Abstracts 63

produced polymorphic restriction fragments of 10.0 and 9.2 kb and was subse- quently used to analyze 59 members of eight families. Polymorphic fragments segregated in six of eight families; in many cases maternal and/or paternal CT/3 haplotypes could be assigned. Recent data concerning the structure of the Cv/3 region suggest that the polymorphic BgllI site lies 5' to the CT32 gene. This polymorphism should serve as a marker for the complex of CT/3 and linked variable region components allowing genetic studies of this immunologically im- portant gene family.

The families studied here have been extensively characterized using HLA class I, II, and III gene probes. Each family includes at least one individual with a recombinant HLA haplotype making it possible to analyze the role of discrete HLA regions as well as CT/3 haplotypes in allogeneic and HLA restricted immune responses. Detection of allelic forms of the CT/3 genes should make it possible to determine whether T/3 haplotypes correlate with heritable variations in T cell function or disease susceptibility.

GENERATION AND REGULATION OF AUTOCYTOTOXIC CELLS IN MIXED LYMPHO- CYTE CULTURES. Karen Rosenkrantz, Bo Dupont, and Neal Flomenberg; Memorial S/oan Ket- tering Cancer Center, New York, N Y

Using a limiting dilution analysis assay combined with a zero-order Poisson sta- tistical model, we have detected the generation and suppression of autocytoxic cells in autologous and allogeneic mixed lymphocyte cultures. At low responder doses a clear autocytotoxic response is generated. At higher responder doses, this autocytotoxic reaction disappears. The kinetics of the disappearance of au- tocytotoxicity follow a single hit model suggesting that a low frequency but highly active suppressor cell inactivates the autocytotoxic cell. Cells capable of auto- cytotoxicity are present at a 10-100-fold lower frequency than cells capable of allocytotoxicity or natural killer like activity and are dependent on Interleukin- 2 for growth. The autoregulatory cells are present at a tenfold lower frequency than autocytoxic cells in a purely autologous culture, but can in vitro be increased relative to the autocytoxic cells by stimulation with allogeneic cells or allogeneic human serum. Therefore, the lack of an autologous response normally seen following mixed lymphocyte culture does not necessarily reflect the complete elimination of autocytoxic cells during ontogeny. Rather, cells capable of auto- cytotoxicity are present in the peripheral blood of normal individuals but are regulated by a suppressor network.

T CELL CLONES RECOGNIZING (KIDNEY) SPECIFIC ANTIGENS. D. Roth, C. Flaa, V. Esquenazi, L. Fuller, M. Milgrom, G. Kyriakides, and J. Miller; Departments of Medicine, Surgery and Microbiology, University of Miami, Miami, FL

Recent data has suggested that bilateral native nephrectomy (BNN) confers a beneficial effect on subsequent renal allograft survival independent of other fac- tors well known to influence graft survival. Our own experience with 249 recip- ients transplanted since 1979 demonstrates 2-yr actual graft survival of 91% in patients undergoing pretransplant B N N and splenectomy vs. 74% in patients who had splenectomy only or had neither operation. Actuarial 6-yr graft survival is 86% vs. 80%, respectively. We questioned whether removing the native kid- neys might have eliminated a source of a kidney (tissue-specific) immune stimulus that was a component of the immunological response to the graft independent

64 D. Bernard Amos Symposium

of alloantigen disparities. To test this hypothesis, we have studied the mixed lymphocyte kidney culture (MLKC), a lymphoproliferative response using kidney cortical stimulating cells, and responding infiltrating T lymphocytes isolated from: (i) kidneys of nephrectomized patients with end-stage renal disease (autologous MLKC); or (ii) rejected renal allografts (allogeneic MLKC). In these MLKC reactions, we have previously demonstrated evidence supporting a tissue-specific response reflected in an in vitro cellular immune assay. In further studies, we have now cloned by limiting dilution (>2 month) infiltrating lymphocytes ob- tained from kidneys of groups A and B. In addition, we have cloned peripheral blood T cells obtained from normal volunteers primed in allogeneic MLKC using kidney cortical stimulating cells obtained from unused cadaver k idneys /Group C). The specificity of the clones was demonstrated using cortical cells or lym- phocyte targets of differing HLA-DR antigens. Secondary type MLKC kinetics were observed and several clones responded to renal cortical cells with reactivity unrelated to HLA-DR specificities. In group B, in one experiment, 7/9 clones exhibited activity against donor KC while negative vs. donor spleen lymphocytes and a lymphocyte DR panel. These clones expressed both helper (Leu 3) and suppressor/cytotoxic (Leu 2) antigens. Group A also contained autoreactive kid- ney-specific clones. These studies lend further support to the importance of the role of tissue-specific immunity in the response to an allograft and in a damaged native kidney.

SELECTIVE MURINE HUMORAL TOLERANCE TO HUMAN CELLS: TIMING, DOSAGE AND ROUTE OF ADMINISTRATION. Richard J. Sharpe, Robert T. Schweizer, Laine Krisiunas, Patricia A. Michalski, and Laurine M. Bow; Department of Surgical Research, Hartford Hospita/. Hartford, CT 06115 and The University of Conn. School of Medicine Farmington. Connectfiut 06032

There is a large body of literature that shows that tolerance to specific antigen can be induced by exposing neonatal animals to the antigen. Chuma et al. (Cell Immunol 63:193, 1981), working with mice injected with xenogeneic cells neo- natally, were able to induce humoral tolerance to these cells. We have injected neonatal mice with one of a pair of human cell lines within 24 hr of birth either i.p. or i.v. using several dosages. We then immunized the mice with the second cell line at various times and also immunized untreated controls in the same way. Two weeks later the antibody titers of the tolerized and control mice against both cell lines were determined. The results show that sera from a significant number of the mice receiving 10 million or more cells neonatally are strongly reactive towards the immunizing cell yet minimally reactive towards the tolerizing cell. This differential reactivity was most pronounced and consistent in the mice tolerized i.v. and immunized at 21 days. The sera from the control mice react strongly with both cell lines. We hope to use this technique to reduce the number of hybridomas that must be screened to produce monoclonal antibodies to rare cellular determinants.

EFFECT OF ACUTE CYCLOSPORINE ADMINISTRATION ON RENAL HEMODYNAMICS IN THE RAT. B. A. Sullivan, L.J. Hak, and W. F. Finn; Schools of Pharmacy and Medicine, University of North Carolina, Chapel Hill, North Carolina 27514

Cyclosporine has become a valuable drug in the field of organ transplantation. However , the occasional occurrence of unique arteriolar lesions in renal biopsy