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Rearrangement and expression of T cell receptor genes in cloned murine natural suppressor cell lines.

Naturally occurring suppressor cells of the in vitro mixed leukocyte culture reaction and of in vivo graft-vs.-host disease have been identified in the spleens of neonatal mice (1) and of adult mice recovering from total lymphoid irradiation (2), whole-body irradiation (3), and syngeneic marrow transplantation (4), or cyclophosphamide therapy (5). Using both positive and negative selection procedures, the suppressors were reported to be null lymphocytes that did not express mature macrophage surface markers, nor differentiate into mature macrophages in vitro, nor demonstrate natural killer (NK) activity (1). Subsequently, cloned lines of these natural suppressor (NS) cells were derived from either adult mice given total lymphoid irradiation (TLI) (2) or from neonates (6). The cloned NS cell lines expressed a surface phenotype (2, 6) similar to that reported previously for cloned NK cells (Thy-1(+), asialo-GM1(+), Ig(-), Lyt-1(-), Lyt-2(-), Ia(-), MAC-1(-)) (7-9). However, the NS cells did not show NK activity in the standard assay with YAC-1 target cells. The cloned NS lines suppressed the proliferation of responder cells and the generation of cytolytic cells in the mixed leukocyte reaction (MLR), and suppressed lethal graft-vs.-host disease in vivo (10, 11). In view of the unusual function and surface phenotype of the cells, the lineage of these cells remained unclear. To determine the lineage of the cloned NS cells, we searched for expression and rearrangement of the alpha and beta chain genes of the T cell antigen receptor, as well as that of the gamma chain gene. Studies of the phenotypically similar NK cell yielded conflicting results. Thus, cloned lines of murine NK cells were reported to have rearrangements of the beta chain genes, and to express mRNA for all three chains (12). In contrast, freshly purified rat or human large granular lymphocytes (LGL) were shown to express only the 1.0 kb mRNA species of the beta chain gene (13), indicative of D-J joining (14). Thus, some but not all cells with NK function express the T cell receptor and are members of the T cell lineage. The current report shows that the NS lines express full-length mRNA transcripts for the a and beta chain of the T cell receptor, as well as the gamma chain gene.

Naturally occurring suppressor cells of the in vitro mixed leukocyte culture reaction and of in vivo graft-vs.-host disease have been identified in the spleens of neonatal mice (1) and of adult mice recovering from total lymphoid irradiation (2), whole-body irradiation (3), and syngeneic marrow transplantation (4), or cyclophosphamide therapy (5). Using both positive and negative selection procedures, the suppressors were reported to be null lymphocytes that did not express mature macrophage surface markers, nor differentiate into mature macrophages in vitro, nor demonstrate natural killer (NK) activity (1). Subsequently, cloned lines of these natural suppressor (NS) cells were derived from either adult mice given total lymphoid irradiation (TLI) (2) or from neonates (6). The cloned NS cell lines expressed a surface phenotype (2, 6) similar to that reported previously for cloned NK cells (Thy-1 ', asialo-GM I +, Ig, Lyt-I -, Lyt-2-, la-, MAC-I -) (7)(8)(9). However, the NS cells did not show NK activity in the standard assay with YAC-1 target cells. The cloned NS lines suppressed the proliferation of responder cells and the generation of cytolytic cells in the mixed leukocyte reaction (MLR), and suppressed lethal graft-vs .-host disease in vivo (10,11) . In view of the unusual function and surface phenotype of the cells, the lineage of these cells remained unclear.
To determine the lineage of the cloned NS cells, we searched for expression and rearrangement of the a and ,B chain genes of the T cell antigen receptor, as well as that of the 7 chain gene . Studies of the phenotypically similar NK cell yielded conflicting results. Thus, cloned lines of murine NK cells were reported to have rearrangements of the ,B chain genes, and to express mRNA for all three chains (12). In contrast, freshly purified rat or human large granular lymphocytes (LGL) were shown to express only the 1 .0 kb mRNA species of the a chain gene (13), indicative of D-Jjoining (14). Thus, some but not all cells with NK function express the T cell receptor and are members of the T cell lineage. The current report shows that the NS lines express full-length mRNA transcripts for the a and ,8 chain of the T cell receptor, as well as the y chain gene .

Materials and Methods
Cell Lines. Three clones NS lines TLI-2.4C, 4BA4, and Al were derived from the spleens of adult BALB/c mice treated with total lymphoid irradiation, neonatal BALB/c mice, and normal adult BALB/c mice using techniques described in detail previously (2,6) . These lines have been propagated continuously for at least I yr in tissue culture medium containing RPMI-1640 supplemented with 10 mM Hepes buffer, 2 mM glutamine (Gibco, Grand Island, NY), 5 X 10-5 M 2-ME (Sigma Chemical Co ., St. Louis, MO), supernatants from Con A-stimulated rat spleen cells (10-20% vol/vol) and FCS (Hyclone, Logan, UT) (10% vol/vol).
Immunofluorescence Staining of Cells. A two-stage immunofluorescent staining procedure was used to detect surface markers on the NS cell lines as described previously (2,6) . Rat anti-mouse mAbs that were used in the first stage include anti-Thy-1 . The probes were labeled by the hexamer-labeling technique (19) . Hybridization conditions were 50% formamide, 10% dextran sulfate, and 5X SSPE (saline sodium phosphate/EDTA buffer) (15) at 42°C for 16 h. Filters were washed twice for 30 min in 2X SSPE, 1% SDS at 55°C, and 30 min in 0.2X SSPE at 55°C . Southern Blot Analysis. DNA from BALB/c liver cells, BALB/c NS cell lines, 4BA4, and TLI-2 .4C were prepared by standard methods (15), digested with the Pvu II restriction endonuclease, electrophoresed through 0.7% agarose gels for 36 h and blotted onto nitrocellulose . Hybridization was performed using the 0 chain gene containing cDNA clone 86T5 (18) . Hybridization and washing conditions were similar to those above.

Results
Function and Surface Markers of Three Cloned NS Cell Lines. All three NS cell lines added to the MLR between normal adult BALB/c responder spleen cells (5 X 10 5) and irradiated (3,000 rad) C57BL/Ka stimulator spleen cells (5 X 10 5) suppressed peak [sH]thymidine incorporation by at least 80% when the ratio of responder to NS cells was 10 :1 (data not shown) . In addition, the TLI-2.4C and 4BA4 cells (1 .5 X 10 7) were able to suppress lethal graft-vs.-host disease in vivo when coinjected with 5 X 106 C57BL/Ka spleen cells into sublethally irradiated weanling BALB/c mice such that >80% of recipients survived at least 100 d (11) . <15% of recipients coinjected with a control BALB/c derived T cell line (HT-2) survived during the same time interval (11) . The TLI-2 .4C and 4BA4 lines do not express NK function, since they fail to kill YAC-1 cells in vitro (2,6) . Table I shows a representative example of the stable immunofluorescent staining pattern of the three cell lines . All were strongly Thy-1 + , but showed minimal staining with antibodies directed to L3T4, Lyt-1, Lyt-2, IgM, and MAC-1 surface markers .
Rearrangement and Expression of T Cell Receptor Genes in NS Lines . The three cloned NS cell lines described above were used for the investigation of the T cell receptor expression of the « and # chain, as well as that ofthe y chain gene . The autoradiograph depicted in Fig . 1 shows that a, #, and y chain mRNA of appropriate sizes were present in all NS lines . In the case of the S chain gene, the full-length mRNA of 1 .3 kb, attributed to V-D J rearrangements, was present in all cell lines . This indicated that the NS cells may assemble a functional T cell antigen receptor .
In addition, the NS cell lines TLI-2 .4C and 4BA4 were examined for rearrangement of the 0 chain locus. Fig . 2 represents a Southern blot of Pvu 11digested DNA from the NS cell lines hybridized with a nick-translated insert

Discussion
The experiments reported herein demonstrated that natural suppressor cells were of the T cell lineage because of the # chain locus rearrangement and the expression of the a and ,B chain genes of the T cell antigen receptor. The NS cells also expressed the^y chain gene . Immature, Lyt-2-, L3T4-thymocytes derived from fetal or adult mice express high levels of the .y chain mRNA (21,22) . However, unlike the NS cells, these cell populations did not express the a chain gene . Mature cytotoxic T cell lines and hybridomas carrying the phenotype Lyt-2 + , L3T4-also showed high-level expression of the .y chain gene (23,24) . The NS cells do not express the Lyt-2 surface marker and, therefore, do not belong to this group of cells . Finally, high y chain gene expression was reported for Lyt-2-, L3T4 + T cells activated by allogeneic stimuli in vitro (25) and for LGL lines with or without NK cell function (12) . It is possible that the LGL lines without NK activity express NS function, and that some NK and NS cells are subsets of an LGL lineage that are derived from pre-T cells . Thus, members of the T cell lineage with NS and/or NK cell function(s) may represent parallel lines of nonspecific suppressor and cytolytic cell development similar to that of the antigen-specific Ts and Tc cell development .
In conclusion, cloned NS cells are members of the T cell lineage that express the a, ,B, and y chain genes. Similar studies are necessary to determine whether freshly purified NS cells also express these genes .