Intra-epithelial lymphocytes in the normal epididymis. A mechanism for tolerance to sperm auto-antigens?

Using a monoclonal immunoperoxidase technique on frozen tissue sections of normal testis and epididymis, a distinctive distribution of lymphocytes has been identified. Almost all of the intra-epithelial lymphocytes were T cells and the majority of these belonged to the T8+ ("suppressor/cytotoxic") subset. The distribution contrasted with the predominance of the T4+ ("helper/inducer") subset in the interstitial tissues and in normal peripheral blood. The predominance of the "suppressor/cytotoxic" subset within the epithelium of the epididymis may exist in order to prevent the development of the immune response to auto-antigens on spermatozoa.     

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.     

Circulating T-lymphocyte sub-subsets and NK-like cells in renal transplant patients: patterns associated with rejection

The patterns of circulating T sub-subset and NK-like cells were monitored in 87 renal transplant patients on a total of 122 occasions. Combinations of monoclonal antibodies were used in two-colour flow cytometric (FACS) analysis to define the major T helper/inducer and T suppressor/cytotoxic subsets and their activated counterparts, the T suppressor inducer and T suppressor effector sub-subsets, NK-like cells and their activated counterparts. In patients with rejection, the proportions of T suppressor inducer and T suppressor effector cells were less and the proportion of activated NK-like cells greater compared with cases exhibiting stable graft function, and these group differences were highly significant. Analysis with stepwise multivariate logistic regression showed that combinations of several functional/activated lymphocyte subsets allow a predictive diagnostic power which is superior to the use of single variables.     

Multidisciplinary therapy using interferon and immunological evaluation for glioma patients: two-color analysis of T cell subsets]

We have employed IAR therapy [combination of postirradiation, chemotherapy and interferon (IFN)] for malignant glioma patients. Changes of lymphocyte fractions in patients were evaluated before and after IAR therapy, using a recently developed two-color analysis. Eight malignant glioma patients received irradiation, chemotherapy (ACNU) and immunotherapy (OK-432 and IFN-beta). Peripheral blood lymphocytes taken during hospitalization with IAR therapy (first half and latter half), and every 3 to 6 months for 2 years at the longest after IAR therapy were double-stained with FITC- and PI-labelled antibodies and two-color analysis was conducted by a FACS Analyzer. Six patients out of 8 survived for 6 months to 2 years, 2 died after 3 and 6 months, respectively. Leu-2a (suppressor/cytotoxic T), especially Leu-2a+ 15- (cytotoxic T) showed a high value. Leu-2a level decreased during treatment, and both Leu-2a+ 15- and Leu-2a+ 15+ (suppressor T) values decreased. Two thirds of the patients showing an increased Leu-2a+ 15+ level died. Leu 3a (helper/inducer T), especially Leu-3a+ 8+ (inducer T) level decreased, but Leu-3a+ 8- (helper T) level increased during treatment. The level decreased in the worse patients. Leu-3a/Leu-2a ratio was low, but it increased during treatment as compared with the results of conventional therapy. Leu-7, Leu-11a, NK activity, and gamma-IFN productivity were further studied. Treatment combined with IFN revealed an influence on the T cells resulting in an increase of helper T level and suppression of suppressor T level.     

A clinical study of immunological status in patients with gastric cancer: with special reference to T-cell subsets, interleukin-2 in the lymphocytes of peripheral blood

Peripheral blood lymphocytes of 63 patients with gastric cancer were studied by using different monoclonal antibodies and flow cytometry. Used monoclonal antibodies were OKT3 (total T cell), OKT4 (helper/inducer), OKT8 (suppressor/cytotoxic), Leu 7 and Leu 11 (NK/K cell). Interleukin-2 was measured by tritium-labelled thymidine CTLL assay on the supernatant of peripheral blood lymphocytes after 24 hours stimulation with phytohemagglutinin. Interleukin-2 receptor was also studied by using monoclonal antibody (for Tac antigen) and flow cytometry. The results were as follows: among peripheral blood lymphocytes; 1. the number of OKT3, OKT4 cells and the percentage of OKT4 cells decreased significantly with more advanced stage of cancer. 2. production of interleukin-2 also decreased with the progression of the cancer. 3. decreases in the OKT4/OKT8 ratio were found with cancer progression. 4. the percentage and the number of OKT8 cells increased. 5. the percentage and the number of Leu 11 cells and the number of Leu 7 cells were increased significantly in the stage III (moderately advanced cancer). These results suggested that the activated helper T cells decreased, the induction capability of cytotoxic T cells decreased and the suppressor T cells increased with the progression of cancer. Quantitative and qualitative change in T-cell subsets in advanced stage may be one factor responsible for immunosuppression.     

The immunologic profile of anesthetists

Reports in the literature have suggested possible impairment of immunocompetence in operating theater personnel. In a group of 18 physician anesthesiologists the following were determined: hemoglobin concentration; white blood cell count; numbers of T, B, and natural killer (NK) lymphocytes; number of T-active cells; and numbers of T-helper/inducer (Th) and T-suppressor/cytotoxic (Ts) cells; and the Th/Ts ratio. Function of T lymphocytes was evaluated using the local xenogeneic graft-versus-host reaction and spontaneous suppressor or helper activity of T cells. The same parameters were determined in a group of 18 age- and sex-matched healthy controls. It was found that no matter what their age or how long they have been engaged in anesthetic practice, anesthetists show no immunosuppression as evidenced by these parameters.     

Lymphocyte subsets in renal carcinoma--a sequential study using monoclonal antibodies

Using monoclonal antibodies in conjunction with flow cytometry, circulating lymphocyte subsets with distinct functions in the regulation of the immune response were enumerated in 32 patients with proven renal carcinoma. Analyses were performed at presentation and sequentially during the clinical course of the patients. Untreated patients with advanced disease had a deficit of T cells with the "helper/inducer" phenotype (Leu-3a+) and this resulted in abnormal T "helper/suppressor" (Leu-3a+/Leu-2a+) ratios. Following nephrectomy, performed in 26 patients, there was a significant increase in the number of T cells with the "helper/inducer" phenotype and a significant increase in T "H/S" ratios. Subsequent follow-up at a minimum of 2 months after nephrectomy showed that the increase in T cells with the "helper/inducer" phenotype was maintained (with the exception of 6 patients with disease progression) and was then accompanied by a significant increase of the T cell subset with the "suppressor/cytotoxic" phenotype (Leu-2a+). Pre-operative renal arterial embolisation resulted in an early transient lymphopenia. The response to embolisation combined with nephrectomy was little different when compared with nephrectomy alone. These observations represent a novel view of the immunosuppressive effects of renal carcinoma and their relation to anaemia and disease progression are discussed.     

Depressed immune response in burn patients: use of monoclonal antibodies and functional assays to define the role of suppressor cells.

Recent experimental evidence has suggested that circulating suppressor leukocytes play an important role in mediating the suppression of immunity seen in burn patients. In order to shed further light on the relationship between suppressor cells and depressed cellular immunity 22 patients were studied (mean age 37) who had suffered severe burns of greater than 30% body surface area. Simultaneous studies were performed on 14 control laboratory personnel (mean age 32). Monoclonal antibodies were used to identify T-lymphocyte subsets known to have suppressor/cytotoxic (OKT8) and helper/inducer (OKT4) function, respectively. In addition, serial measurements were made of the response of circulating lymphocytes to the T-cell mitogen phytohemagglutinin (PHA). An inversion of the normal ratio between suppressor/cytotoxic and helper/inducer subsets (normal 0.55:1, postburn 1.4:1; p less than 0.001) occurred soon after burn injury, reached a peak in five to seven days and then returned gradually to normal levels by 14 days. A diminished response of patients' lymphocytes to PHA (57 +/- 10% SD suppression as compared with normal controls at five to seven days) corresponded with high suppressor to helper cell ratios and returned to normal at the same time. Functional assays, which recognize only high levels of activity, demonstrated circulating suppressor cells in nine patients during this same period but became negative by 14 days. These early immunologic modulations were not predictive of morbidity or mortality. Later in the postburn course, systemic sepsis in eight patients was associated with a return of increased suppressor to helper cell ratios and decreased mitogen (PHA) responsiveness. At this time functional assays demonstrated circulating suppressor cells in six patients. Five of these six patients died of sepsis. It was concluded that severe burn injury regularly induces an early transient increase in circulating suppressor cells accompanied by a depression of lymphocyte activation. A later (greater than 14 days postburn) increase in suppressor cells to levels detectable by functional assays is closely correlated with mortality from sepsis.     

Influence of operative trauma on circulating blood mononuclear cells: analysis using monoclonal antibodies

The influence of operative trauma on the quantity and proportion of cooperating mononuclear cells in peripheral blood of patients with stage 0 uterine cancer who underwent hysterectomy was studied using monoclonal antibodies. The percentage of OKM1+ cells (monocytes) had increased on day 1 by 12.4%, on day 3 by 24.8%, and had returned to normal by day 7. The percentage of large OKIa1+ cells (expressing Ia antigen) on day 1 was increased by 186.9%, while on day 3 it had returned to a normal level. There was a decrease on day 1 of the number and percentage of OKT3+ cells (all T lymphocytes) by 17.4%, OKT4+ cells (helper/inducer cells) by 27.9%, OKT8+ cells (suppressor/cytotoxic cells) by 24.6%, and small OKIa1+ cells by 49.4%. The number and percentage of T lymphocytes remained below the preoperative level throughout the observation period. However, the number of helper cells had increased significantly on day 5, while suppressor cells remained below the preoperative level during the entire observation period. The rising ratio of OKT4+/OKT8+ cells reflected the kinetics of recovery of the helper over the suppressor cell population.     

Cellular infiltrate in renal graft rejection: T lymphocyte subsets detected by monoclonal antibodies

We have examined the interstitial cellular infiltrate using monoclonal antibodies against T cells (Cris 1), helper/inducer T cells (OKT4) and suppressor/cytotoxic T cells (OKT8) by indirect immunofluorescence in renal biopsies taken from 14 transplanted patients during clinical episodes suggestive of acute (n = 9), chronic (n = 2) and no rejection (n = 3). Infiltrating T cells and T cell subsets were found to be significantly increased during all types of rejection (n = 11) as compared to no rejection (n = 3). Two types of biopsies could be distinguished according to the predominance of T cell subsets. In some biopsies (n = 6), OKT8+ cells were significantly more numerous that OKT4+ cells. In the remaining biopsies (n = 5), OKT4+ cells were more common that OKT8+ cells, the OKT4/OKT8 ratio being significantly higher. No association was observed between HLA mismatch and predominating T cell subset, neither for type nor outcome of graft rejection. Our results suggest that the OKT4+ cells may play a more important role than previously reported in renal graft rejection.     

T lymphocyte subsets and antilymphocyte antibodies in lupus nephritis

T lymphocyte subsets were determined in 12 patients with untreated systemic lupus erythematosus (SLE) and in 14 healthy controls. Six out of 8 (75%) patients with lupus nephritis had reduction in the percentage of T helper cells and low helper: suppressor cell ratios compared with controls. None of the 4 patients without nephritis had low ratios. Cold-reactive anti-lymphocyte antibodies cytotoxic to both the helper and the suppressor cells were detected in 7 of the 8 patients who had nephritis. Low T helper: suppressor cell ratio in SLE seems to correlate with the presence of active nephritis.     

Down regulation of CD45 expression on CD4 T cells during acute renal allograft rejection: Evidence of a decline in T suppressor/inducer activity

Acute rejection is associated with the activation of helper and cytotoxic cells. A shifting balance between the suppressor/inducer CD45+ CD4+ and T helper/inducer (CD4+CD45–) cells may be responsible for the transition from quiescence to overt rejection. We examined the kinetics of CD45 expression on CD4+ T cells in renal allograft recipients from pretransplant values to acute rejection and after reversal of rejection, searching for a shift in balance between helper/inducer and suppressor/inducer cell subsets. Using two color flow cytometry, the peripheral blood levels of CD4+, CD4+CD45– [T helper/inducer (Thi)], CD4+CD45+ [T suppressor/inducer (Tsi)], CD3+, and CD8+ T cells subsets and their interrelationships, were determined in 49 patients prior to transplantation, and in 10 of them, during acute rejection and after its reversal. Results were analyzed and compared to data obtained from 10 healthy blood donors. Acute rejection was associated with a significant decline in CD45+ CD4+ expression compared to quiescent phase (22% ± 3.7%vs. 26.5% ± 3.2%, p = 0.05) and controls (29.5% ± 6.2%, p = 0.01). No difference was observed compared to pretransplant levels (19.9% ± 3.2%, p = ns). CD45–/CD45+ (Thi/Tsi) ratio was lowest during quiescence (0.75) compared to rejection (0.97, p = 0.05), in controls (0.98, p = 0.05) and pretransplant values (1.4, p = 0.01). Acute rejection was characterized by higher Thi/CD8+ and lower Tsi/CD8+ ratio (103 and 88 respectively, p = 0.045), compared to clinical quiescence (104 and 116 respectively, p = 0.039). These data suggest that acute rejection is associated with down regulation of CD4+CD45+ suppressor/inducer subset. This shift may account for the transition from quiescence to overt rejection, concurring with reports on CD4+CD45 regulatory function.     

Alterations in T-lymphocyte subpopulations in type I diabetes. Exploration of genetic influence in identical twins

To evaluate factors influencing the alteration in subsets of T-lymphocytes, we studied 24 pairs of identical twins discordant for insulin-dependent (type I) diabetes mellitus. Subsets were assessed by monoclonal antibodies and a pure preparation of peripheral blood mononuclear cells obtained by centrifugation of heparinized whole blood with a Ficoll/Triosil gradient. In 12 pairs studied within 5 yr of diagnosis, we observed a reduction in the percentage of cells reacting with OKT8 (recognizing the CD8 antigen present on the suppressor/cytotoxic subset) (P less than .05), but a similar level was detected in their nondiabetic cotwins. In 12 pairs studied greater than 5 yr after the diagnosis and in whom the nondiabetic twin is less likely to develop diabetes, the percentage of cells reacting with OKT8 was reduced in both the diabetic (P less than .05) and the nondiabetic (P less than .01) twins. Reductions were also seen with OKT3 (recognizing the CD3 antigen present on the total T-lymphocyte population) and OKT4 (recognizing the CD4 antigen present on the helper/inducer subset), but only in the diabetic twins from the group with longer discordance. We conclude that a reduced percentage of suppressor/cytotoxic cells is associated with type I diabetes, but the reduction appears to be genetically determined. Total T-lymphocytes are also reduced but mainly in the helper/inducer subset and only in diabetic patients of long duration. Such a reduction cannot therefore be primarily genetically determined.     

Analysis of murine lymphocyte subpopulations by dual-color flow cytometry: technical considerations and specificities of monoclonal antibodies directed against surface markers

We performed detailed phenotypic analysis of murine lymphocytes from thymus, spleen, lymph node, and peripheral blood using commercially available monoclonal antibodies, each with specificities for membrane surface markers and dual-color flow cytometry. Erythrocyte lysis techniques were utilized for lymphocyte preparation so that inherent difficulties with gradient techniques would be avoided, such as the potential for loss of abnormally sized cells. These studies demonstrated that the specificities of each monoclonal must be carefully determined; for example, the Lyt-1 monoclonal, frequently utilized to identify helper/inducer T cells, also reacts with suppressor/cytotoxic (Lyt-2+) cells; helper/inducer cells are better studied with a more recently available monoclonal, L3T4. Cells from different tissues may differ greatly not only in the presence of surface markers, but also in the surface density of each marker; this density can be studied and quantitated using appropriate analytic software. We also show that larger and more granular lymphocytes appear to be enriched for surface Ia antigen, indicating that these cells may be activated or regulatory subsets; these large, Ia+ T-cells will be lost from analysis if standard, narrow gate settings are used for analyzing forward and side-scatter characteristics or for cell sorting.     

The effect of in vivo T helper and T suppressor lymphocyte depletion on wound healing.

The role of T lymphocytes in wound healing is still not well-defined. Because it had been previously shown that in vivo depletion of T cells leads to impaired wound healing, the effect of depleting T cell subsets on subsequent fibroplasia was studied. T helper/effector cells were depleted by the use of the monoclonal antibody GK1.5, reactive against the L3T4 antigen (CD4). T suppressor/cytotoxic lymphocytes were depleted by using the 2.43 monoclonal antibody reactive against the Lyt 2 antigen (CD8). In the first experiment, Balb/c mice were treated with the antibodies starting at 24 hours before wounding was performed, and weekly thereafter. Depletion of the T helper/effector cells had no effect on wound-breaking strength or hydroxyproline deposition in sponge granulomas, whereas depletion of T suppressor/cytotoxic cells significantly enhanced both of these healing parameters. In a second experiment, T cell subset depletion was started on Days 0, 3, 7, 10, and 14 postwounding, and treatments were continued weekly thereafter. Once again, depletion of T helper/effector cells had no effect on wound healing, whereas depletion of T suppressor/cytotoxic cells markedly increased both wound-breaking strength and collagen synthesis. In conclusion, the data show that T suppressor/cytotoxic cells have a counter-regulatory role in wound healing, whereas the T cell subset responsible for up-regulating wound healing remains to be identified.     

Analysis of tissue lymphocytes by double fluorescent staining--gastric cancer tissue and regional lymph nodes

An immunohistochemical study was performed on human lymphocytes in the tissue of gastric cancer, and also in the regional lymph nodes, by double fluorescent staining, using monoclonal antibodies. Leu3a+8+ cells (inducer T cells) which consist about 30 per cent of Leu 3a+ cells were seen in the tissue surrounding the gastric cancer. The other 70 per cent of Leu 3a+ cells were Leu3a+8- cells (helper T cells). In the lymph nodes they were noted in T cell areas in almost the same proportions, while in germinal centers, only Leu3a+8- cells were found. On the other hand, OKT8+Leu15- cells (cytotoxic T cells) were noted in a large number, while OKT8+Leu15+ cells (suppressor T cells) were few. Further, an increase of OKT8+Leu15- cells was seen around gastric cancer or metastatic cancer in lymph nodes. These immunohistochemical findings suggest that cytotoxic T cells are the main component in the tissue of gastric cancers and the regional lymph nodes. Increases in inducer T cells and helper T cells are probably required to induce cytotoxic T cells around the cancer tissue.     

A clinical study of immunological status in gastric cancer patients--with special reference to T-cell subsets in the lymphocytes of regional lymph nodes

Non-metastatic regional lymph node lymphocytes of 41 patients with gastric cancer were studied by using different monoclonal antibodies and flow cytometry. Used monoclonal antibodies were OKT3 (total T; CD3), OKT4 (helper/inducer T; CD4), OKT8 (suppressor/cytotoxic T; CD8) and Leu11 (NK/K cell; CD16). The results were as follows: 1. The percentage of CD3 cells and CD4 cells were about ten point fewer in lymph nodes than in peripheral blood. 2. CD8 cells were found to be one half or one third lesser in lymph nodes than in peripheral blood. 3. CD16 cells were found to be rare in lymph nodes. 4. The percentage of CD3, CD4 and CD8 cells were higher in distal lymph nodes than proximal ones. 5. The percentage of CD3, CD4 and CD8 cells were not different with progression of the cancer, whereas CD3 cells and CD8 cells were decreased in lymph nodes of stage IV. 6. The percentage of CD8 cells was higher in distal nodes of stage III. Regional lymph nodes are necessary to protect against cancer metastasis, and killer T cells and cytotoxic T cells were fewer in lymph nodes. These results suggested that killer activity and cytotoxicity of the lymph node lymphocytes are inactive and anergy.     

Inflammatory infiltrate in prostatic hyperplasia--evidence of a host response to intraprostatic spermatozoa?

The inflammatory infiltrate was characterised in 20 samples of prostatic hyperplasia using cell surface specific monoclonal antibodies. The infiltrate was composed predominantly of CD-3+ T-lymphocytes. The distribution of the T-lymphocyte subsets (CD-4 and CD-8) between the epithelial and stromal components of the gland was significantly different, with most of the intra-epithelial infiltrate being CD8+, T cytotoxic/suppressor cells. This may be a necessary immunological barrier, as in the epididymis, to prevent the development of autoimmunisation to sperm antigens. Large numbers of CD4+ T helper/inducer cells and significant numbers of CD-11c+ macrophages were demonstrated in the extra-glandular stromal tissues, suggesting a delayed type hypersensitivity reaction. Intraprostatic spermatozoa have been demonstrated recently and we have confirmed this finding on post mortem specimens. It is postulated that the presence of an intra-epithelial cytotoxic barrier and the marked stromal infiltrate may be due, in part, to the intermittent presence of spermatozoa within the prostate.     

小儿肾母细胞瘤患者细胞免疫状况与生物学特征的关系

应用单克隆抗体碱性磷酸酶抗碱性磷酸酶法（ＡＰＡＡＰ法）检测３５例小儿Ｗｉｌｍｓ瘤患者外周血总Ｔ细胞（ＣＤ３＋）、Ｔ辅助／诱导细胞（ＣＤ４＋）、Ｔ抑制／杀伤细胞（ＣＤ８＋）。按ＮＷＴＳ标准，对患者进行临床分期、组织学分型、组织分化程度等病理学分类。把患者的免疫状况与其形态学特征联系分析，结果显示：晚期肿瘤、胚细胞型、组织分化差的不顺利型细胞免疫抑制明显较重（Ｐ＜０．００１），预后也明显较差。提示小儿Ｗｉｌｍ瘤细胞免疫状况与其生物学特征有一定内在关系。     

Density of lymphocytic infiltration of primary breast cancer does not affect short-term disease-free interval or survival

Lymphocytic infiltration of breast cancer is often associated with a favourable prognosis. Seventy-seven women with operable breast cancer were followed for a minimum of 3 years. Tumours were frozen and sectioned by cryostat before staining with monoclonal antibodies using an immunoperoxide technique for total lymphocytes, helper/inducer, suppressor/cytotoxic, natural killer and B subsets. Lymphocyte density was assessed by light microscopy at x400 and divided at the 50th percentile to less than 30 and greater than or equal to 30 cells per high power field to give scanty and dense lymphocytic infiltration. The helper/suppressor lymphocyte ratio was greater than 1 in 45 patients but without improvement in survival or cancer recurrence. Natural killer and B lymphocytes were rarely seen in tumour sections. Poorly differentiated tumours excited a more marked lymphocytic infiltration (P less than 0.01). Neither the density of lymphocyte infiltration nor the ratio of helper to suppressor lymphocytes correlated with improved short-term survival or recurrence. These data suggest that the immune defence is ineffective in preventing spread from the primary tumour in breast cancer patients.