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This study compares the cellular events in excised rejected renal allografts (RKT), with concurrent data in the peripheral blood (PB) in the same patients. The kidney transplants were obtained from recipients after rejections crises that were refractory to treatment. Two-color flow cytometry data was used for quantitation of cell subset profiles and quantitation of the density of individual lymphocyte surface antigen(s). The level of CD3DR-positive T cells in RKT was significantly higher than in PB. This difference ranged from 3 to 19-fold increases. There was an even more pronounced increase in CD8CD57 cell subset levels in RKT versus PB. In parallel with these findings, there was a 15-fold greater mean density of the CD8CD57 markers on T cells in RKT, when compared with the same cells in PB. The density of CD8 and of DR markers on CD3+ cells was also significantly higher (4-fold in each instance) in RKT. These data point to a homing of CD8CD57 cells with corresponding increases in the density of these markers in acutely rejected renal allografts, with only a relative decrease of this cell subset in peripheral blood. The appearance of a high preponderance of CD8CD57 cells in the renal allograft at the time of a rejection crisis may constitute a particularly severe prognostic sign regarding the reversibility of the response after treatment with steroids and/or monoclonal antibodies.

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gamma irradiation (GR) or mitomycin C (MC) treatment of stimulator cells is frequently used to achieve unidirectionality of response in the mixed lymphocyte reaction. As GR differs from MC in the pathways used to block lymphocyte replication, this study analyzes the effects of these modalities upon the expression of various differentiation and Class II major histocompatibility antigens on lymphocytes cultured for 24, 48, and 72 hr. There was a decrease in the mean density of HLA-DR expression on CD3+ and on CD8+ cells at 24, 48, and 72 hr after exposure to GR (42 and 35, 60 and 69, and 26 and 49%, respectively) or to MC (26 and 11, 26 and 18, and 46 and 30%, respectively). There was a parallel decrease in the levels of the corresponding cell subsets when compared with control cultured cells not exposed to GR or MC. In contrast, the density of HLA-DR markers on CD3-negative cells was increased at 24, 48, and 72 hr of culture following exposure to GR (73, 82, and 102%, respectively) or to MC (9, 45, and 80%, respectively). There was a more profound decrease in CD3+, CD8+, and CD19+ cell subset levels and in the density of the corresponding markers in GR-treated cells than in those of cells exposed to MC when the results were compared with those of untreated cultured control cells. Although GR appears to exert a more profound effect than MC, the results indicate that both modalities have the capacity to reduce the density of polymorphic determinants on Class II (HLA-D region)-encoded molecules on T (CD3+ and CD8+) and B (CD19+) cells which are known to trigger potent MLR responses. Both modalities may therefore affect profoundly the relative strength of MLC responses and the derived measurements of the degree of HLA Class II compatibility between stimulator and responder cells.

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We describe 12 acute rejection episodes in 11 cadaver donor renal allograft recipients who required OKT3* rescue treatment for steroid-resistant acute rejection (9) or for severe vascular (antibody-mediated) rejection (3). There were 3 treatment failures with subsequent graft loss. Using 2-color flow cytometry the total T (CD3), B (DR+), activated T (CD3DR), T helper/inducer (CD4), T cytotoxic/suppressor (CD8) and activated T cytotoxic cell (CD8DR) subsets were analyzed before, in mid course (5 to 7 days) and at the end of 12 to 14 days of therapy with 5 mg. OKT3 intravenously daily. In parallel changes in the density of such T cell associated antigens were analyzed. Significant decreases in the mean levels of the CD3 (p less than 0.001), CD3DR (p less than 0.05), CD4 (p less than 0.05), CD8 (p less than 0.05) and CD8DR (p less than 0.05) subsets were observed at mid course. A significant decrease in the density of CD3 was observed (p less than 0.0001). The surface antigen density of CD3DR, CD4 and CD8 had decreased by 160% (p less than 0.002), 383% (p less than 0.001) and 260% (p less than 0.001), respectively. At the end of treatment CD3 and CD4 subset levels increased by 425% and 240% (p less than 0.001 and p less than 0.005), respectively. In contrast, the CD3DR and CD8DR subset levels continued to decrease (p less than 0.05). A higher pre-treatment level of CD3DR and a less sharp decrease in CD3, CD4 and CD8 subsets were associated with a higher risk of treatment failure (p less than 0.05, p less than 0.01, p less than 0.05 and p less than 0.05, respectively). The mean decrease in the density of the CD3 marker in the lost grafts was significantly smaller compared to successful outcomes (p less than 0.001). The results of this preliminary study suggest that OKT3 affects T cell associated antigens other than CD3. Such may provide a sensitive prognostic index for the effectiveness of OKT3 therapy, and permit the identification of those patients who might require higher doses and/or duration of OKT3 therapy to enhance renal allograft salvage rates.

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The kinetics of expression of activation-linked T-cell surface markers were analyzed in T-cells obtained from normal donors. The cells were cocultured in triplicate for 0, 1, 24, 48, and 72 hr in the presence of Con A in RPMI 1640. The density of HLA-DR, interleukin 2, and transferrin receptors (IL-2-R and TR, respectively) on the surface of CD3- and CD8-positive cells was computed by a mathematical model based on fluorescence intensity vector analysis, adjusted for cell size, utilizing two-color flow cytometry. The results were compared with controls obtained at the same time with control cells cultured in RPMI 1640 alone. There was a significant increase in the mean density of HLA-DR on the surface of CD3- and CD8-positive cells as early as 1 hr after exposure to Con A when compared with controls (250 and 300%, respectively; P less than 0.0001). The mean density of IL-2-R and TR on CD3+ cells increased by 265 and 208%, respectively; P less than 0.06 and P = n.s., respectively, when compared with control cells. The mean density of Class II MHC products on CD3+ and CD8+ cells treated with RPMI alone increased by 202 and 468%, 234 and 540%, and 1375 and 2442%, respectively, at 24, 48, and 72 hr of culture. In contrast, the mean cell surface density of these markers in cells treated with Con A increased by 614 and 1962%, 3304 and 7231%, and 8665 and 22,619%, respectively (P less than 0.00001) at the corresponding times following exposure to Con A. The density of IL-2-R and TR on CD3+ cells exposed to Con A also increased significantly at 24, 48, and 72 hr (P less than 0.0001). At the same times, the relative percentage of cell subsets bearing these particular markers increased by 78, 138, and 175% at 24, 48, and 72 hr, respectively. The data suggest that objective quantitative evidence of lymphocyte activation after exposure to Con A may be obtained as early as 1 hr after antigen stimulation, and before significant changes in cell numbers occur. Measurement of cell surface marker densities may provide a useful index for the detection and quantitation of cell activation in the early phase of antigenic stimulation.

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Parameters of cell-mediated immune function were determined in 76 patients with end-stage renal disease. Lymphocyte subpopulations (OKT3, OKT4, OKT8, OKIa1, OKM1, OKT9, OKT10), natural killer (NK)-cell activity (percentage 51Cr release from K562 targets), and delayed cutaneous hypersensitivity were measured and correlated with other variables. The results indicate that (1) uremic patients have a significant diminution in the OKT4-lymphocyte subpopulation and OKT4/OKT8 (helper/suppressor) ratio compared to normal controls; (2 blood transfusions do not induce significant alterations in the helper/suppressor-cell ratio; (3) uremic patients have a significant increase in OKM1 cells compared to normal controls; (4) the majority of uremic patients in this series developed delayed cutaneous hypersensitivity responses to recall antigens and could be de novo sensitized to 2,4-dinitrochlorobenzene (DNCB); (5) skin-test reactivity could not be correlated with total circulating T cells or levels of any lymphocyte subpopulations; and (6) NK-cell activity in uremic patients is not significantly different from that in normal controls. These results highlight the varying levels and function of different lymphocyte subsets in patients with end-stage renal disease when they are treated with chronic maintenance hemodialysis.

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