SummaryFunctional interactions between T and B lymphocytes are necessary for optimal activation of an immune response . Recently, the T lymphocyte receptor CD28 was shown to bind the B7 counter-receptor on activated B lymphocytes, and subsequently to costimulate interleukin 2 production and T cell proliferation . CTLA-4 is a predicted membrane receptor from cytotoxic T cells that is homologous to CD28 and whose gene maps to the same chromosomal band as the gene for CD28 . It is not known, however, ifCD28 and CTLA-4 also share functional properties . To investigate functional properties of CTLA4, we have produced a soluble genetic fusion between the extracellular domain of CTLA-4 and an immunoglobulin Cy chain . Here, we show that the fusion protein encoded by this construct, CTLA4Ig, bound specifically to B7-transfected Chinese hamster ovary cells and to lymphoblastoid cells. CTLA4Ig also immunoprecipitated B7 from cell surface 125 1-labeled extracts of these cells . The avidity of 1251-labeled B7Ig fusion protein for immobilized CTLA4Ig was estimated (Kd -12 nM) . Finally, we show that CTLA4Ig was a potent inhibitor of in vitro immune responses dependent upon cellular interactions between T and B lymphocytes . These findings provide direct evidence that, like its structural homologue CD28, CTLA4 is able to bind the 137-counter-receptor on activated B cells. Lymphocyte interactions involving the B7 counter-receptor are functionally important for alloantigen responses in vitro .

SummaryA successful immune response requires intercellular contact between T and B lymphocytes . We recently showed that CD28, a T cell surface protein that regulates an activation pathway, could mediate intercellular adhesion with activated B cells by interaction with the B7 antigen . Here we show that CD28 is the primary receptor for B7 on activated peripheral blood T cells, that CD28 binds to B7 in the absence of other accessory molecules, and that interaction between CD28 and B7 is costimulatory for T cell activation . To characterize the binding of CD28 to B7, we have produced genetic fusions of the extracellular portions of B7 and CD28, and immunoglobulin (Ig) Cy1 chains . 121 I-labeled B7 Ig bound to CD28-transfected Chinese hamster ovary (CHO) cells, and to immobilized CD28 Ig with a Kd -200 nM . B7 Ig also inhibited CD28-mediated cellular adhesion . The function of CD28-B7 interactions during T cell activation was investigated with soluble fusion proteins and with B7-transfected CHO cells. Immobilized B7 Ig and B7+ CHO cells costimulated T cell proliferation . Stimulation of T cells with B7+ CHO cells also specifically increased levels of interleukin 2 transcripts. These results demonstrate that the CD28 signaling pathway could be activated by B7, resulting in increased T cell cytokine production and T cell proliferation . Cellular interactions mediated by B7 and CD28 may represent an important component of the functional interactions between T and B lymphoid cells. I t has long been known that interactions between T and B lymphocytes play a central role in regulating an immune response (1, 2). More recent studies have shown that activation and differentiation of both T and B lymphocytes is dependent upon direct intercellular (cognate) interactions between these cell types. While the specificity of T. cell-B cell interactions is determined by interaction between the TCR/ CD3 complex (3-5) and antigen associated with class II MHC molecules on B cells (6), interactions between other (accessory) cell adhesion molecules are also necessary for a full immune response (7-9). Interactions between accessory receptors and their counter-receptors may increase the avidity of cellular interactions (7) ; control lymphocyte localization and migration (10); and have direct signaling functions (7,11,12) during lymphocyte activation. Accessory receptors and their counter-receptors involved in T-B cell interactions include (reviewed in reference 7): CD2 and LFA 3, CD4 and class II MHC molecules, LFA1, and ICAM-1 and ICAM-2 .The T cell homodimer, CD28, a member of the Ig superfamily (.13), has been shown in studies using mAbs to have an accessory function during T cell activation (14). Anti-CD28 mAbs have been shown to costimulate T cell proliferation induced by a number of polyclonal stimuli (reviewed in reference 14) . These mAbs also inhibited alloantigen and soluble antigen-specific T cell responses (15, 16), indicating that CD28-mediated signalling may be crucial during these responses. CD28-mediated T cell activation...

SummaryT cell costimulation by molecules on the antigen presenting cell (APC) is required for optimal T cell proliferation. The B7 molecule on APC binds the T lymphocyte receptor CD28, triggering increased interleukin 2 (IL-2) production and subsequent T cell proliferation. CTLA-4 is a predicted T cell membrane receptor homologous to CD28, which also binds the B7 counter receptor, but whose distribution and function are unknown. Here we have developed monodonal antibodies (mAbs) specific for CTLA-4 and have investigated these questions, mAbs were produced that bound CTLA-4 but not CD28, and that blocked binding of CTLA-4 to B7. CTLA-4 expression as measured by these mAbs was virtually undetectable on resting T cells, but was increased several hundred-fold during T cell activation. On activated lymphocytes, CTLA-4 was expressed equally on CD4 + and CD8 + T cell subsets and was coexpressed with CD25, CD28, and CD45RO. CTLA-4 expression was lower than that of CD28, reaching a maximum of "~1/30-50 the level of CD28. Despite its lower expression, CTLA-4 was responsible for much of the B7 binding by large activated T cells. Anti-CTLA-4 mAb 11D4 and anti-CD28 mAb 9.3 acted cooperatively to inhibit T cell adhesion to B7, and to block T cell proliferation in primary mixed lymphocyte culture. When coimmobilized with anti T cell receptor (TCR) mAb, anti-CTLA-4 mAbs were less effective than anti-CD28 mAb 9.3 at costimulating proliferation of resting or activated T cells. However, coimmobilized combinations of anti-CD28 and anti-CTLA-4 were synergistic in their ability to augment anti-TCR-induced proliferation of preactivated CD4 + T cells. These results indicate that CTLA-4 is coexpressed with CD28 on activated T lymphocytes and cooperatively regulates T cell adhesion and activation by B7.

5 g tumor mass). In contrast, unconjugated CalichDMH, unconjugated G5/44, and an isotype-matched control conjugate, CMA-676, were ineffective against these BCL xenografts. Thus, CD22-targeted delivery of CalichDMH is a potent and effective preclinical therapeutic strategy for BCLs. The strong antitumor profile of CMC-544 supports its clinical evaluation as a treatment option for B-lymphoid malignan-

One of the P2-integrins found on hematopoietic ceUs is lymphocyte function-associated antigen 1 (LFA-1), a lymphocyte/myeloid cell-specific receptor that binds to members of the intercelular adhesion molecule (ICAM) family on antigen-presenting cels. Stimulation of LFA-1 with antibodies or purified ICAMs induces augmentation of T-cell antigen receptor (TCR)-directed T-cell responsiveness. In the present study, LFA-1 was shown to be linked to the tyrosine kinase signalig pathway that stimulates tyrosine phosphorylation and activation of phospholipase C-y1 (PLC-y1