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Immune mechanisms in leukemia role of the Ia antigens.

We have shown that the selective removal of cells possessing Ia determinants coded by the I-A, I-B, and I-J regions of the H-2 gene complex completely abrogates the protective capacity of nylon-wool- purified T lymphocytes against leukemic challenge. This suggests that the Ia antigen bearing T cells play an important role in tumor immunity.

Immunization of Mice. Procedures for immunizing mice to Ib cells have been described (1). In brief, fresh Ib cells prepared as described above and at a final concentration of 1 × 107 cells/ml were incubated in a 0.02% formalin solution for 18 h at 4°C. Mice received a single i.p. injection (1.0 ml) of the formalinized cells and were observed for 15 days to assure that none died of the immunizing preparation. Mice thus immunized could survive a challenge dose of 107 viable Lo cells and served as donors of immune spleen cells (12).
Adoptive Cell Transfer. Mice were immunosuppressed by cytoxan treatment (250 mg/kg body weight); 24 h later they were injected with a counted number of Ib-primed T cells; 24 h after this injection they were challenged with a partially formalin-killed preparation of Ib cells. These preparations contained approximately 103 viable cells and are lethal to immunosuppressed, unprotected mice (1,2).
Spleen Cell Preparation. Immune spleen cells (ISC) were obtained from mice immunized 15 days previously. Donor mice were killed by cervical dislocation, and a single cell suspension was prepared as described for Ib cells.
Nylon Wool Columns. Thymus-derived lymphocytes were prepared from whole ISC preparations using nylon-wool (LP-1 Leukopak Leukocyte Filter, Fenwall Laboratories Inc., Morton Grove, Ill.) as described by Julius et al. (13). Such cells were routinely 90-95% Thy-1 positive as determined by anti-Thy 1.2 serum and complement treatment. Less than 5% of these cell populations are Ig ÷ by immunofluorescence.
Antisera. Antisera specific for the Ia antigens were produced as previously described (14). Cell experiments were performed using a single-peol antiserum (A.TH × B10.HTT)F~ anti-A.TL which contains antibodies against the I-A k, I-B k, and I-J k regions, but not the I-E and I-C regions. This antiserum might contain anti-Ia.1, 2, 3, 19, plus the specificity coded by Ia-4. This serum has cytotoxic activity for Ia.3 and against promoter T cells (15).
Antiserum Treatment of Cells. For blocking experiments nylon wool-purified T cells were incubated with 2.0 ml of a 1:10 dilution of the anti-Ia serum for 20 min at room temperature and for an additional 20 min at 4°C. Appropriate viability counts (trypan blue exclusion) were made at the end of each procedure. Cells were then washed by centrifugation in MEM-FCS and resuspended at the appropriate concentration.
To determine if the protective cells carried Ia determinants, 1 × 107 cells were incubated with 2.0 ml of a 1:20 dilution of the anti-Ia serum for 20 min at room temperature and 20 min at 4°C, centrifuged, and resuspended in 2 ml of guinea pig complement (Microbiological Associates, Bethesda, Md.) diluted h2 in MEM, and incubated for 30 min at 37°C. The cells were then washed twice by centrifugation in media, counted in a hemocytometer, and adjusted to the appropriate concentration.

Results and Discussion
Protective Capacity of T Lymphocytes Bearing the Ia Determinants. To ascertain the role of Ia + T lymphocytes in protecting immunosuppressed mice against leukemic challenge, we treated nylon wool-purified T-cell populations with antisera against a restricted portion of the I region of the mouse H-2 complex. We tested graded log doses ofT cells treated with such antisera or with normal mouse serum (NMS), with and without addition of exogenous complement, for their ability to protect against Ib cell challenge (Table I). 10 6 untreated, nylon-wool-purified T cells were able to protect immunosuppressed recipients. This is in agreement with our previous findings showing the protective cell bears Thy 1.2 +, Ig-, and Ly 4.2-cell surface antigens (4). Treatment with specific antisera alone, NMS alone, complement alone, or with NMS and complement does not alter the protective capacity of the transferred T cells.  We have not completely eliminated the possibility that the depletion ofIa + macrophages may be responsible for the lack of protection seen by the treated cell population. This seems unlikely in view of our preliminary findings (unpublished) that T-cell populations depleted of macrophages by glass wool filtration are competent in providing protection. Whether this protective capacity is relegated to cells bearing determinants ceded by one, two, or all three of the regions tested is yet to be determined. The role of Ia determinants as a marker for functional T-cell subsets has recently been explored. It has been shown that the Ia determinants are expressed on suppressor T cells and map in the I-J region (16). Further, we have recently shown the Ia antigens expressed on concanavalin A-reactive T cells to be restricted to the I-J subregion (15). Stout et al. have studied Ia on Fc receptorpositive T cells by blocking of Ig binding and have determined that a portion of the FcR ÷ T cells are blocked by antibodies directed at I-A-or I-C-coded determinants (17). The relationship between the Ia-positive T cells capable of protecting mice against leukemic challenge and Ia ÷ T cells with these other functions is yet to be determined.
It is of interest to point out that normal cells, i.e., cells from nonimmune donors, possess the capacity to protect against line Ib leukemia, but only with a 100-fold increase in cells transferred in this assay (1). Studies are now underway to determine if this protective capacity of normal cells can also be abrogated by specific antisera. If it cannot, it would suggest that the Ia determinants are expressed as a result of antigenic stimulation. This seems a real possibility since Ia expression on T cells appears more easily detected on activated T cells (18,19).
It is important to consider the results reported here with two other pieces of information. It has been accepted that Ia antigens are not expressed on cytotoxic T cells specific for MHC-coded determinants using negative selection by anti-Ia and complement such as presented here (20), although a contradictory result has appeared (21). The second bit of information is that although it is clear that cytotoxic T cells specific for MHC determinants express the phenotype Ly-l-, Ly-2+3 + (22), a recent report suggests that the effectors for syngeneic tumor cells are Ly-l+2+3 + and therefore represent a distinct subset from the effectors specific for MHC alloantigens (23). The effectors that function in the adoptive transfer system reported here for protection against the syngeneic Ib leukemia are clearly Ia + in contrast to the cytotoxic T cells directed at the MHC-coded antigens. This together with the previous report provides strong evidence that the effectors in immunity against syngeneic tumors are a different lymphocyte class than those directed at allogeneic cells. Assuming we are not eliminating a putative Ia + amplifier population, we would predict the effector cells in this system would be Ly-l+2+3+ and Ia + and represent a uniquely marked T-lymphocyte subset distinct from both suppressors Ly-l-2+3 +, Ia +, and helpers Ly-1+2-3 -, Ia-. Further studies to determine which Ly antigens are expressed and any possible subregion restriction of the Ia antigens expressed on the effectors are underway.

Summary
We have shown that the selective removal of cells possessing Ia determinants coded by the I-A, I-B, and I-J regions of the H-2 gene complex completely abrogates the protective capacity of nylon-wool-purified T lymphocytes against leukemic challenge. This suggests that the Ia antigen bearing T cells play an important role in tumor immunity.
We are grateful to Eileen Gannon and Glenn Matsushima for expert technical assistance.
Received for publication 3 January 1977.