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

Abstracts 191

through which these autocytotoxic cells are regulated. We therefore isolated and analyzed a series of T-cell clones from 7- and 10-day microcultures which had been established with culture conditions favoring the development of autocy- totoxicity. Four clones were isolated which exhibited an amplifier function for cytotoxic responses. Like previously described helper-inducer populations, all four clones expressed the CD3 + CD4 + 4B4 + 2 H 4 - phenotype. These clones were not directly cytolytic toward either autologous of allogeneic target cells. They similarly failed to proliferate when stimulated with autologous or allogeneic cells, though they proliferated briskly when stimulated with CD3 antibodies coupled to sepharose beads. When added on day 0 to freshly mixed lymphocyte microcultures, however, these clones produced a threefold increase in cytolytic activity compared to control cultures. This increased cytolytic activity was ob- served toward both autologous and allogeneic target cells. Amplifier activity for autocytotoxic responses required the physical presence of the relevant clones and could not be reproduced with supernatants from resting cultures. Amplifier activity was radioresistant up to doses of 5000 rads. In limiting dilution conditions in which autocytotoxic responses are normally suppressed (i.e., high responder cell dose) the addition of amplifier clones could abrogate suppression. Such amplifier cells might be expected to play a role in the pathogenesis of autoimmune disorders.

REGULATORY PROPERTIES OF HUMAN MONOCYTES. Nancy E. Goeken and Tina Staggs; VA +'vJedical Center, Iowa City, Iowa

It is well known that antigen pulsed, washed monocytes (Mo) can effectively activate naive T cells. In a series of experiments designed to examine the kinetics of this phenomenon, we have observed that such Mo also can downregulate T- cell responses. Purified human T cells were cocultured with plastic adherent Mo that had been pulsed overnight with a soluble antigen (tetanus toxoid). The T cells were removed after various time intervals, cultured separately, and prolif- eration was measured by ~H thymidine uptake on day 6. T-cell responses in- creased up to 3 days of coculture (65,000 cpm) and then fell to low levels by day 6 (12,000 cpm). That is, when T cells and Mo were cocultured for the entire 6-day period, responses were much lower than when the T cells were removed on day 3 and cultured alone in media for an additional 3 days. Addition of exogenous IL-2 did not reverse this phenomenon. This appears to relate to the development of two separate suppressive activities in the adherent Mo popula- tion. When fresh T cells were cocultured for four days with Mo that had been previously cultured for 1, 4, or 6 days, the responses were 16,008, 4,955, and 740 cpm, respectively. Further, supernatants (sup) from 6 day cultured Mo in- hibited the membrane bound IL-1 activity of 24-hr Mo in the murine thymocyte costimulator assay. Interestingly, these sups did not inhibit a known source of secreted Mo IL-1 in the same assay. These data suggest that Mo develop sup- pressor activities after antigen exposure and may act directly on T cells and/or inhibit the activity of membrane bound IL-1 but not of soluble IL-1.

ANTI LFA=I ANTIBODIES INHIBIT ACTIVATION OF CLONED ALLOSPECIFIC T CELLS BY CLASS II POSITIVE HUMAN DERMAL FIBROBLASTS. David H. Maurer, Jeffrey H. Hanke, Timothy A. Springer, Robert R. Rich, and Marilyn S. Pollack; Baylor College of Medicine, Houston, TX