Previous studies (1-9) have suggested that peripheral human T cells can be divided into two mutually exclusive functional subsets by the two monoclonal antibodies, OKT8 and OKT4. The OKT8+ subset does not provide helper activity but contains cells capable of suppressing B cell differentiation . Importantly, the suppression observed with OKT8+ cells requires the presence of radiosensitive OKT4 + cells . Potent helper activity is found in a radiosensitive OKT4' subset . Irradiated OKT4+ cells also induced B cell differentiation, but only at high ratios of T cells to B cells . In our previous studies (7), we also found that the addition of graded numbers of radiosensitive OKT4 + cells to B cells eventually decreased the net helper activity observed . These experiments raised the possibility that precursors of suppressor cells may be contained within the OKT4 + population .The current study was undertaken to further investigate the functional heterogeneity within the OKT4+ population. In particular, we asked whether OKT4 + cells could be induced to differentiate into immunoregulatory cells capable of suppressing B cell differentiation . In the experiments reported here, we observed that although in vitro pokeweed mitogen (PWM) -activated' OKT4+ cells can function as radioresistant helper cells, these activated OKT4+ cells could also exert potent feedback suppression . This suppression mediated by activated OKT4+ cells required the presence of radiosensitive cells contained within the resting OKT4 + population. These data emphasize the potential role of interactions of T cell subsets contained exclusively within the OKT4 + population in the immunoregulation of B cell differentiation . Volume 154 August 1981 459-467 Materials and MethodsLymphocyte Preparation and Isolation ofHuman T and B Cells . Fresh peripheral blood lymphocytes were isolated from consenting healthy human volunteers by Ficoll-Hypaque density gradient centrifugation . Highly enriched population of T and B cells were then isolated by methods previously described in detail (10) . In brief, human mononuclear cells were washed in minimum essential medium (Grand Island Biological Co ., Grand Island, N . Y .) containing 5% fetal calf serum (FCS ; Microbiological Associates, Bethesda, Md .) and then separated into surface Ig* Supported in part by grants AI-14969 and AI-11524 from the National Institutes of Health, and by The Robert Wood Johnson, Jr . Charitable Trust and The Arthritis Foundation .'Abbreviations used in this paper: C, complement ; E+ , E rosette positive ; FCS, fetal calf serum ; PFC, plaqueforming cells ; PWM, pokeweed mitogen. J . Exp . MED .

Knockout and blocking studies have shown a critical role for CTLA-4 in peripheral tolerance, however, it is unknown whether augmenting CTLA-4 expression actually promotes tolerance. Here we demonstrate a specific and requisite role for CTLA-4 and its up-regulation in tolerance through anti-CD45RB. First, long-term murine islet allograft survival induced by anti-CD45RB is prevented by CTLA4-Ig, which interferes with B7:CTLA-4 interactions. Second, anti-CD45RB is ineffective in recipients lacking CTLA-4, B7-1, and B7-2. In contrast, CTLA4-Ig, which targets B7 on allogeneic cells, promotes long-term engraftment in these mice. Moreover, anti-CD45RB was effective in B7-deficient controls expressing CTLA-4. Finally, in wild-type mice, CTLA-4 expression returned to baseline 17 days after receiving anti-CD45RB, and was refractory to further increase. Transplantation and anti-CD45RB therapy at this time could neither augment CTLA-4 nor prolong engraftment. These data demonstrate a specific role for CTLA-4 in anti-CD45RB-mediated tolerance and indicate that CTLA-4 up-regulation can directly promote allograft survival.

Water transport across plasma membranes is a universal property of cells, but the route of such transport is unclear. In this study, volume changes of cells of the J774 murine macrophage-like cell line were monitored by recording the intensity of light scattered by the cells. We investigated the effects of several inhibitors of glucose transport on cell membrane osmotic water permeability as calculated from the rates of cell volume change. Cytochalasin B (2.5 ,ug/ml), phloretin (20 FM), and tomatine (3 jzM) reversibly blocked glucose uptake into these cells. All three inhibitors reversibly decreased the osmotic water permeability of J774 cells from 89.6 ± 3.2 to 27.2 ± 1.4 ,um/sec. We conclude that a major component of the osmotic water flow across the plasma membranes of these cells is accounted for by water traversing their glucose transporters.Water transport across plasma membranes is thought to occur both by diffusion across the lipid bilayer and through specific channels. Although the lipid bilayer of cell membranes has a relatively low intrinsic permeability to water, it may suffice for water exchanges in most cells (1). Some cells [i.e., urinary tract epithelial cells (2) and erythrocytes (3, 4)] display osmotic water permeabilities (P) much higher [up to 350 ,um/sec (5)] than can be accounted for by osmotic water passage through lipid bilayers [P = 4-37 gm/sec (6)], suggesting that they possess membrane channels specialized for water transport. In the human erythrocyte, Solomon et al. (3) proposed that the anion transporter (band 3) serves as a water channel. However, other possible transmembrane water routes in this cell have been suggested (4, 7). Jung et al. (8) used the kinetics of hydrogen-tritium exchange in erythrocytes to suggest that the glucose transporter contains a water pore. Experimental data and theoretical analysis from others (9-11) also suggest that the dimensions and physical properties of the glucose transporter are consistent with its functioning as a water channel. Nonetheless, direct evidence identifying the water channel has been lacking.The identification of water channels in cells has been hindered by the lack of inhibitors that selectively block water transport. We report here that a specific inhibitor of glucose transport [cytochalasin B (CytB)] (12) reversibly slows osmotic water transport across the plasma membranes of J774 cells, a murine macrophage-like cell line (13). For these experiments, we used the intensity of light scattered (Is) by cells to monitor transient cell volume changes. From our evidence, we conclude that glucose transporters serve as the major water channel in these cells. Some of our initial results have appeared in abstract form (14). METHODSCell Culture. J774 cells were maintained as suspension cultures in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% (vol/vol) heat-inactivated calf serum (GIBCO). Cells were plated and maintained overnight as adherent cultures in the above medium on either 12-mm round coverslips (for solut...

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8αα homodimers with a 10-fold higher affinity than H-2Kb class I molecules. To understand the molecular basis for this difference, we created a panel of CD8α mutants and tested the ability of the CD8αα homodimers to bind to H-2Kb tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the α3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2Kb class I sequences are highly conserved in the α3 domain of MHC class I, this suggests that CD8 contacts the α3 domain of TL and H-2Kb in a similar manner. In contrast, mutations in residues on the A and B β strands of CD8 that are involved in contact with β2-microglobulin affected interaction with the H-2Kb tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2Kb. The unique high affinity binding of TL with CD8αα is most likely a result of amino acid differences in the α3 domain between TL and H-2Kb, particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8αα-H-2Kb cocrystal.

The role of CTLA-4 in tolerance is primarily inferred from knockout and blocking studies. Anti-CD45RB mediates allograft tolerance in mice by inducing CTLA-4 expression on CD4 cells, providing a novel opportunity to determine how therapeutic enhancement of CTLA-4 promotes tolerance. We now show that induced CTLA-4 expression normally resolves by day 17. Although thymectomy prolongs enhanced CTLA-4 expression, long-term engraftment is unaffected. To address the temporal relationship between increased CTLA-4 expression and engraftment, transplantation was delayed for various times after anti-CD45RB treatment. Delaying transplantation for 7 days (when CTLA-4 expression had peaked but treatment mAb was no longer detectable), resulted in long-term engraftment comparable to transplantation with no delay (day 0). Delaying transplantation from 10 to 18 days led to a progressively poorer outcome as CTLA-4 expression returned to baseline. This suggested that Ag exposure while CTLA-4 expression is enhanced is sufficient to induce long-term engraftment. To substantiate this, on day 0, anti-CD45RB-treated mice received BALB/c vs unrelated alloantigen, followed by transplantation of BALB/c islets 10 days later. Whereas recipients exposed to unrelated Ag experienced acute rejection, recipients exposed to donor Ag achieved long-term engraftment. Anti-CD45RB-treated mice exposed to alloantigen exhibited anergic CD4+CD25− effector cells and regulatory CD4+CD25+ cells. Moreover, CD25 depletion in the peritransplant period prevented anti-CD45RB-mediated engraftment. Thus, exposure of CD4 cells expressing CTLA-4 to donor Ag is necessary and sufficient to induce long-term engraftment which appears to be mediated by both regulation and anergy.

Our knowledge of human T cell differentiation and function has expotentially increased during recent years. With this growth in knowledge there has been an increase in our appreciation of the complexity of the T-T interactions which initiate and control immune responses. A great deal remains to be learned concerning the mechanisms of these complex cellular interactions. In particular, it will be important to precisely understand the clear heterogeneity of functions within isolated subsets of OKT4+ and OKT8+ T cells. Perhaps, as importantly, it will be necessary to define more clearly the functions of the T4 and T8 molecules as well as the precise function of the other defined glycoproteins on the T cell surface. The evidence is clearly emerging that many of those molecules are not solely markers of unique functional subsets and are intimately involved in the functions of T cells.

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