Antibodies to the T3 antigen complex on human T cells render thymocytes unresponsive to mitogen.

Human thymocytes differed from peripheral blood T cells, in that they did not proliferate in response to mouse monoclonal antibodies specific for the T3 antigen complex (UCHT1 and OKT3), even in the presence of T-cell growth factor (IL-2) or with monocytes plus IL-2. The failure to respond was not the result of inhibition by cortical thymocytes, since OKT6-negative cells also did not respond when tested under conditions of limiting dilution. Of more importance, these antibodies rendered human thymocytes unresponsive to the lectin phytohaemagglutinin when added before, during, or immediately after addition of the lectin, an effect which was much more profound than the decrease in mitogenesis caused with blood mononuclear cells. These results illustrate a clear difference between medullary thymocytes and peripheral T cells in their ability to respond to signals transduced through the T3 antigen complex.     

Immunologic unresponsiveness in leprosy is mediated by modulation of E-receptor

By using an indirect immunofluorescence technique with OKT3 and OKT11 monoclonal antibodies, the percentage of CD2 positive cells was found to be reduced in the peripheral blood of bacterial index positive lepromatous leprosy patients; however, in these patients, CD3 positive cells were at the normal level. Further CD2 positive cells attained the normal proportion in lepromatous patients when mycobacterial load was reduced. Both CD2 and CD3 receptors were expressed at the normal level in tuberculoid leprosy patients. Prior treatment of peripheral blood mononuclear cells from healthy controls with Mycobacterium leprae significantly decreased the percentage of CD2 but not CD3 positive cells. Such a modulation of CD2 on T cells also resulted in blocking the lymphoproliferative response induced by mitogen and antigen. These results suggest that there is a strong correlation between CD2 modulation and immunologic unresponsiveness in leprosy.     

WUT6-抗人胸腺细胞共同性分化抗原来交瘤细胞系的建立

本文报导了用人胎儿胸腺细胞免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞系NS-1融合,获得一株能稳定分泌抗人共同性胸腺细胞单克隆抗体的杂交瘤细胞株,W_(?)T_(?)。它与72％胸腺细胞、皮肤Langerhan's细胞及部分T细胞系呈阳性反应;外周血T、D细胞、粒细胞、红细胞、血小板及B细胞系、Null细胞系均阴性。FACS检测结果及W_(?)T_(?)阳性细胞的组织分布特点均同HIT_6相似。在胸腺、皮质细胞及髓质中少数胞体较大呈树突状的细胞呈阳性反应。在扁桃体实质中少数散在细胞阳性。上皮基底层中的Langerhan's细胞则呈强阳性反应。用W_(?)T_(?)亲和层析柱提取胸腺细胞膜上相应的靶抗原分子,证明W_(?)T_(?)识别分子量为51KDa的抗原。免疫竞争抑制试验表明,W_(?)T_(?)同抗原的结合可被HIT_6完全阻断。说明W_(?)T_(?)与HIT_6识别相同的抗原决定簇。     

Concanavalin A Induction of Suppressor Activity in the T-Helper Subset Defined by Monoclonal Antibodies

Human monocyte-depleted peripheral blood mononuclear cells were separated in T4+ and T8+ populations by a panning technique. Petri dishes coated with goat anti-mouse antibodies were plated by peripheral blood mononuclear cells coated by monoclonal antibodies, either T4 or T8. The cell populations were separated into adherent and non-adherent populations based on binding to the goat anti-mouse-coated plastic dishes. The purity of the adherent populations was 96%. T4+ and T8+ populations were used as effector cells in the concanavalin A-induced suppressor test. The T4+ population revealed a pronounced suppressor activity similar to that exhibited by the T8+ population. This finding was independent of two different sources of monoclonal antibodies, T4/T8 and OKT4/OKT8. The registered suppressor activity in the monoclonal antibody-defined helper population could not be explained either by a switch of the membrane phenotype from T4+ to T8+ cells or by an increased interleukin 2 consumption of the concanavalin A-treated cells.     

Pharmacological modification of immunoregulatory T lymphocytes . II. Modulation of T lymphocyte cell surface characteristics.

Human peripheral blood lymphocytes were fractionated into non-T lymphocyte, T lymphocyte, theophylline resistant (TR) and theophylline sensitive (Ts) T lymphocyte subpopulations. The proportion of cells bearing surface membrane immunoglobulin (sIg), Clq, Ia antigen, beta 2 microglobulin and T lymphocyte specific antigens detected by monoclonal antibodies OKT3, OKT4, OKT5 and OKT8 was studied using immunofluorescent techniques. Incubation of T lymphocytes or TR lymphocytes with adenosine or impromidine, an H2 histamine agonist, under conditions previously shown to increase Fc gamma receptors and radioresistant suppressor cell activity, was found to increase the proportion of cells expressing readily detectable surface beta 2 microglobulin and the antigen detected by OKT8. Cells expressing OKT4 antigen declined and there was no change in OKT3, OKT5, Ia, Clq, sIg or Es receptor expression. These data indicate that the expression of T lymphocyte Fc gamma receptors, beta 2 microglobulin and the antigens detected by the monoclonal antibodies OKT4 and OKT8 are, at least in part, regulated by agents acting upon adenosine and H2 histamine receptors.     

Helper T cell activation for the human B cell response to trinitrophenylated polyacrylamide beads: involvement of the T4 antigen

The monoclonal antibody (mAb) OKT4A (but not OKT4) inhibits the in vitro antibody response of human peripheral blood mononuclear cells. The target of OKT4A mAb is the helper T cell, as the helper cells for the antibody response to trinitrophenylated polyacrylamide beads (TNP-PAA) are exclusively in the T4 subset. The OKT4A mAb is still suppressive when the anti-TNP response of cultures of monocyte-depleted cells is supported by purified interleukin 1. Both the OKT4A mAb and anti-DR mAb suppress the non-specific T cell proliferation in the cultures leading to the in vitro mAb response. A parallel inhibition is observed for the autologous mixed lymphocyte reaction, the nonspecific B cell response, but the T cell response to mitogens is not affected. These results suggest that the recognition of self major histocompatibility complex class II determinants by the T4 molecule plays a major role in the activation of T helper cells for antibody production to this particulate antigen.     

Detection of CD1 positive cells in the peripheral blood of patients suffering from cancer-associated malnutrition

The immunological phenotyping of peripheral blood mononuclear cells (PBMC) was assayed in a series of 22 patients suffering from severe cancer-associated malnutrition. A marked decrease of the T-lymphocyte subsets (CD3+, CD4+, CD8+) and of the CD20+ B lymphocytes occurred; there was however an increased percentage of monocytes but their absolute number was normal. Interestingly, 5% of the PBMC expressed "activated T-cell antigens". The specificity of two different monoclonal antibodies (MoAbs) towards CD1 epitopes (OKT6 and D47) was assessed by indirect immunofluorescence (IIF): about 5% of CD1+ thymocytes were detected with no antigen (Ag) cross reactivity with the small subset of activated T cells. It is hypothesized that some relationship may exist between such cells and malnutrition and/or cancer.     

In vitro immune cell function in six cases of immunoproliferative small intestinal disease after long term remission

We have studied several parameters of in vitro immune cell function in peripheral blood mononuclear cells from six patients with immunoproliferative small intestinal disease after long term remission. We have observed two groups of patients with different patterns of response. (a) After stimulation with pokeweed mitogen (PWM) and Staphylococcus aureus, three patients showed a significant reduction of the Ig synthesis (A, G and M) and the proliferative response. In two of them, we found increased spontaneous suppressor T cell activity. In the third case, the diminished response could not be attributed, according to our assays, either to suppressor T cells, lack of T helper activity (although the number of OKT4+ cells was diminished) or an intrinsic B cell defect. The three patients showed normal or augmented NK activity and an inversion of the OKT4+/OKT8+ ratio was detected in two of them. (b) The remaining three patients showed a normal Ig synthesis after stimulation with PWM and a slightly depressed IgM synthesis in response to S. aureus. They expressed a normal T helper cell function and did not show increased spontaneous suppressor T cell activity. They had low levels of natural killer cytotoxicity and the OKT4+/OKT8+ ratio was not significantly altered. Taken together, our data indicate that significant alterations of the in vitro immune response can be found in peripheral blood mononuclear cells of some immunoproliferative small intestinal disease patients after long term remission.     

Characterization of the Blood Mononuclear Leucocytes Producing Alpha Interferon after Stimulation with Herpes Simplex Virus in Vitro, by Means of Combined Immunohistochemical Staining and in Situ RNA-RNA Hybridization

A procedure is described for combined immunohistochemical staining and in situ RNA-RNA hybridization of human peripheral blood mononuclear leucocytes (PBMC). These cells were first stimulated in vitro by herpes simplex virus (HSV)-infected fibroblasts and after 6 h fixed and stained with a panel of antibodies against differentiation antigens. Alpha interferon (IFN-alpha)-producing cells (IPC) were identified by in situ hybridization by means of a 35S-labelled IFN-alpha 2 cRNA probe. The IPC were infrequent, one in 200-5000 PBMC, but heavily labelled with the cRNA probe. They lacked antigens typical of T and B lymphocytes, and were also essentially negative for the Leu-M5 antigen, present on a majority of monocytes. However, 50% of IPC expressed OKM5 antigens, corresponding to the thrombospondin receptor. The IPC lacked the antigens present on null lymphocytes detected by OKT16, but most of them expressed HLA-DR, -DP and -DQ antigens. The IPC may represent a small subpopulation in the monocyte/macrophage lineage, resembling cells described as antigen presenting and stimulators of autologous mixed lymphocyte reactions. Alternatively, they constitute a subpopulation among the null lymphocytes.     

T cell binding to B lymphoid cell lines in humans: a marker for T-B cell interaction?

Binding of human circulating T cells to established normal and malignant B cell lines results in rosette formation. The percentage of B cells, circulating T cells, and thymocytes able to bind to the B-LCL Raji were 0%, 59 +/- 4% and 61 +/- 6%, respectively. The percentage of rosettes formed between Raji cells and circulating mononuclear cells from 92 normal individuals was 27.8 +/- 5.3%, and remained stable over several months. This phenomenon seems to involve relatively mature B cells, and a T cell marker which appears early in T cell ontogeny. In the peripheral blood, most of the B-LCL binding T cells exhibit a 'helper-inducer' phenotype, as determined with the monoclonal antibodies Leu 3a and OKT4. However, a significant percentage of T cells with so-called 'cytotoxic-suppressor' markers (Leu 2a and OKT8) also bind to B-LCL. The T cells involved in this morphological interactive reaction with B cells might conceivably be specifically involved in regulating B cell functions. Enumeration of this particular subset may be useful in conditions where abnormal T-B cell interactions are suspected.     

Monocyte-regulated Hyporesponsiveness of Human Cord Blood Lymphocytes to OKT3-monoclonal-antibody-induced Mitogenesis

OKT3 monoclonal antibody recognizes surface antigenic structures present on all human mature T lymphocytes and is mitogenic for resting peripheral T cells. Recent reports suggest that these structures are linked to the specific antigen receptor of the T cells and play an important role in T-cell activation. We have tested the mitogenic action of OKT3 on resting lymphocytes from human newborns, their mothers, and unrelated adults. We found that the proliferative response of cord T cells to OKT3 is significantly lower than the response of maternal and adult cells at all doses of the antibody tested (5-1000 ng/ml). This difference was not dependent on culture conditions (source of serum, kinetics induced by the OKT3 antibody, or different proportions of adherent cells in peripheral blood mononuclear leukocytes), and could only to some extent be accounted for by differences in the proportions of OKT3-binding cells between these populations. Removal of adherent monocytes largely diminished the OKT3-induced proliferation of maternal and adult cells, by an average of 70-80%. In contrast, it significantly enhanced the proliferation of cord cells. The proliferative response of cord T lymphocytes to the two polyclonal T-cell activators phytohaemagglutinin and concanavalin A was similar to or greater than that of mothers and other adults.     

Isolation of T lymphocyte subsets from peripheral blood using monoclonal antilymphocyte antibodies

Monoclonal antisera against helper (serum OKT4) and suppressor (serum OKT8) T cells were employed in a complement-dependent lymphocytotoxicity reaction to isolate helper and suppressor T cells from peripheral blood mononuclear cells. When prepared by this method, helper and suppressor T cells had a purity of 67.8 percent and 78.8 percent, respectively. Approximately 56 percent of viable suppressor and 33 percent of viable helper T cells were lost during the procedure.     

Human in vivo antigenic modulation induced by the anti-T cell OKT3 monoclonal antibody

The anti-pan T cell monoclonal antibody OKT3 was administered daily for 2 weeks in four human renal allograft recipients. The antibody induced a dramatic and immediate depletion of peripheral T cells followed by an in vivo antigenic modulation of the OKT3-defined membrane antigen: after three injections, OKT3-treated patients showed a limited but significant number of OKT3- cells of T cell nature (as defined by OKT4 and OKT8) which recovered the OKT3 receptor after an overnight in vitro incubation in the absence of the monoclonal antibody.     

Lymphocyte subpopulations in chronic Chagas' disease

Monoclonal antibodies of the Orthoclone series were used to identify total T lymphocytes (OKT3) and their helper-inducer (OKT4) and cytotoxic-suppressor (OKT8) subsets in 25 patients with chronic Chagas' disease and 25 healthy controls. No significant difference in the number of total T cells (OKT3+) circulating in the peripheral blood of patients and controls was found. However, in contrast to normal subjects, chagasic patients show quantitative alterations in both helper (OKT4+) and suppressor (OKT8+) T cell subsets. The chagasic patients have abnormal helper/suppressor ratios, with low and high values scattered around the mean. Surprisingly, high ratios were found within females while almost all males have low ratio values. These findings suggest that the putative immunoregulatory disfunctions in patients with chronic chagasic cardiomyopathy may involve both helper and suppressor T cell subsets.     

Selection and continuous growth of antigen-specific human T cells by antigen-treated monocytes

Antigen-specific human T lymphocytes were selected by adsorption of peripheral blood mononuclear cells on monolayers of antigen-treated autologous monocytes. The enriched cell populations were propagated in interleukin 2-containing medium for 60-80 days. Their activity was tested by a proliferative response against the sensitizing antigen in the presence of irradiated autologous lymphocytes. Continuous cultures of enriched T cells responded specifically to three different protein antigens. The specific reactivity induced in vitro against at least one of the antigens was probably a primary response. In addition, the selected cultures were found to be enriched for OKT4-positive cells, in contrast to cultures of nonselected T cells which were enriched for OKT8-positive cells. These properties of the selected T lymphocyte populations were obtained following only one exposure to the sensitizing antigen, which was concomitant with adsorbance on monocytes and without cloning of the reactive cells. We suggest that specific adsorbance of lymphocytes on antigen-treated monocytes may result in a T cell population which combines both properties of antigen specificity and enrichment for the helper proliferative subset of T cells.     

Specific inhibition of OKT8 binding to peripheral blood mononuclear cells by jacalin

Human T-specific monoclonal antibodies were used to study the interactions between the binding of jacalin to peripheral blood mononuclear cells (PBMC) and the immunoregulatory molecules displayed at the surface of T cells. Jacalin inhibits the binding of OKT8 (anti-CD8) to both fresh PBMC and jacalin-induced T cell blasts. In both cases the binding of anti-CD3 (OKT3) or anti-CD4 (OKT4) was not affected by the lectin. The effect of jacalin on OKT8 binding is abolished by 1-O-alpha-D-methylgalactopyranoside, suggesting its mediation by the lectin saccharide combining sites. Preincubation experiments indicated that the inhibitory effect of jacalin is due to a competition between the lectin and the monoclonal antibody. The effect of the lectin could also be reversed by increasing concentrations of the monoclonal antibody. Taken together this data demonstrates a specific inhibition of OKT8 (anti-CD8) binding by jacalin. This effect is mediated by the binding of the lectin to structures on the cell surface, perhaps the CD8 antigen. The data also points to the discovery of a new mitogen that could be useful for studying the physiological role of CD8 on T cell responses.     

Immunological parameters in the aged and in Alzheimer's disease.

Peripheral blood from patients with Alzheimer's disease, elderly normal subjects and young (normal subjects) was examined with respect to leucocyte phenotypes and proliferative responses to lectins. Whole blood cell analysis showed that the neutrophil count was similar in all three groups. However, the lymphocyte count was depressed in the Alzheimer group. The monocyte count was reduced in the healthy aged and further reduced in the Alzheimer group. Analysis of peripheral blood mononuclear cells showed no change in the proportion of E+ cells, total T determined with the monoclonal antibody UCHT1 (similar to OKT3) and the T cell subsets: active E and T dot (discrete esterase staining). However, the proportion of T cells bearing the antigen detected by UCHT3 monoclonal antibody (similar to Leu 1 and OKT1) was reduced in the healthy aged and further reduced in the Alzheimer group. Proliferative responses to PHA, Con A, PA, and PWM were similarly depressed in both the aged and Alzheimer groups.     

Heterogeneity of immunological abnormalities in ataxia-telangiectasia

Peripheral blood mononuclear cells from five patients with ataxia-telangiectasia were evaluated for their reactivity with a panel of monoclonal antibodies directed against T-cell subsets and for theirin vitro functions in a pokeweed mitogen-induced immunoglobulin biosynthesis assay. All the patients had significantly reduced proportions of cells identified by monoclonal antibodies to subpopulations of T lymphocytes with helper activity (OKT4 and 5/9) and produced low amounts or no IgA and IgGin vitro. Immunoglobulin biosynthesis was increased by the addition of normal x-irradiated peripheral blood mononuclear cells in one of three patients, suggesting a helper T-cell deficiency in this patient and intrinsic B-cell defects in the other two. Two patients had increased proportions of cells identified by a monoclonal antibody to a subpopulation of T lymphocytes which includes suppressor T cells (OKT8), and their cells were able to suppress immunoglobulin biosynthesis by peripheral blood mononuclear cells from normal donors. These findings indicate heterogeneous disturbances of immunoregulatory mechanisms in ataxia-telangiectasia.     

Identification of an Anti-Human Osteogenic Sarcoma Monoclonal-Antibody-Defined Antigen on Mitogen-Stimulated Peripheral Blood Mononuclear Cells

An anti-human osteogenic sarcoma monoclonal antibody (mouse IgG2b) termed 791T/36 was found to exert complement-dependent cytotoxicity against phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear (PBMN) cells. This reaction was examined by flow cytofluorimetry using indirect membrane immunofluorescence to detect cell-bound antibody and by measurement of the binding of fluorescein-isothiocyanate-conjugated 791T/36 antibody to cells. The antibody reacted strongly with peripheral blood blast cells induced by PHA, and there was negligible reactivity with resting lymphocytes. Maximum binding was observed after 3 days' culture with PHA, coinciding with maximum DNA synthesis, and this represented of the order of 2 X 10(5) antibody molecules bound per cell. After cell surface radioiodination of PHA-stimulated PBMN cells, detergent lysis and immunoprecipitation of antigen with 791T/36 antibody and Sepharose-protein A, the apparent molecular weight of this antigen was determined to be 72,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This is identical to that of the 791T/36-defined antigen expressed on various osteogenic sarcoma cell lines [3, 17], and by this criterion the antigen is distinguishable from other cell surface markers of activated human lymphocytes.     

Identification of lymphocyte subsets in peripheral blood and thyroid gland in Graves' disease by alpha-naphthyl-acetate-esterase reaction

T lymphocyte subsets were prospectively examined in the peripheral blood and thyroid aspirates of 10 patients with hyperthyroid Graves' disease before and after treatment with methimazole and attainment of euthyroidism. T lymphocyte subsets were identified with monoclonal antibodies and pattern of alpha-naphthyl-acetate esterase (ANAE) staining pattern in the case of peripheral blood and ANAE staining pattern with thyroid aspirate smears. Before treatment, OKT8+ lymphocytes were significantly decreased (18.4% +/- 4.8) (S. D.) in the patients compared to control (28.8 +/- 6.7%, p less than 0.05), the OKT4/OKT8 ratio was increased (2.92 vs 2.11). Percent OKT8+ lymphocytes were not different from the controls when the ten patients had been rendered euthyroid. ANAE mononuclear cells with a diffuse pattern (presumed suppressor cells) were 4.2% +/- 1.8 before treatment and 8.3 +/- 2.4 (p less than 0.05) after treatment and 11.5% +/- 2.2 in controls. ANAE mononuclear cells with diffuse pattern represented 4.2% +/- 1.8 of the mononuclear cells infiltrating the thyroid gland of untreated patients and rose to 8.3% +/- 2.4 after the patients had become euthyroid. ANAE negative cells (B cells and some T cells) were increased in the thyroid of untreated patients. It is concluded that mononuclear cells with presumed suppressor T cell phenotype are decreased in the blood and thyroid glands of patients with active Graves' disease and that this defect is corrected when euthyroidism has been established.