Abstracts 189

RESTRICTED ALLOREACTIVE T-CELL RECEPTOR REPERTOIRE AMONG CD4+/9.3 - GRANULAR LYMPHOCYTES. Y. Morishita, P.J. Martin, and J.A. Hansen; Fred Hutchinson Cancer Research Center; Puget Sound Blood Center; and the University of Washington, Seattle. WA

We have identified a population of CD4 + lymphocytes having granular mor- phology and reduced ability to produce IL-2. When stimulation with alloantigen in the presence of exogenous IL-2, these cells generate specific allocytotoxicity. In the present investigation, we have examined T-cell receptor (TCR) 3 chain rearrangements in cloned CD4÷/9.3 + and CD4+/9.3 cells. Flow microfluori- metry was used to separate these two distinct CD4 + populations prior to allo- stimulation. D N A was prepared from CD4+/9.3 + and CD4+/9.3 clones and digested with EcoRl, HindlII , or BamHI for analysis by Southern blot hybrid- ization with the C /32 probe "Jur/32." All clones tested showed rearrangement of TCR3. There were, however, many repeat rearrangement patterns among 9 .3- clones, whereas each 9.3 + clone showed an individual rearrangement. In one donor, all ten 9 .3- clones examined had the same pattern of rearrangement, and in another donor only three different patterns were observed among twelve 9 .3- clones. Moreover, CD4+/9 .3 - cells stimulated in a series of bulk cultures with various HLA-D homozygous cells (HTC) exhibited distinct rearranged bands on hybridization within 20 days, indicating selective outgrowth of certain clonal populations. In contrast, bulk cultured CD4 +/9.3 + cells demonstrated a diverse range of rearrangements with no single clone dominating to the point that a single rearrangement band was detectable by Southern blotting. These findings suggest that certain CD4+/9.3 - "granular lymphocytes" may preferentially proliferate after allostimulation. Alternatively, this cell population may contain less clonal diversity and thereby have a more limited repertoire of TCR/3 gene rearrange- ment patterns than conventional CD4+/9.3 + T-helper cells.

ALLOREACTIVE T-CELL CLONES RECOGNIZE DR2-ASSOCIATED DQ POLYMORPHISM DEFINED BY DQce AND DQ/3 RFLP. Adriana Zeevi, Loretta Di Vecchia, Sharon Rosenshine, Isabella Cascino, Massimo Trucco, and Rene Duquesnoy; Department of Pathology, University of Pittsburgh, and Central Blood Bank, Pittsburgh, PA

We have previously described two alloreactive clones DS6 and DS9 that recognize a cellular determinant expressed on a subgroup of DR2 cells. Since the PLT activity of these clones was inhibited by anti-DQ but not anti-DR monoclonal antibodies, it seemed that they recognized a D Q gene product. In order to learn more about the genetic basis of this cellular DR2-associated, DQ-encoded gene product, we have studied the corresponding DQce and DQ/3 allelic forms defined by RFLP analysis. With the restriction enzyme Pstl we observed that DR2 cells generated the same 15.2 kb DQce band (called D Q ap l ) in combination with either a 4.5 kb DQ/3 band (DQbp l ) or a 13.0 kb D Q 3 band (DQbp4). Only those DR2 cells that were D Q a p l , b p l stimulated clones DS6 and DS9. However , certain DR1 and DRw6 cells were also DQap 1,bp 1 and neither of them stimulated DS6 or DS9. This suggested that these clones recognized a determinant corre- sponding to a D Q gene allelic combination which could not be defined by Pstl alone. By using a second restriction enzyme Taql , it was possible to define DQce and DQ/3 gene allelic forms which corresponded with the PLT specificity of DS6 and DS9. Taq 1 digestions yielded two DQap 1 associated bands, DQap l . r l (with DR1 cells) and DQapl . t2 (with DR2 cells). Also, D Q b p l could be "subdivided" by Taq 1 into DQbp 1.t I (with DR 1 cells), DQbp 1.t2 (with DR2 cells) and DQbp 1. t6

190 Abstracts

(with DRw6 cells). DS6 and DS9 only reacted with those DR2 cells which are D Q a p l . t 2 , bp 1.t2. We have recently identified another DR2 associated allo- reactive T-cell clone DB29 which recognized a cellular determinant found on those DR2 cells that failed to stimulate DS6 and DS9 clones. The RFLP pattern of DR2 cells which stimulated DB29 was DQap l . t 2 , D Q b p 4 . t l . These studies demonstrate that DR2 associated with two allelic D Q forms DQap 1.t2, D Q b p 1.t2 and D Q a p l . t 2 , D Q b p 4 . t l which can be defined at both the molecular and the cellular level.

ANTIGEN SPECIFICITY OF MLR INDUCED SUPPRESSOR CELLS. Edgar L. Milford, John E. Leggat, Eleanor L. Ramos, Laurence A. Turka, and Charles B. Carpenter; Brigham and Women's Hospital. Boston, MA

Modulator cells with suppressor function (SCs) can be generated in a primary 10-Day MLR (R + Sx). These SCs (RS) are capable of inhibiting ~HTdR incor- porat ion in a test MLR, but the question of SC specificity is unresolved. Prelim- inary experiments in our laboratory showed that HLA class I or II did not account for the reproducible polymorphic patterns of suppression. Therefore, we inves- tigated the specificity of SCs using a larger panel of modulator cells and test cultures. Twenty-five different SCs were generated and assayed in cultures with HLA typed stimulators for a total of 194 assays. A significant decrease (p < 0.03) in experimental vs. control 3HTdR incorporation (CPM) was used to distinguish suppressed ( + ) f rom unsuppressed (0) cultures. Mean percent suppression ([1 - exp/ctrl] × 100) in ( + ) and (0) cultures was 66 .8% + 24% and - 1 . 2 % +-- 27%, respectively. A representative panel is shown below. Twenty of 25 SCs showed polymorphic patterns of suppression. As a null hypothesis, we postulated no correlation between suppression and presence of appropriate HLA antigens in the test culture (those present on the stimulator in the generating culture but not shared by the responder). Chi-square analysis of actual vs. ex- pected suppression was per formed for class I, DR, and D Q antigens alone or together. There was significant correlation for class I (X 2 = 4.2, p < 0.04) and D R (X 2 = 5, p < 0.018), but not DQ. Despite this, 33% of positive assays could not be accounted for by any of these classical HLA antigens. These results suggest that SCs recognize non-HLA determinants.

Test Stimulator Test

SC resp A B C D E F G H I J K L M N

A'B A + 0 0 0 0 0 + 0 0

A'C A 0 + + 0 + 0 + 0 + D'E D 0 0 0 + 0 0 0 0 F'D F 0 0 + + + 0 + + + + + + G'D G '+ + + + + + + + +

T-CELL CLONES AMPLIFYING AUTO- AND ALLOCYTOTOXIC RESPONSES. Karen Ro- senkrantz, Soo Young Yang, Carolyn Keever, Donna Williams, Karim Bhimani, Bo Dupont, and Neal Flomenberg; Memorial Sloan-Kettering Cancer Center, New York, N Y

Autocytotoxic cells can be detected in normal peripheral blood through the use of limiting dilution techniques. Little is presently known about the mechanisms