Relationships among the histologic pattern, intensity, and phenotypes of T cells infiltrating renal allografts

The evaluation of renal allograft rejection by routine histologic evaluation of transplant biopsy is often a diagnostic problem. Advances in monoclonal antibody and immunohistochemical technology have led to their application in characterizing the phenotype of peripheral blood lymphocytes in transplant recipients, as well as the infiltrating lymphocytes in renal biopsy specimens (both tissue and aspiration cytology samples). Recent reports suggest that the relative number of infiltrating T cells and the T cell phenotypic subset ratio may have some association with graft survival. However, most allograft biopsy studies have not examined the relationship of the intensity and phenotype of T cells to the histologic pattern of infiltration, while fine-needle aspiration cytology (FNAC) and peripheral blood analysis do not allow for this assessment. To examine the association of histologic pattern with the intensity and phenotype of T cell infiltrates, immunoperoxidase labeling of cells using monoclonal antibodies was evaluated in 66 renal allograft biopsies performed because of clinical suspicion of rejection. Our findings indicate that the ratio of T helper-inducer (Leu 3) cells to T suppressor-cytotoxic (Leu 2) cells is significantly lower in the pattern of diffuse renal cortex infiltration (cortical diffuse [CD] pattern) when compared with other histologic patterns (cortical aggregate or perivascular), regardless of the overall intensity of the T cell infiltrate. A significant difference in T cell phenotypes was also found among different histologic patterns as expressed by Leu 3/Leu 2 ratios and by the number of Leu 2 cells. An increase in the overall intensity of T cell infiltrate was not associated with significant changes in T cell phenotype seen in any pattern, but an increase in the intensity of the cortical diffuse T cell infiltrate was associated with a significant decrease in the Leu 3/Leu 2 ratio (P less than 0.04), which was due to an increase in the population of Leu 2 cells (P less than 0.002). Interestingly, increases in the intensity of either cortical aggregate or perivascular T cell infiltrates were associated with significant increases in both Leu 3 and Leu 2 cells, as well as a tendency towards higher Leu 3/Leu 2 ratios. These findings indicate that evaluation of T cell infiltrates by phenotype in renal allografts is dependent upon the pattern as well as the intensity of infiltrate.     

Mononuclear cells in renal allografts. Correlation with peripheral blood T lymphocyte subpopulations and graft prognosis

Cellular infiltrates in 20 renal allograft biopsies taken at the time of acute rejection episodes were analyzed with antibodies reactive with monocytes (BRL antihuman monocyte monoclonal antibody), T lymphocyte subpopulations (OKT- and Leu-series of monoclonal antibodies), and B lymphocytes (heterologous antihuman IgM antibodies). For demonstration of the various mononuclear cells, an indirect immunoperoxidase technique was used. The number of monocytes in the infiltrates varied from 10-20%; the number of B lymphocytes was always below 10%. The T lymphocytes accounted for 50-90% of the total number of mononuclear cells. The OKT4/OKT8 ratios for the T lymphocytes in the graft infiltrates were correlated with the peripheral blood OKT4/OKT8 ratios measured by indirect fluorescence and flow cytometry. The OKT4/OKT8 ratios for perivascular infiltrates correlated far better with peripheral blood OKT4/OKT8 ratios (r = 0.72) than did the OKT4/OKT8 ratios for interstitial infiltrates (r = 0.58). Low or inverted OKT4/OKT8 ratios and low or inverted Leu 3a/Leu 2a ratios were associated with a high risk of irreversible graft rejection (P less than 0.001 for perivascular infiltrates).     

Immune mechanisms in organ allograft rejection. II. T helper cells, delayed-type hypersensitivity, and rejection of renal allografts

The cellular requirements for renal allograft rejection have been assessed by adoptive transfer studies in a rat model. Sublethally irradiated (780 rads) LEW (RT1l) recipients of WF (RT1u) renal allografts were selectively reconstituted with spleen cells from specifically sensitized donors. In some experiments the reconstituting inocula were depleted of SIg+ cells by passage over anti-Ig columns or subjected to additional depletion of cytotoxic T cells (Tc) and their precursors reactive with monoclonal antibody OX8. WF renal allografts underwent acute rejection in the unmodified LEW recipient with day 7 serum creatinines of 6.8 +/- 0.9 mg/dL (mean +/- SD; n = 7), graft histology characterized by marked mononuclear cell infiltration and evidence of a brisk humoral response (complement-dependent cytotoxicity (CDC) titer greater than 2(6] and generation of Tc demonstrable by in vitro monitoring. Sublethally irradiated recipients mounted no detectable immune response, and day 7 serum creatinine and graft histological findings were not significantly different from those obtained in isograft controls. Renal allografts were, however, rejected in sublethally irradiated recipients reconstituted with unfractionated immune spleen cells, as evidenced by functional and histologic criteria (day 7 serum creatinine of 5.5 +/- 1.2 mg/dL; histology characterized by extensive interstitial hemorrhage, fibrinoid necrosis of blood vessels, and polymorphic infiltration). Neither antibody nor Tc appear, in this model, to be required to effect rejection, because recipients reconstituted with inocula depleted of SIg+ cells (day 7 CDC titer less than 2l) or subjected to additional depletion of Tc and their precursors (day 7 lymphocyte-mediated cytotoxicity assay: % specific chromium release at 100/1 E/T ratios less than 5%) underwent acute rejection with a day 7 serum creatinine of 5.0 +/- 1 and 4.3 +/- 1.5 mg/dL, respectively, and histological findings were characterized by marked mononuclear cell infiltration and a paucity of hemorrhage.     

肾移植受者外周血辅助性T淋巴细胞17、调节性T细胞表达及其平衡关系的研究

目的 探讨肾移植慢性排斥反应(CR)患者外周血辅助性T淋巴细胞(T helper,Th)17、调节性T细胞(regulatory T cells,Treg)、Th17/Treg变化及意义.方法 采用流式细胞术检测24例肾移植CR患者(CR组)和22名健康志愿者(正常对照组)外周血中的Th 17占CD4+T淋巴细胞的比例...     

Identification of the cellular subpopulations infiltrating rejecting cadaver renal allografts. Preponderance of the T4 subset of T cells

In the rejection response against renal allografts, the relative importance of helper/inducer T cells mediating a delayed-type hypersensitivity response and of T cells with direct cytotoxicity has not been defined. These subpopulations were identified with commercially available monoclonal antibodies and an indirect immunoperoxidase technique in 31 renal biopsies from patients undergoing acute rejection episodes and in 9 rejected nephrectomy specimens. T lymphocytes were the predominant cell population in all biopsies and in 8 of 9 nephrectomies. The T4 helper/inducer subset was equal to, or greater than, the T8 cytotoxic/suppressor subset in 28 of the 31 biopsies and in the 8 nephrectomy specimens that had histological evidence of cellular rejection. T4 lymphocytes were found predominantly in large areas of cellular infiltrate. T8 lymphocytes had a more diffuse interstitial distribution and were a minority of the cells in the large areas of cellular infiltration. These results show that helper/inducer T lymphocytes are often more frequent than cytotoxic/suppressor cells in acute renal allograft rejection in humans and they suggest that helper/inducer T cells may play an important role in the mediation of graft destruction.     

Assessment of tumour-infiltrating lymphocytes, regional lymph node lymphocytes and peripheral blood lymphocytes and their reaction to interferon-gamma in patients with renal carcinoma

The immunological distribution of tumour-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL) was evaluated by means of immunohistochemical staining using monoclonal antibodies of each subset of lymphocytes (stored in frozen sections) in a series of 22 patients with renal carcinoma. The immunological effect of IFN (interferon)-gamma on these immunocompetent cells was also investigated. The effect of IFN-gamma on TIL was an increase in CD3 (pan-T cells), especially an increase in CD8 (cytotoxic/suppressor-T cells). When examining these cells according to stage and grade, a marked increase in CD3 was found in low stage and low grade patients. With regard to RLNL, there was a tendency towards a decrease in CD3 and an increase in CD20 (B-cells) following the administration of IFN-gamma. No specific effect on stage and grade was observed apart from a reduction in T cell subset ratios in high grade patients. With regard to PBL, no specific trend was noted except for an increase in CD16 (NK cells) when IFN-gamma was administered.     

Rejected human renal allografts: recovery and characteristics of infiltrating cells and antibody.

Viable infiltrating host leukocytes have been isolated from 10 rejected human renal allografts, removed 1 to 67 months after transplantation. The cell populations have been identified by surface characteristics and their cytotoxic capacities were assessed. A heterogenous population of cells of host origin accumulated in the grafts, including T and B lymphocytes, Fc+ cells, and macrophages. Using a 51Cr release assay, specific cytotoxicity against donor alloantigens was determined. Cytotoxicity of the infiltrating cells was almost invariably greater than cytotoxicity mounted by recipient peripheral blood lymphocytes. Deletion studies confirmed previous work and suggested that T cells were primarily responsible for cytolysis in early acute rejection; non-T cells more often in late chronic rejection. Antibodies eluted from the grafts demonstrated both specific antidonor and nonspecific activity as well as cross-reacting anti-HLA activity. Allograft morphology was examined and cellular and humoral host responses were assessed. These studies emphasize the complexities of immune responses produced by the host against transplanted tissues.     

Induction of transplantation tolerance in rats by spleen allografts. I. Evidence that rats tolerant of spleen allografts contain two phenotypically distinct T suppressor cells

We have examined suppressor cell activity in transplantation tolerant (TT) rats bearing vascularized spleen allografts in several different donor-recipient combinations. More than 60% of WAG (RT-1u) and 65% of AGUS (RT-1l) spleen allografts were permanently accepted when transplanted to AGUS and PVG (RT-1c) rats, respectively. All (WAG X AGUS)F1 to AGUS and (AGUS X PVG)F1 to PVG spleen allografts survived indefinitely. Unseparated LNC, TDL, and whole T cell or W3/25+, OX8- T cell populations obtained from AGUS rats bearing (WAG X AGUS)F1 spleens exhibited reduced mixed lymphocyte reaction (MLR) responses to the spleen donor, and to some extent to BN(RT1n) third-party stimulators, but responded normally to PVG.A(RT1a) stimulators. Coculture experiments demonstrated that lymph node cells (LNC) and thoracic duct lymphocytes (TDL) of TT rats contain RT1 specific suppressor cells. Furthermore, T cells isolated from all donor-recipient combinations contained two phenotypically distinct suppressor cell populations: a radiosensitive W3/25+, OX8- (Th/i) and a relatively radioresistant W3/25-, OX8+ (Ts/c). These Ts may be responsible for the maintenance of TT.     

The effects of donor-specific blood transfusion enhancement of rat renal allografts on cytotoxic activity and phenotypes of peripheral blood lymphocytes, splenocytes, and graft-infiltrating cells

Donor-specific blood transfusion prolongs survival of fully allogeneic ACI (RT1a) renal grafts in PVG (RT1a) recipients from 6-8 days to greater than 100 days. To determine how DSBT alters effector cytotoxic cell responses, we tested freshly isolated peripheral blood lymphocytes, spleen cells, and graft infiltrating cells (GIC) from pairs of PVG recipients of ACI kidneys pretreated with DSBT or autologous blood transfusion (ABT) for cell-mediated lympholysis, antibody-dependent cellular cytotoxicity (ADCC), and natural killer activity at the day of transplantation (day 0) and days 3 and 6 posttransplantation. PBL and GIC from the same pairs of animals were examined for their phenotypic profile (CD4, CD8, 3.2.3 NK cell marker, IL-2 receptor). CML, ADCC, and NK activity were higher in PBL than splenocytes of GIC of both ABT and DSBT groups at all time points examined. CML activity of PBL at day 3 was significantly higher in DSBT vs. ABT recipients (P less than 0.01), while at day 6 both groups were equally elevated. Splenocytes demonstrated significantly lower CML activity in DSBT vs. ABT recipients at day 6 (P less than 0.05). CML activity of GIC eluted from ABT and DSBT kidneys was not detected at day 3, but was significantly elevated and equivalent in both groups at day 6. ADCC and NK activities of PBL did not differ between ABT and DSBT groups, and were negligible in splenocytes. GIC demonstrated higher NK activity in DSBT vs. ABT recipients at day 3 (P less than 0.05), while ADCC activity was not detectable at any time in either group. Phenotypic analysis of PBL and GIC at day 3 showed no significant differences between ABT and DSBT groups in the percentage of CD4 or CD8 cells. However, in both ABT and DSBT groups the ratio of CD4:CD8 cells was markedly lower in GIC than PBL. By day 6, PBL from both ABT and DSBT groups showed equivalent and significant decreases in CD4+ cells and increases in CD8+ and 3.2.3+ (NK) cells. The percentage of IL-2R+ cells remained low (less than 5%) in both groups at day 3. In contrast, at day 6 there was a significant increase in IL-2R+ (and to a lesser extent CD4+) GIC in ABT- but not DSBT-treated recipients, while CD8+ cells were significantly increased in both groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Graft-versus-host reactivity and renal allograft survival in rats given allogeneic spleen cells or spleen allografts.

Selective recruitment of antigen-sensitive cells (ASC) into the spleen as a method of inducing specific suppression was attempted by intravenous injection of either DA or Lewis spleen cells 24 hr before a (DA X Lewis)F1 renal allograft into a Lewis or DA recipient, either with or without a splenectomy. This led to suppression of rejection in the DA recipient and delayed rejection in the Lewis recipient. Splenectomy produced a minimal augmentation effect. Assay of graft-versus-host (GVH) reactions in (DA X Lewis)F1 rats by a popliteal node assay showed that injection of allogeneic DA or Lewis spleen cells 48 hr before the assay significantly reduced the reaction produced by node lymphocytes but not spleen lymphocytes, suggesting a loss of ASC from the lymph nodes. Lewis spleen allografts did not produce such a significant reduction in the GVH reactivity of DA node lymphocytes as intravenous Lewis cells, whereas DA spleen allografts led to an increased GVH reactivity of Lewis node lymphocytes. From these studies, it is not possible to attribute the suppression produced by the intravenous injection of allogeneic cells to selective recruitment of antigen-sensitive cells to the spleen.     

Effect of irradiation on the immunocompetence of peripheral blood and regional lymph node lymphocytes in bladder cancer

This study was undertaken to examine the effects of procaine (P) (2 g/liter) in multidose crystalloid (KCP) and blood (BCP) potassium (25 meq/liter) cardioplegia solutions during 2 hr of hypothermic (22°C) aortic occlusion. Four groups were studied: Group I KCP, P(?) n = 8, Group II KCP, P(+) (n = 8), Group III BCP, P(?) n = 6, Group IV BCP P(+) n = 8. Change in left ventricular (LV) function was defined as percentage change in center of mass between pre- and postarrest function curves. Regional myocardial flow was measured with microspheres and myocardial metabolism monitored by lactate and oxygen utilization. Ventricular biopsies were serially obtained for myocardial ATP, creatine phosphate, calcium, water content, and ultrastructure study. LV function percentage recovery was 62 ± 4% in Group I, 79 ± 5% in Group II, 81 ± 4% in Group III and 72 ± 4% in Group IV. Myocardial regional flow did not show significant differences between groups P(?) and P(+) though KCP and BCP groups showed different patterns of reperfusion. Lactate and oxygen utilization did not show significant differences between groups. In all groups ATP and calcium were well preserved and mild edema formation was observed at reperfusion. Ultrastructural preservation was good except in Group I where myofilament breakdown was present. Procaine was effective in obtaining spontaneous recovery of normal electrical activity. In conclusion, BCP without procaine significantly improved myocardial protection over similar KCP. Procaine had a significant additive effect in KCP and no additive effect in BCP.     

Phenotype and function of cells infiltrating rat renal allograft

Phenotype and function of graft infiltrating cells (GIC) from rat renal allografts were investigated in comparison with those of spleen (SP) cells, peripheral blood mononuclear cells (PBMC), and regional lymph node (LN) cells of the recipient. Relative proportions of all T cell, suppressor/cytotoxic T cell, helper T cell, and antigen-activated cell displayed significant increases in GIC during ongoing rejection assessed by flow cytometry. Cytolytic activity (using 51Cr release assay) of GIC on day 3 was much higher (20.2%) than those of SP (6.0%), PBMC (3.8%), and LN (3.2%) on BN target cells and this activity gradually increased during ongoing rejection up to 53.1% (GIC), on day 6. In vitro production of cytokines (IL-2, IL-3, gamma-IFN, and BSF-2) from these groups of cells were investigated. GIC demonstrated the most remarkable increases of cytokine production from day 3 to day 6. Especially, GIC on day 6 produced higher amount of BSF-2 compared with SP cells, PBMC and LN cells. These results demonstrated that alloactivated Th cells as well as Tc cells accumulated within the allografts and that the subtype of Th cells which produce BSF-2 preferentially assembled to the allograft.