurate method (Fig. 2). Table 2 summarizes the data. 90- TL.l TL.2 TL-5 IL4 0 :3 D 10 or ~I Z 0 ~ a2an* n* C O U),) Cm I o H 2 90_ H H-2b H-2b H-20 H-20 e U) [ 70- (D end) (Kend) (D end) (K end) H Number of Cells Used for Absorption (x 10-6) 0- Unselected Line Variant FIG. 2.-Estimation of H-2 and TL antigens by quantitative absorption.7 Each point represents a separate determination of cytotoxicity for an aliquot of antiserum absorbed with the numbers of unselected cells or variant cells indicated on the abscissa. * n is used to calculate relative amounts of antigen (see Table 2). The results enlarge our view of variant formation and confirm the picture we have built up of the Tla locus. The implications of each antigenic change will be considered in turn. H-2: The apparently complete loss of H-2a antigen was expected.2 It is noteworthy that the quantity of H-2b antigen was unchanged, for an increase in antigen of the remaining haplotype has been reported in other variants.9 Our finding shows that compensation by the remaining haplotype is not an integral feature of variant formation.

4 936 GENETICS: BOYSE ET AL. PROC. N. A. S. TABLE 2. Estimation of H-2 and TL antigens on an H-2a1loss variant of an H-2b/H-2a leukemia. Relative amount (compared Antigen* with unselected line) t TL TL TL.3 Absent TL.4 12 H-2b (D-end) No change H-2b (K-end) No change H-2a (D-end) Absent H-2a (K-end) Absent O-C3H (control) No change Based on the data illustrated in Fig. 2. * Serology: see ref. 8. t Calculation: Amount of antigen on variant cells relative to cells of the unselected line = n(unselected cells)/n(variant cells). The value of n is given by the absorption curve (see Fig. 2). Note: The variant, like the unselected line, was sensitive to therapy with -asparaginase. TL.4: This antigen is found only on leukemia cells, not on thymocytes. The structural gene is carried by C57 but not by A.10 Therefore no decrease would be anticipated from H-2a deletion and there was none, in fact there was a remarkable 12-fold increase. (This does not necessarily imply a corresponding increase in the number of antigenic determinant sites, because the TL antigens are clustered together on the cell surface' so that the disappearance of some specificities may facilitate the accommodation of antibody at the remaining sites and mimic a real increase in antigen in the absorption test, see refs. 5, 8, and 11. The presence of TL.4 in the unselected leukemia shows that the Tla locus of C57 origin in the hybrid leukemia has been derepressed, for the A strain Tia locus does not include the gene for TL.4. This excludes a possibility that a leukemogenic process involving only the A strain Tla region was responsible for malignancy in this case. TL.1 and 2: The fact that the TL.4 structural gene of the C57 chromosome is derepressed suggests that the neighboring TL.1 and 2 structural genes are derepressed also, for leukemic derepression has so far been seen to affect Tla haplotypes in their entirety. Thus complete loss of TL.1 and 2 antigens from the variant is not expected. A decrease, as was actually observed, is consistent with loss of the "constitutive" TL.1 and 2 of the A locus, and suggests that the Tia locus is included in the deletion process leading to variant formation. TL.3: If the Tla locus is included in the deletion process, then total loss of TL.3 is expected because the gene is not carried by C57. Thus the deletion of TL.3 observed is a critical finding, and in line with loss of constitutive TL.1 and 2 produced by the A haplotype. O-C3H (Control). Assay of this antigen was included because of the possibility that variant formation might entail more general cell surface antigenic changes than those which might be anticipated from events affecting H-2 and Tia. The 0 locus'2 is not linked with H-27 and so there is no reason to suspect that it would be involved in variant formation. In fact the content of 0-C3H antigen on the variant cells was unaltered, which tells against the occurrence of changes in cell surface antigens unrelated to chromosome IX.

5 VOL. 65, 1970 GENETICS: BOYSE ET AL. 937 Conclusions. Variant formation: There is little doubt that in this variant the A strain Tla locus has been functionally deleted as well as the A strain H-2 locus. For the following reason this must have occurred in the absence of selection against cells bearing TL antigens.-when TL + leukemias are passed in hosts immunized against TL, they undergo antigenic modulation,"3 a process (whose mechanism is unknown) whereby the phenotype of the cell changes from TL + to TL- as long as exposure to TL antibody continues, the phenotype reverting to TL+ when antibody is withdrawn. Antigenic modulation is excluded in the case of the variant by the permanence of the new phenotype TL.1,2,4 in hosts that are not producing TL antibody. With regard to mechanism, the implications of this particular variant are only slight. The conflict between the reported position of the centromere in chromosome IX and chromosomal theories of variant formation based on terminal deletion or somatic crossing-over prompts consideration of other mechanisms on a smaller, subchromosomal scale. Interstitial genetic deletion is not a satisfactory explanation because it does not account for orientation of the H-2 losses. The operon is an obvious model that could account for this, although not one that so far has any precedent in the cells of higher organisms. Particularly if it were to be shown that variant formation involves only the products of the H-2 locus, the hypothesis would be tenable. However, it is complicated in the case of the variant described in this report by the deletion of TL antigen, since this appears to require the inclusion of both H-2 and Tla in the postulated region of coordinate genetic control, and these are two loci whose genes are repressed and derepressed under very different physiological conditions. Thus the operon is seemingly excluded, at least as a general model applicable to all parental H-2-loss variants. Nevertheless there is some evidence of interrelated function by these two loci,8 suggesting possibly that they are under some form of coordinated regulation and that this regulatory system is the mediator of variant formation. We include this tenuous suggestion only because present hypotheses based on genetic deletionl all either conflict with the reported location of the centromere or fail to account for orientation of the H-2 losses. Relevance to leukemia: Turning to the implications of this variant in relation to leukemogenesis, one statement seems worth making. The continued malignancy of the variant, despite the functional deletion of one Tla locus, tends to exclude one of several possible conditions relating the locus to the TL+ type of malignancy. This possibility, seemingly excluded, is that two aberrant Tla loci are necessary for TL+ malignancy. One of the remaining possibilities is that a single aberrant Tla locus, which may be either of the two alleles, is necessary to TL+ malignancy. In the variant under discussion this would have to be the C57 allele. This possibility also will be excluded if it is found possible to derive the reciprocal variant H-2a+b-:TL.1,2,3 from the same hybrid leukemia, as we are now attempting to do. The answer cannot be inferred from previous observations that variants of both parental types can often be obtained from heterozygous lines, for in no instance is it known that the unselected line was TL +. * This investigation was supported by grant CA from the National Cancer Institute and a grant from the John A. Hartford Foundation, Inc.

6 938 GENETICS: BOYSE ET AL. PROC. N. A. S. 1 Boyse, E. A., and L. J. Old, Annual Rev. Genetics, 3, 269 (1969). 2 Bjaring, B., and G. Klein, J. Nat. Cancer Inst., 41, 1411 (1968), provide a key to an extensive literature on the subject. 3 Lyon, M. F., J. M. Butler, and R. Kemp, Genet. Res., Camb., 11, 193 (1968). 4Green, M. C., and J. Stimfling, personal communication. 5 Boyse, E. A., L. J. Old, and E. Stockert, these PROCEEDINGS, 60, 886 (1968). 6 Leukemia EARAD1 which is referred to in various reports from this laboratory. See Boyse, E. A., M. Miyazawa, T. Aoki, and L. J. Old, Proc. Roy. Soc. B, 170, 174 (1968), for details of the use of cytotoxic antisera in demonstrating cell-surface antigens and of the performance of quantitative absorption as illustrated in Fig The serological assays for each antigens were those tabulated in Boyse, E. A., E. Stockert, and L. J. Old, J. Exptl. Med., 128, 85 (1968), with these changes and additions: (a) For TL.2 the serum was A(TL-) anti-a strain spontaneous leukemia ASMi: test cells 129 thymocytes. (b) For TL.4 the serum was C57(TL+) anti-c57 radiation leukemia ERLD: test cells ERLD. A(TL-) and C57(TL+) are reciprocal congenic stocks derived from serial backcrosses of 2 crossovers between H-2 and Tla in the cross C57 X A, introducing the Tla- locus of C57 into the TL+ A strain and vice versa. (c) The serum C57BL/6 anti-a strain leukemia ASL1, used with H-2H and H-21 lymphocytes for H-2a (K-end) and H-2a (D-end) antigens, respectively, was first absorbed in vivo in (C57BL/6 X A.SW)F, mice. This removes antibody to antigens shared by H-2a and H-2' and thus obviates possible interference in the absorption assay by antigen carried on any (C57 X A.SW)F1 host cells included in the suspension of leukemia cells. 9 Klein, E., in Cold Spring Harbor Symposia on Quantitative Biology, vol. 29 (1964), p Boyse, E. A., Stockert, E., and Old, L. J., Int. Convoc. on Immunology (1968), in press; where it is reported that TL+ leukemias of C57 have TL.4 whereas TL+ leukemias of the crossover congenic stock C57(TL+), derived from a C57 X A cross, do not. We have since shown that TL+ leukemias of strain A do not have TL.4 whereas TL+ leukemias of the congenic crossover stock A(TL-), derived from the same cross, do. This establishes that the gene for TL.4 belongs to the Tla locus and is carried by C57 but not by A. 11 Boyse, E. A., E. Stockert, and L. J. Old, these PROCEEDINGS, 58, 954 (1967). 12 Reif, A. E., and J. M. V. Allen, J. Exptl. Med., 120, 413 (1964). 13 Old, L. J., E. Stockert, E. A. Boyse, and J. H. Kim, J. Exptl. Med., 127, 523 (1968).