Eur. zyxwvutsrqpon J. Immunol. 1989.19: 747-755 CD45R molecule and CD8 cells 747 Tsutomu Takeuchia, Christopher E. Rudd', Shinsuke Tanaka, David M. Rothstein, Stuart F. Schlossman and Chikao Morimoto Division of Tumor Immunology, Dana- Farber Cancer Institute, Boston Functional characterization of the CD45R (2H4) molecule on CD8 (T8) cells in the autologous mixed lymphocyte reaction system* In the present study, we have investigated the molecular basis for the immunoregula- tory function of CD8 cells after autologous mixed lymphocyte reaction (AMLR) activation. We demonstrated that the CD8+CD45R+, but not the CD8+CD45R- subset of cells effected suppression following AMLR activation. In contrast, cytotoxic activity against alloantigens resided in both the CD8+CD45R+ and CD8+CD45R- subsets of cells. Biochemical analysis showed that on CD8 cells, the 220-kDa isoform of the LCAK200 antigen family was better represented than the 200-kDa isoform, when compared to CD4 cells. The density of the CD45R antigen increased on CD8 cells following activation in AMLR and treatment of AMLR-activated CD8 cells with either anti-CD45R antibody or anti-CD3 antibody abolished the suppressor function of these cells. In contrast, treatment of AMLR-activated CD4 cells with anti-CD45R, but not anti-CD3 antibody, abolished the suppressorhnducer function of these cells. The results suggest that the CD45R antigen as well as CD3 T cell receptor complex have an important role in the suppressor function of AMLR-activated CD8 cells. 1 Introduction Considerable evidence exists to support the notion that the immune system is comprised of a network of cells bearing unique differentiation antigens which perform a variety of reg- ulatory and effector functions zyxwvutsr [ 1, 21. Monoclonal antibodies (mAb) have been used to identify antigens on the surface of T cells, such as CD4 and CD8, which can subdivide lymphocytes into discrete subsets [l-41. Recently, it has become evident that the CD4+ lymphocytes can be further subdivided into functionally and phenotypically distinct populations of cells based on their expression of unique isoforms of the leuko- cyte common antigen (LCA)/T200 family of antigens [5-91. Anti-2H4 (CD45R) antibody has been shown to define the 200-kDa and 220-kDa isoforms of the LCAK200 family of antigens [6, 71, whereas UCHL-1 defines the 180-kDaisoform of this family [9]. Importantly, the distinction between CD4+CD45R+(2H4+) and CD4+CD45R-(2H4-) cells is based on the differential expression of the 200-kDa and 220- kDa isoforms of the LCA/T200 antigen, zyxwvutsr i.e. the CD4+CD45R+ population possesses these isoforms and the CD4+CD45R- subset lacks them [6]. Although the CD8+ population contains precytotoxic, cytotoxic, natural killer, pre-suppressor and suppressor/effec- tor cells, these distinctions still rest mainly on the use of func- [I 69981 * This work was supported by NIH grant A1 12069, CA 25369 and AR 33713. Recipients of post-doctoral fellowships from the Cancer Research Institute, New York. Correspondence: Chikao Morimoto, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA Abbreviations: AMLR: Autologous mixed lymphocyte reaction Con A: Concanavalin A FITC: Fluorescein isothiocyanate LCA: Leukocyte common antigen mAb: Monoclonal antibody(ies) MLR: Mixed leukocyte reaction zyxwvuts HA: Phytohemagglutinin PWM: Pokeweed mitogen TcR: T cell receptor tional assays [l, 21. Attempts have been made to define the heterogeneity of the CD8+ population of cells with mAb [lo, 121. A distinction can be made between pre-cytotoxic and cytotoxic effector cells [12], but little has been done to define suppressor/effector cells. In an earlier study, we showed that there exists two types of suppressor cells within the CD8+ population, as defined by the CDll molecule [13]. The CD8+CDllf subset, having NK activity and a large granular morphology, could directly suppress B cell immunoglobulin (Ig) production in the absence of CD4+CD45R+ suppressor/ inducer cells, while the CD8'CDll- subset could only sup- press Ig synthesis in the presence of CD4+CD45R+cells [12]. The latter studies suggested that for authentic suppression to be generated, CD8 suppressor effectors had to be triggered by CD4+CD45R+ suppressor/inducer cells. In the present study, we investigated the functional charac- teristics of CD8 cells lacking the CDll antigen, further sub- divided by the anti-CD45R antibody, anti-2H4. We show that the CD8+CDll-CD45R+ but not CD8+CDll-CD45R- sub- set of cells is the subpopulation with the majority of suppressor activity after autologous mixed lymphocyte reaction (AMLR) activation. In contrast, cytotoxic activity against alloantigens resided in both subsets. Following activation of CD8 cells in AMLR, the density of the CD45R (2H4) antigen increased, and more importantly, both anti-CD3 and anti-CD45R anti- bodies could block the suppressor function of these activated CD8+ cells. These results suggest that both the CD45R (2H4) antigen and the CD3/T cell receptor (TcR) complex have an important role in the suppressor/effector function of AMLR- activated CD8+ cells. 2 Materials and methods zyx 2.1 mAb Seven mAb termed anti-2H4 (CD45R; IgGI), anti-T3 (CD3; IgM), anti-T8A (CD8A; IgGI), anti-T8B (CD8B; IgG2), anti- T4 (CD4; IgG2), anti-T6 (CD1; IgG1) and anti-Mol (CD11; IgG2) were used in the present study. The production and characterization of these mAb was described elsewhere [l, 2, z 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1989 001 4-2980/89/0404-0747 $02.50/0