Direct visualization of T lymphocytes bearing Ia antigens controlled by the I-J subregion

Previous studies have demonstrated that I-J-subregion-controlled Ia antigens are only expressed on a small subpopulation of peripheral T lymphocytes which includes the suppressor T cells of antibody responses (6). This subpopulation of T cells cannot be detected by conventional dye-exclusion cytotoxicity tests. A sensitive rosetting procedure therefore was developed for detecting the binding of anti-Ia antibodies to T lymphocytes. This assay system, unlike the complement lysis technique, has a low background and since it represents a direct binding assay could detect noncomplement-fixing antibodies in the antisera. Anti-Ia sera were absorbed with B cells and using the rosetting procedure in genetic mapping studies the remaining antibodies were found to be directed against I-J-subregion-controlled determinants. These determinants were shown to be highly haplotype specific for H-2(k) and H-2(s) and appeared to be exclusively expressed on Ly-l.l(-), Ly2.1(+), T lymphocytes, at least some of which were suppressor T cells. Lymphoid organs differed in their content of anti-I-J-reactive cells, the hierarchy being spleen, lymph node more than thymus, bone marrow. In contrast, on a T-cell basis, a high proportion (35 percent) of the T cells in bone marrow reacted with anti-I-J antibodies, a substantial proportion (13 percent) of T cells from spleen were reactive, whereas the lymph node and thymus T-cell populations contained only a small proportion of positive cells (1-4 percent).     

Association between Ia antigens and the Fc receptors of certain T lymphocytes

The Fc receptors of thymic and splenic T lymphocytes were detected using indirect immunofluorescence and soluble antigen-antibody complexes. 10-20% of thymocytes and 40-50% of Thy-1-positive splenic lymphocytes bound antigen-complexed Ig. The binding to thymocytes was partially inhibited (45-74%) by antibodies against antigens determined by the I region of the H-2 complex, but not by antibodies against K- or D-region antigens or Thy-1 antigen. The inhibition did not require the Fc portion of the inhibiting antibody. These results provide evidence that Ia antigens and the Fc receptors of some T lymphocytes are associated, and that the populations of T cells which bear these moieties at least partially overlap.     

Direct T helper-B cell interactions induce an early B cell activation antigen

We have explored the consequences for the B cell of cognate interaction with T cells. Early expression of the B cell-restricted cell surface activation antigen, BLAST-2, has been used as an assay system to measure direct T-B cell collaboration. BLAST-2 is preferentially expressed by allogenic B cells cultured with MHC class II antigen-restricted Th clone cells matched to the DR specificity of the target B cells. B cells cultured with DR-mismatched allospecific Th cells express minimal BLAST-2. Th cell-induced BLAST-2 expression appears to be accessory cell independent and occurs as early as 8 h after initiation of culture, with peak expression at 18 h. Direct T-B cell contact, rather than Th-derived lymphokines, provides the most efficient stimulus for BLAST-2 expression. Crosslinking of sIg on B cells is a poor stimulus for BLAST-2 expression. The BLAST-2 assay permits the evaluation of early events associated with B cell activation through cognate interactions, and may facilitate subsequent studies of the mechanism of B cell differentiation.     

Cell interactions between histoincompatible T and B lymphocytes. VII. Cooperative responses between lymphocytes are controlled by genes in the I region of the H-2 complex

The results of this study provide compelling evidence for the existence of the gene or genes controlling optimal T-B-cell cooperative interactions in the designated I region of the H-2 gene complex. Previously, we have speculated that the relevant gene(s) involved may well be located in this region based on several observations from our earlier work in this area (3, 5, 6). Thus, in the preceding paper, we showed that T and B cells from B10.BR and A strain mice developed effective cooperative interactions in vitro to DNP-KLH in a system identical to the one reported herein. Since these mice differ for genes in the S and D regions of H-2 but are identical for K and I region genes, we were able to localize the critical genes to the K-end of H-2.     

Blocking and redistribution ("capping") of antigen receptors on T and B lymphocytes by anti-immunoglobulin antibody

Antigen-binding T and B lymphocytes were studied by combined autoradiography and immunofluorescence; mouse spleen lymphocytes binding the antigens, [¹²⁵I]MSH or [¹²⁵I]TIGAL, were incubated with rhodamine-labeled anti-Ig reagents or with a rhodamine-labeled IgG fraction of anti-θ serum. B cells were identified as Ig+ or θ-, T cells as Ig- or θ+. It was found that: (a) 20% (1–2 mo after priming) to 30% (3.5–4 mo after priming) of the antigen-binding cells were T cells. (b) The range of antigen molecules bound by B and T cells was similar. (c) Binding of antigen to B and T cells was inhibited by polyvalent anti-Ig, anti-µ, or anti-L reagents. Binding to T cells was more readily inhibited than to B cells. Normal rabbit serum, antimouse lymphocyte serum, or anti-θ did not inhibit antigen binding. (d) When Ig at the surface of B cells was induced, by noninhibiting concentrations of anti-Ig reagents, to redistribute into polar caps and the cells subsequently exposed to [¹²⁵I)antigen under noncapping conditions, the [¹²⁵I]antigen silver grains were distributed in caps superimposed on the Ig fluorescent cap. Of crucial importance, antigen was found in cap in the same proportion of T cells as B cells. Significant capping of antigen receptors was not induced in B or T cells with normal rabbit serum or by anti-Ig reagents absorbed with mouse Ig. The main conclusions of this series of experiments using direct visualization of antigen-binding B and T lymphocytes is that T cells have antigen-specific receptors, probably of IgM nature, and that the number of these receptors appears to range in the order of thousands.     

LDH isozymes of human T and B lymphocytes

Peripheral lymphocytes were obtained from five normal healthy adults. T and B lymphocytes were separated by rosette formation. LDH (L-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27) isozymes were studied on both populations after agar gel electrophoresis.

The mean B lymphocyte pattern was: LDH-1, 7.2%; LDH-2, 25.0%; LDH-3, 39.2%; LDH-4, 21.9%; LDH-5, 6.6%.

The mean T lymphocyte pattern was: LDH-1, 25.8%; LDH-2, 35.1%; LDH-3, 28.3%; LDH-4, 9.4%, LDH-5,1.3%.

This makes the T lymphocytes look more aerobic than the B lymphocytes.

Daudicells had the following pattern: LDH-1, 8.4%; LDH-2, 19.1%, LDH-3, 26.6%, LDH-4, 24.9%; LDH-5, 20.9%.     

Polyclonal stimulation of resting B lymphocytes by antigen-specific T lymphocytes

Resting B lymphocytes are activated, proliferate, and differentiate into antibody-secreting cells when cultured with long-term lines of major histocompatibility complex (MHC)-restricted, antigen-specific T cell in the presence of the antigen for which the T cells are specific. Under optimal conditions, essentially all B cells are activated and approximately 35% enter S phase in the absence of antigens for which the B cells are specific. Activation and proliferation are observed in cells from both normal mice and mice with the xid-determined immune defect. Highly purified B cells bearing Ia molecules for which the T cells are "cospecific" can present antigen to T cells with the resulting T cell stimulation leading to the activation and proliferation of the antigen-presenting B cells. However, B cells that do not bear Ia molecules for which the T cells are cospecific are also activated and proliferate if antigen and a source of antigen-presenting B cells or macrophage-rich cells of proper histocompatibility type are present. Thus, resting B cells, both normal and "xid", can be activated by non-MHC restricted factors without receptor cross-linkage. Experiments are presented that support the concept that local production and action of such unrestricted activating factors may be responsible for the MHC-restriction of T cell-B cell interaction seen in many circumstances.     

Hapten-specific IgE antibody responses in mice. II. Cooperative interactions between adoptively transferred T and B lymphocytes in the development of IgE response

The present studies have established conditions for the demonstration of cooperative interactions between specific T and B lymphocyte populations in the development of IgE antibody responses in vivo in mice. This has been accomplished by utilizing a system which permits the successful adoptive transfer to irradiated recipients of DNP-specific secondary IgE responses with spleen cells from suitably primed syngeneic donor mice. Thus, adoptively transferred DNP-KLH or DNP-ASC-primed spleen cells produced high levels of anti-DNP antibodies of both IgE and IgG antibody classes in response to challenge with the appropriate homologous priming conjugate but failed to develop more than meager responses to the reciprocal heterologous conjugate. However, when spleen cells from donors primed to the second carrier were concomitantly transferred with hapten-primed lymphocytes, secondary IgE ant-DNP responses were consistently obtained upon challenge with the heterologous conjugate. Moreover, we have been able to elicit augmented primary IgE anti-DNP antibody responses to either DNP-ASC or DNP-KLH after adoptive transfer of spleen cells from donors primed only to the carrier, ASC or KLH, respectively. This adoptive transfer system has enabled us to provide direct proof for the participation of θ-bearing T lymphocytes in antibody responses of the IgE class. Thus, the capacity of ASC-primed spleen cells to effectively cooperate with the DNP-KLH-primed lymphocytes in the adoptive secondary response to DNP-ASC could be abolished by in vitro treatment of such cells with anti-θ serum plus complement. This was true not only for the anti-DNP response of the IgG antibody class, but for the IgE antibody class as well. These studies have, furthermore, demonstrated the capacity to stimulate secondary anti-DNP antibody production in vivo by the concomitant administration of the DNP and relevant carrier determinants on separate molecules. This was more readily seen in the IgE than in the IgG antibody class. Thus, DNP-ASC-primed cells developed significant IgE, but more variable IgG, anti-DNP responses upon challenge with DNP-KLH plus unconjugated ASC. Antibody responses of both classes elicited in this manner were appreciably improved by the transfer of additional carrier (ASC)-primed cells. These and other results presented herein suggest that IgE B lymphocyte precursors may be inherently more sensitive than IgG B cells to at least certain of the functions of T lymphocytes concerned with regulatory mechanisms involved in antibody production.     

T lymphocytes possess receptors for brain myelin small protein

In immunomediated demyelinating diseases, T cells are found in chronic lesions. To discover whether immunocompetent cells may interact with some myelin proteins, we purified myelin proteins in the lipid-bound native state and evaluated their binding to peripheral blood mononuclear cells (PMBC) isolated from healthy donors. To this end, myelin proteins were conjugated to biotin and added to PBMCs or purified CD4+ and CD8+ cells; then binding was detected using fluoresceinated avidin. In this article, we describe experiments carried out with a myelin protein recently discovered in the central nervous system. Our results show that this small, phosphatidylserine-binding protein can bind to human T cells.     

慢性乙型肝炎虚实病机与病毒复制及T细胞关系的研究

目的：通过对虚实分类的慢性乙型肝炎(乙肝)患者进行乙肝病毒DNA(HBV—DNA)定量、T淋巴细胞亚群检测分析，探讨中医虚实病机与现代医学免疫发病机制之间的关系。方法：将HBeAg阳性的慢性乙肝轻、中度的患者分为虚证组、实证组、虚实夹杂组，检测上述指标并对各组进行比较分析。结果：与正常对照组比较，各组慢性乙肝患者CD4^ 水平明显下降，CD4^ /CD8^ 存在明显差异。HBV—DNA定量实证组＞虚实兼夹组＞虚证组(P＜0．05)。CD4^ ／CD8^ 比值虚证组＞虚实兼夹组＞实证组(P＜0．05)。结论：慢性乙肝患者存在着免疫调节紊乱;HBV—DNA定量水平可作为实证的参考指标，CD4^ ／CD8^ 值可作为虚实变化的参考指标，CD8^ 可能是虚证的参考指标。     

Activation of T and B lymphocytes in NZB mice

Since NZB mice manifest evidence of intense B cell activation, the state of activation of their T cells was explored. Electron microscopic examination and automated laser light scatter analysis demonstrated the presence of many blast lymphocytes in the spleens of aging NZB mice but not in controls. Only 45% of these large cells were B cells by the criterion of stainable surface IgM. NZB T and B cells were separated using the fluorescence-activated cell sorter; cytographic analysis revealed that both T and B cell populations contained substantial numbers of blast cells. The data thus indicated that T cells, as well as B cells, undergo activation as autoimmunity develops in NZB mice.     

Antigen heterogeneity of human B and T lymphocytes.

Rhesus monkeys were immunized with normal human lymphoid cells, cultured lymphoid cells, and chronic leukemic lymphocytes. Antisera were analyzed by cytotoxicity and immunofluorescence techniques to study the antigenic characteristics of human lymphocytes. In an attempt to obtain a reagent specifically reactive with T (thymus-derived) lymphocytes, an antispleen antiserum was absorbed with cellf from five B- (bone marrow-derived) cell lines. After absorption, the antiserum killed 60-75% of peripheral blood lymphocytes and 40-50% of tonsil cells, so that there was a relationship between the percentage of killed cells and the proportion of T lymphocytes. However, when cells after cytotoxic treatment were assayed for rosette formation with sheep erythrocytes (a T-cell marker) 5-20% of viable rosette-forming lymphocytes were found. Therefore, this antiserum was cytotoxic for only 75-90% of T cells. From studies performed with antisera prepared against spleen and B-cell lines, we conclude that lymphoblastoid cells are antigenically different and deficient in comparison to normal B lymphocytes. In addition, cultured B-cell lines appear to be antigenically heterogenous, as shown by the cytotoxic activity remaining in antispleen and anti-B-cell lines sera after absorption with various numbers and types of lymphoid cell lines. After absorption with normal lymphocytes, an antiserum produced against chronic lymphatic leukemia cells had specific activity associated with 12 chronic lymphatic leukemia cells tested. Absorption of the same antiserum with leukemic cells from two patients showed that a certain degree of antigenic heterogeneity also exists among chronic leukemic lymphocytes.     

Antigen-induced aggregation and modulation of receptors on hapten- specific B lymphocytes

Mouse spleen cells were subjected to a fractionation procedure designed to enrich for 4-hydroxy-3-iodo-5-nitro-phenylacetyl (NIP)- or DNP-specific B lymphocytes, which depended on adherence of specific cells to a layer of hapten-gelatin at 4 degrees C, recovery of bound cells by melting, and digestion of adherent antigen by collagenase. A population of cells resulted which contained 90% typical B cells and 37% of cells capable of binding a fluorescent, haptenated polymeric protein. Fractionated cells were reacted in vitro with fluorescent conjugates of the specific haptens with polymerized flagellin [NIP-polymerized flagellin (POL)-tetramethylrhodamine isothiocyanate conjugate or DNP-POL-fluorescein isothiocyanate conjugate] under a variety of conditions, with the aim of investigating the behavior of Ig receptors on B lymphocytes after exposure to antigen; Experiments were performed with immunogenic and tolerogenic concentrations of antigen. Furthermore, four experimental designs were used, namely: (a) brief labeling with fluorescent antigen followed by culture without antigen (pulse design); (b) culture in the continuous presence of fluorescent antigen (continuous-labeling design); (c) culture in the continuous presence of nonlabeled antigen followed by labeling of unoccupied receptors by fluorescent antigen (receptor status design); and (d) culture with nonlabeled antigen for 2 h followed by incubation without further antigen for 20 h and labeling with fluorescent antigen (modulation design). Further insight into receptor occupancy and distribution was gained by the use of fluorescent antihapten and antiglobulin reagents. It was found that both immunogenic and tolerogenic antigen concentrations caused rapid patching and capping of the receptors to which they attached, followed by endocytosis and probably some shedding of Ig receptors. However, a proportion of cells continued to bear some cell surface antigen for 24 h. The immunogenic antigen concentration failed to completely remove the receptor coat from the cell surface. At all stages of immunogenesis, plentiful unoccupied receptors could be demonstrated. The tolerogenic concentration nearly saturated available receptors, and in its continuous presence, only few unoccupied or antigen-occupied surface receptors could be detected after 24 h of culture. Experiments of the modulation design showed that brief incubation with the tolerogenic concentration appeared to suppress receptor resynthesis, as few new receptors could be demonstrated after 20 h of further culture without antigen. Experiments were performed to determine whether fractionated cells prepared from spleens of 8-day-old mice showed an unusual tendency for modulation, even with immunogenic antigen concentrations. They were found to behave essentially like adult fractionated cells. The results are discussed in the framework of current theories of B-lymphocyte activation and tolerization.     

Properties of a subpopulation of T cells bearing histamine receptors.

C57BL/6 mice immunized i.p. with alloantigen (P815 mastocytoma cells) develop cytolytically active thymus-derived (T) splenic lymphocytes. The definition of specific histamine receptor sites on effector T cells has been studied by measuring the in vitro effects of the hormone on cytolytic activity. Histamine was found to inhibit cytolysis reversibly and to increase lymphoid cell cyclic AMP levels. Both of these histamine activities were reversed by burimamide and metiamide; neither activity was affected by diphenhydramine or pyrilamine. These findings indicate that modulation of effector T cell activity by histamine is mediated only by one of the subtypes of tissue histamine receptors, designated a histamine-type 2 receptor. This receptor appears to be present on cytolytically active cells; there is no evidence for activation by histamine of auxiliary or "suppressor" cells. The estimated dissociation constant (KB) for the burimamide-receptor complex (9 times 10-minus 6 tm) and for the metiamide-receptor complex (8 times 10-minus 7 M) indicated that the histamine receptor on T cells is quite similar to histamine-type 2 receptors in other tissues. Cells bearing such receptors could not be isolated by passage through a column of histamine-coated tsepharose beads. The cytolytic activity of spleen cells taken from mice early (days 7-9) after immunization is virtually unaffected by histamine in vitro. In contrast, the activity of spleen cells taken from mice later in the immune response is progressively more susceptible to inhibition by histamine. After reaching a maximum at day 11, the spleen cell cytolytic activity falls in a pattern that parallels the increase in susceptibility to histamine. The susceptibility of effector T cells to histamine appears also to reflect their site of origin, for peritoneal exudate effector cells were found to be significantly less sensitive than spleen cells to inhibition by histamine. The progressive increase in inhibition by histamine apparently reflects the appearance of greater numbers of specific histamine-type 2 receptors, and is probably a general phenomenon, for spleen cells from A/J or C3H mice immunized with either P815 mastocytoma (H-2d) or EL-4 (H-2b) cells showed the same effect. However, the appearance of histamine receptors could be altered by prior immunization with an unrelated alloantigen: thus, when A/J mice are preimmunized with EL-4, a subsequent immunization with mastocytoma cells results in peak spleen anti-H-2d activity at day 9 instead of days 11-13, and the appearance of significant (greater than 40 percent) inhibition by histamine as early as day 8 instead of day 16. The physiological role of the histamine receptors is as yet undefined, though their unexpected rate of appearance on effector T cells, coincident with a decline in the number of lytically active cells in vivo, may be a significant hint that hormone receptors play a role in the control of T-cell proliferation.     

Restricted nucleocytoplasmic relationship in activation of T and B lymphocytes

Nuclei of murine T lymphocytes or B lymphocytes were purified and transferred into lethally irradiated whole spleen cells or B or T lymphocytes by means of polyethyleneglycol-mediated cell fusion. Transfer of lymphocyte nuclei could save the irradiated cells from cell death, and such reconstituted cells could respond to mitogens. The present study showed that nuclei of T cells could be activated in the concanavalin A-stimulated T cell cytoplasms but not in the lipopolysaccharide-stimulated B cell cytoplasms. On the other hand, nuclei of B cells were activated in the lipopolysaccharide-stimulated B cells but not in the concanavalin A-stimulated T cell cytoplasms. These data suggested that a specific interaction between cytoplasm and nucleus might exist in the activation of nuclei of each lymphocyte subset.     

B lymphocytes may escape tolerance by revising their antigen receptors

To explore mechanisms that prevent autoreactivity in nonautoimmune mice, endogenous immunoglobulin (Ig) light (L) chains that associate with a transgenic anti-DNA heavy chain were analyzed. The antibodies from splenic B cell hybridomas of such mice did not bind double- stranded DNA (dsDNA) and their L chain sequences showed a biased use of V kappa and J kappa gene segments. The 44 L chains in this survey were coded for by just 18 germline genes. Six of the genes, each belonging to a different V kappa group, were used more than once and accounted for three fourths of all sequences. Based on the distribution of V kappa genes, the L chain repertoire in this line of transgenic mice was estimated at 37 V kappa genes. The most frequently observed gene, a member of the V kappa 12/13 group, was identified in 16 hybrids. In addition, the majority of V kappa genes used J kappa 5. We interpret the skewed representation of V kappa and J kappa gene segments to result from negative selection. Based on the data, we suggest that V kappa rearrangements giving rise to anti-dsDNA reactivity are removed from the repertoire by a corrective mechanism capable of editing self- reactive Ig.     

Inhibitory receptors for HLA class I molecules on cytolytic T lymphocytes

In recent years, the molecular mechanism by which natural killer cells lyze, or fail to lyze, different target cells has been elucidated. Natural killer cells express receptors which recognize MHC class I molecules on target cells. This interaction leads to inhibition of cytolytic activity, thus preventing lysis of target cells. The receptors belong to two distinct molecular types: (1) the Ig superfamily which includes receptors (p58.1, p58.2, p70, and p140) which recognize specific HLA allotypes; (2) CD94 molecules which display a broad specificity for HLA class I molecules. Recently, a subset of cytolytic T lymphocytes has been shown to express the various natural killer cell receptors. Such T cells are detectable in peripheral blood, spleen, tonsils, and lymph nodes, but not in the thymus and cord blood. In some instances, two or more natural killer receptors can be coexpressed at the single cell level. Surface marker analysis has revealed that natural killer cell receptor-positive T cells always express a memory phenotype. Moreover, they are characterized by a skewed T cell receptor VΒ repertoire. Further analysis of the T cell receptor VDJ sequences revealed that natural killer cell receptor-positive, CD3-positive cells isolated from a given individual are oligoclonal or monoclonal in nature. Crosslinking of natural killer receptors leads to inhibition of different T cell functions, including non-specific lysis of appropriate HLA class I-negative target cells, T cell receptor mediated cytotoxicity, and cytokine production. The inhibitory effect on T cell receptor-mediated function has important implications. Thus, the expression of natural killer cell receptors as a consequence of chronic antigen stimulation may result in functional impairment of specific cytolytic T lymphocytes. Preliminary data indicate that this phenomenon may occur in tumor or virally infected patients. Remarkably, various patients with large granular lymphocyte expansions characterized by a CD3-/ natural killer receptor-positive phenotype had chronic viral infections. The fact that antigen-specific cytolytic T lymphocytes may simultaneously express T cell and natural killer cell receptors, both recognizing HLA class I molecules but mediating opposite signals, offers new perspectives in our appreciation of the regulation of T cell responses and offers new clues for understanding the immunopathological events involved in certain diseases.