Biomedical subjects

G Pobor

Publications and source records attributed to G Pobor.

14 recordsLinked to original sources

PubMed1985

Functional analysis of pokeweed mitogen-dependent cell interactions in murine spleen cells. I. Lack of B-cell mitogenicity and low frequency of effector helper T cells.

The nature of lymphocyte responses on addition of pokeweed mitogen (PWM) to normal murine spleen cells was studied in low cell density cultures. PWM, over a wide range of concentrations, stimulated proliferation in a set of cells roughly 10-fold smaller than the lymphocyte populations responding to either concanavalin A or lipopolysaccharide. PWM also induced a relatively small number of B lymphocytes in these cultures to mature to Ig-secreting plaque-forming cells (PFC). Proliferative and PFC responses were completely abrogated by T-cell removal from normal spleen cell cultures. Moreover, cell mixture and irradiation experiments demonstrated that B lymphocytes do not proliferate in response to PWM, even in the presence of an excess of normal T cells, suggesting that PFC development results from terminal maturation without proliferation. Finally, parallel titrations of cloned helper cells, normal splenic T cells or T-cell blasts induced by PWM showed that the poor B-lymphocyte responses in normal spleen cell cultures is due to the very low frequency of competent helper cells in these populations. PWM, however, was competent to activate and expand this set of helper lymphocytes in primary cultures.

Animals↗

PubMed1984

B lymphocyte activation upon exclusive recognition of major histocompatibility antigens by T helper cells.

This study has investigated whether exclusive recognition of I-A or I-E molecules on the B cell surface by T helper cells is sufficient to activate resting B cells. Lines and clones of long-term-cultured T helper cells with specificity for I-A or I-E antigens have been derived from mixed lymphocyte cultures between spleen cells from major histocompatibility complex (MHC)-congenic mouse strains. These cells were tested for helper activity and proved competent to induce resting B lymphocytes expressing the specific MHC antigens to polyclonal expansion and maturation to Ig secretion. B cell activation was shown to require direct recognition of I-A/E antigens by the helper cells on the responding B lymphocyte surface and it could not be achieved by soluble factors released by "third-party" helper cell activity ongoing in the same cultures. Since B lymphocyte activation occurs in the absence of antigen recognition by the responding B cells, these observations suggest that I-A and I-E molecules expressed on the B cell surface participate in the functional reception of T helper cell-derived induction signals.

Animals↗

PubMed1984

Functional and chemical characterization of B-cell growth factor produced by normal cloned T helper cells.

Media conditioned by clones of normal helper T cells exposed to appropriate antigen-presenting cells contain growth-promoting activity for B-cell blasts induced either by lipopolysaccharide or on direct interaction with competent helper cells. This B-cell growth factor (TH-BGAPet) is recovered on sodium doecyl sulphate polyacrylamide gel electrophoresis corresponding to mol. wt of 15,000-20,000 displays no mitogenicity for small, non-induced B lymphocytes and is completely devoid of the ability to activate immunoglobulin secretion in proliferating B cells. These results are ascribed to the activity derived from normal T cells, with the same characteristics as BSF-p1 previously obtained from lymphomas and hybridomas. Since hybridization of these T helper cells results in the constitutive production of BSF-p1 in the absence of macrophages, these experiments demonstrate that BSF-p1 is a normal T-cell product.

Animals↗

PubMed1984

A quantitative assay detecting small numbers of effector helper T cells, regardless of clonal specificity.

We have developed a new assay for quantitative detection of all helper T cells that can induce normal B lymphocytes to proliferation and Ig secretion. To establish the optimal assay conditions, we have used cloned T helper cells of defined specificities that had previously been shown to activate normal B lymphocytes expressing the specific antigen(s) on direct cellular interactions. As shown in this paper, 'irrelevant' B lymphocytes--that is, those that do not express either antigen or restriction elements recognized by the effector helper T cells--can also be induced in the presence of appropriate concentrations of pokeweed mitogen which are not mitogenic for the 'target' B lymphocytes. 'Nonspecific' plaque-forming cell responses are of the same magnitude as those provided to specifically triggered targets and equal or better than those induced by lipopolysaccharide. The assay is highly sensitive and enables 'semi-quantitative' detection of less than 20-30 effector T cells per culture. Since effector helper T cells can be detected regardless of the clonal specificity, the assay appears useful for quantitative studies of various populations of T helper cells displaying mixed specificities and for the classification of cells with unknown functions.

Animals↗

PubMed1984

T cell-dependent B cell activation.

T cell-dependent induction of small, resting B lymphocytes requires direct recognition of antigen and/or I-A/E molecules on the B cell surface by the inducing helper cell, and it does not require the participation of Ig receptors on the responding B cell. Triggering B cell receptors, therefore, are either the I-A/E molecules themselves, or other structures with complementarities on helper cell membranes that become available for productive interactions upon I-A/E recognition. It would appear that signal delivery by such triggering receptors can be regulated by a membrane complex of molecules, involving immunoglobulins, Class II MHC molecules and other classes of receptors, which in selective and distinct manners control the quantitative levels of expression and/or availability of the relevant structures. Classical in vivo observations and our in vitro experiments led us to conclude that induction of B cells does not occur upon binding of T cell-dependent antigens to Ig receptors and, consequently, that B lymphocyte activation by anti-receptor antibodies has no physiological counterpart. Induced B lymphocytes proliferate and mature to high rate secretion of antibodies under the influence of selective growth and maturation factors produced by helper cells which are MHC-unrelated, act polyclonally and have no influence in normal, resting cells. Specific ligand interactions with the membrane molecules participating of that functional complex may also regulate reactivity to either growth or maturation factors, and, thus, control clonal performances and the fate of activated cells.

Animals↗

PubMed1984

The role of I-A/E molecules in B lymphocyte activation. I. Inhibition of lipopolysaccharide-induced responses by monoclonal antibodies.

A panel of 22 different monoclonal antibodies, including specificities against various antigenic clusters of I-A and I-E molecules, were probed over a wide range of concentrations for their ability to inhibit lipopolysaccharide-induced B lymphocyte proliferation and maturation to immunoglobulin-secreting plaque-forming cells (PFC). Most antibodies were competent to inhibit up to 80 to 100% of the response of appropriate target cells, although having little or no effect on irrelevant spleen cell cultures. Mixtures of either anti-I-A or anti-I-E specificities were more efficient inhibitors than individual antibodies, as shown by the average concentrations required for 50% inhibition (30 and 300 ng/ml, respectively). The selective role of I-A/E molecules in B cell activation was demonstrated by the failure of anti-K antibodies of the same isotype, bound in comparable amounts to target cells, to modulate B cell responses in parallel cultures. Fc receptor-mediated inhibitory effects were further excluded by equivalent inhibition obtained with anti-I-A antibodies of the IgM class. Anti-I-A/E antibodies appear to inhibit the inductive phase of B cell responses, as suggested by limiting dilution experiments performed in the presence of 50% inhibitory concentrations of antibodies: 50% of the control number of reactive clones were found to respond, but those that escaped inhibition developed to control sizes of progenies producing PFC.

Animals↗

PubMed1983

Differential macrophage requirements for T helper cell and T helper cell-induced B lymphocyte proliferation.

Major histocompatibility complex-restricted helper T cell clones against "minor" antigens expressed on B cell and macrophage surfaces, when confronted with appropriate T cell-depleted spleen cells, are induced to proliferation and, in turn, activate "target-responder" B cells to polyclonal growth and maturation. Irradiation of helper cell populations, however, demonstrates that their effector functions (and B lymphocyte responses) are independent of proliferative activity. Adherent cell depletion on Sephadex G10 columns, while completely abrogating helper T cell proliferation, does not abolish helper cell-induced B cell responses, demonstrating a remarkable quantitative difference in macrophage requirements for the growth of these two cell types. Because significant B cell responses are detected upon interaction with primed helper T cells under conditions of extreme macrophage depletion, we conclude that the role of macrophages in T-B cell cooperation is limited to expansion of optimal numbers of helper T lymphocytes. It follows that activated helper cells can autonomously produce all B cell-specific growth and maturation factors mediating cooperative antibody responses. In contrast, the profound reduction of LPS-induced responses upon macrophage depletion suggests accessory cell production of such factors in thymus-independent B cell growth and/or maturation.

Animals↗

PubMed1983

Distinct helper activities control growth or maturation of B lymphocytes.

A clone (C-11) of C3H/HeJ Lyt-1+2-T cells with specificity for "minor" antigens of C3H/Tif has been isolated which, in contrast to other similarly derived clones, did not activate polyclonal plaque-forming cell (PFC) responses in T cell-depleted "target" spleen cells. This clone, however, showed unaltered proliferative responses to the naturally occurring antigen(s) on presenting cells, and strongly synergized with regular helper clones in the induction of PFC responses. Further analysis demonstrated that C-11 cells are competent to stimulate extensive "target" B cell proliferation, but lack the ability to produce (or participate in the production of) maturation factors for activated B cells. Thus, the defective PFC responses could be fully reconstituted with supernatants from regular clones stimulated with antigen, but not by supernatants prepared from the C-11 cells themselves. While it is not clear whether this clone represents a normal helper T cell subpopulation or a variant that has lost maturation-factor production, these results demonstrate that distinct factors control growth and maturation in T cell-dependent B lymphocyte responses.

Animals↗

PubMed1983

Differential requirements for activation and growth of unprimed cytotoxic and helper T lymphocytes.

The requirements for activation and growth of T lymphocytes capable of mediating either cytolytic activity or help to B lymphocytes were studied in unprimed splenic T cell populations. The selectivity of expression of Lyt-2 antigens, the reactivity to soluble concanavalin A (Con A), to partially purified interleukin 2 (IL 2, T cell growth factor[s]) and to lectin-pulsed macrophages (M phi) were used in this analysis. Lectin-dependent cytotoxicity assays and a novel method that allows for the detection of all effector helper cells, regardless of their clonal specificities, were used for the functional identification of the responding T cells. The results show a marked contrast between cytolytic and helper T cells in their growth and activation requirements. Thus, while Lyt-2+ cytotoxic T lymphocyte precursors grow exponentially in IL 2 after a short pulse with soluble Con A in the absence of accessory cells, Lyt-2- helper cell precursors completely fail to proliferate under the same conditions and require the continuous presence of lectin-pulsed M phi for significant growth. Furthermore, addition of IL 2 to M phi-stimulated cultures of Lyt-2- cells has no effect. T cells which produce IL 2 have the same growth characteristics as helper cells. In both cases, effector helper functions could be expanded more than 10-fold on a per cell basis by a 5-day-culture period under those growth supporting conditions. The development of effector helper functions, however, was strongly inhibited by the presence of Lyt-2+ T cells.

Animals↗

PubMed1983

Activation of helper T cells for B lymphocytes in primary mixed lymphocyte cultures.

Normal B10.BR (H-2k, C57B1/6 background) spleen cells, enriched in primary mixed lymphocyte culture (MLC) for antigens of C3H/Tif mice (H-2k, C3H background), induced normal C3H/Tif but not B10.BR B lymphocytes to proliferate and produce Ig. In contrast, normal B10.BR spleen cells enriched in parallel B10.BR anti-C57B1/6 (H-2b) MLC were not able to activate either B10.BR or C57B1/6 B lymphocytes. However, normal B10.BR spleen cells depleted of Lyt2+ cells before initiation of the MLC, and subsequently enriched either for C3H/Tif or C57B1/6 antigens, activated B lymphocytes of the respective mouse strains specifically and equally well. These experiments show that primary MLC gives rise to effector T helper cells that, on recognition of specific alloantigens, activate normal B lymphocytes of the 'stimulator' strain. In response to major histocompatibility complex (MHC) alloantigens, this help is not revealed because of interference by Lyt 2+ lymphocytes. MHC-reactive T helper cells for B lymphocytes, however, participate in these reactions and constitute the predominant population in long-term cultures that are maintained by consecutive in vitro restimulations.

Animals↗

PubMed1982

Ontogenic development of B cell reactivities to cooperative cell signals: dissociation between proliferation and antibody secretion.

Cloned helper T cell lines, with specificity for "minor" antigens expressed on all B lymphocyte (and macrophage) surfaces were used to study the postnatal development of B cell reactivity to helper T cell cooperative signals. Such helper T cell populations, upon physiologic interactions with "target" B lymphocytes, activate them polyclonally, allowing for the analysis of functional ontogeny independent of V gene repertoires. Due to the high frequency of responding cells, these methods also offer a high level of sensitivity and permit measurement of B cell proliferation in the absence of antibody production. The results demonstrate that neonatal spleen cells are fully competent to stimulate adult helper T cell responses and that neonatal and adult B cells are comparable in their proliferative responses to cooperative cell interactions. In contrast, a marked deficiency in the ability of neonatal cells to mature to immunoglobulin secretion was observed in the same responses. Since the presence of suppressive activities in neonatal spleen cells could not be demonstrated, it was concluded that "early" B lymphocytes are intrinsically defective in their ability to secrete immunoglobulin upon cooperative induction, whereas they show full competence to expand clonally.

Aging↗

PubMed1982

Clonal analysis of the specificity of alloreactive cells: "dominance" of E beta reactive clones.

The present experiments analyze the specificity of cells proliferating in murine mixed lymphocyte cultures (MLC). Lymphocytes from the B10.A(5R) strain, enriched by repeated restimulations with B10.A cells, proliferate specifically in response to B10.BR, B10.A and [B10.A(4R) X B10.A(5R)]F1 stimulators, while showing very limited responsiveness to B10.A(4R) cells. The preferential recognition of determinants on molecules other than Kk or I-Ak was directly proven by isolating 21 T-cell clones, all of which were Ek beta specific. This "dominance" seems to reflect a high frequency of Ek beta reactive T cells in the unselected B10.A(5R) repertoire because: (1) it is already observed in blasts isolated in a primary MLC, (2) it is typical of several independently raised B10.A(5R) anti-B10.A cultures, and (3) it is not found in parallel C57BL/6 anti-B10.BR MLCs that show dominant I-A specificity. Results showing the importance of determinant concentrations expressed by stimulator cells suggest that uncloned T-cell lines are heterogeneous in their "affinities" and that our cloning conditions select cells with high "functional affinity".

Animals↗

PubMed1982

MHC restriction of male-antigen-specific T helper cells collaborating in antibody responses.

By priming female C57BL/6 mice with syngeneic male spleen cells and enriching inguinal and paraaortic lymph node cells in long-term culture (LTC) by repeated restimulations, H-Y-specific T helper cells can be produced. In response to male spleen cells carrying I-Ab antigens these cells activate "antigen-expressing" B cells to secrete polyclonal antibody. Before the end of the second week in LTC it was impossible to detect any helper activity. Induction of plaque-forming cells (PFC) also requires simultaneous recognition of antigen and I-A-encoded determinants in the "stimulator-responder" spleen-cell population. The testing of spleen cells from H-2 recombinant strains as "stimulator-responders" to anti-H-Y helper T cells of C57BL/6 origin also revealed that other genes, telomeric to I-A, control the magnitude of both specific T-cell proliferation and helper-dependent B-cell activation.

Animals↗

PubMedDate not supplied

The membrane receptor complex regulating induction and clonal expansion of normal murine B lymphocytes.

Induction of resting B lymphocytes results from the interaction of competent ligands or helper cells with "triggering receptors." Subsequent clonal expansion and performance are thought to be regulated by the interaction of selective growth or maturation factors with specific receptors on induced B cells. A set of membrane molecules of B lymphocytes, including IgM, IgD, IA, IE, lipopolysaccharide receptors, receptors for Fc and C3b, and other non-immunoglobulin structures recognized by some antiidiotypic antibodies, display ligand-induced relationships. Functional studies also indicate that this group of molecules functions as a complex that regulates delivery of inductive signals, the expression of growth and maturation receptors, and/or the outcome of the interactions of these receptors with the corresponding factors.

Animals↗