B cell differentiation induced by helper T cell membranes: evidence for sequential isotype switching and a requirement for lymphokines during proliferation

Small dense B cells are stimulated to proliferate by membranes prepared from activated helper T (Th) cell clones. In combination with Th2 lymphokines, Th membranes stimulate B cells to differentiate to secrete predominantly immunoglobulin (Ig)M, IgG1 and IgE. The activity in Th membrane requires the expression of CD40 ligand by the T cells, and initiation of the B cell response occurs through the ligation of CD40 on the B cell surface. We have further characterized the properties of the B cell response and found that Th membranes stimulated B cell proliferation and Ig secretion in a cell density independent manner and the majority of the stimulated B cells underwent a limited number of division rounds between day 2 and 5 of culture. IgM-secreting cells appeared in culture by day 3 and increased to reach a maximum, comprised of 30% of the viable cells, on day 5. IgG1-secreting cells appeared 12--24h after IgM-secreting cells, and IgE-secreting cells did not appear until day 5. These data are consistent with a sequential model of isotype switching related to cell division. As lymphokines were absolutely required for antibody production we were able to determine when during culture they were essential. Lymphokines needed to be present prior to and during B cell proliferation for differentiation to Ig-secreting cells to proceed. This period corresponded closely to the time of maximum DNA synthesis. Consistent with sequential switching of Ig isotypes, differentiation to IgM secretion required the shortest exposure to lymphokines and IgE the longest. These experiments strongly suggest that the lymphokine-induced commitment to differentiate is made before DNA synthesis begins, although the lymphokine-delivered signals are necessary during DNA synthesis to support Ig class switching.     

Engineered promiscuous T helper peptides for the induction of immune responses

Following recognition of antigens by T helper (Th) lymphocytes, T cell help is elicited to induce humoral and cellular immune responses. These antigens are presented as short peptides, T helper peptides (THP), bound to MHC class II molecules. Since both endogenous THP (from antigens of interest) or exogenous THP (not encompassed by the sequence of the antigen of interest) are able to elicit T cell help, we decided to engineer promiscuous exogenous THP capable of binding to several HLA-DR molecules, in order to cover an important proportion of the human population. Some of these exogenous THP were able to bind to all seven HLA-DR molecules tested and were immunogenic in vivo in HLA-DR4 transgenic mice. Among them, peptides p37, p62 and p45 elicited Th1 cytokine profiles in vivo, providing help for the induction of potent CTL responses. Finally, in vitro stimulation assays carried out using human cells, showed that these peptides could induce T cell responses using cells obtained from individuals with a broad spectrum of HLA-DR molecules. Thus, engineered exogenous THP may be a valuable tool for the induction of immune responses in a large proportion of human population.     

辅助T细胞分化的表观遗传学调控

在抗原的刺激下,CD4^＋辅助性T细胞（Th细胞）可分化为不同的细胞亚群以对抗不同的外界环境。Th细胞的分化主要受细胞因子、细胞特异性转录因子等调控。近年来研究发现,DNA甲基化、组蛋白修饰以及染色质重塑等表观遗传学修饰能调控相关基因的表达,使Th细胞分化成稳定的亚群,发挥相应的功能。同时,这些表观遗传学修饰机制在赋予Th细胞亚群可遗传性、可塑性等方面也发挥着重要的作用。     

The effect of second signals on the induction of B cell tolerance: failure of helper T cells to block tolerance induction

The effect of carrier-primed helper T (Th) cells and T cell-replacing factors on the induction of hapten-specific tolerance in B cells from adult mice has been tested. The 2,4,6-trinitrophenyl conjugate of human gamma-globulin (TNP17HGG) was used as tolerogen in an in vitro tolerance induction system. Tolerance was assessed by the subsequent induction of plaque-forming cell responses using TNP-Brucella abortus and trinitrophenylated sheep red blood cells (TNP-SRBC) plus SRBC-primed Th cells as T-independent (TI) and T-dependent (TD) forms of TNP, respectively. B cells that respond to the different forms of TNP appear to be distinct B cell subpopulations. TNP17HGG induced TNP-specific tolerance in B cells responsive to TI and TD forms of the antigen, although more tolerogen was required to induce unresponsiveness in the TD antigen-reactive B cells. The presence of antigen nonspecific T cell-replacing factors during tolerance induction had no effect on the induction of unresponsiveness in either TI or TD antigen-responsive B cells, although these factors were able to support primary anti-TNP responses in T-depleted B cell populations to subtolerogenic doses of TNP-HGG. Populations of irradiated lymphocytes enriched for HGG-specific Th cells also had no effect on tolerogenesis in TI or TD antigen-responsive B cells, although priming of TD antigen-responsive B cells occurred at subtolerogenic doses of tolerogen. The inability of these Th cells to modulate B cell unresponsiveness was not due to their inability to exert helper function at higher concentrations of HGG. Thus, in this system, tolerance susceptibility is an intrinsic property of B lymphocytes, i.e. immunogenicity vs. tolerogenicity of signals is not determined by a "second signal" provided by Th cells.     

Secondary IgG responses to type 3 pneumococcal polysaccharide. III. T cell requirement for development of B memory cells.

Mice primed with a thymus-dependent form of Type 3 pneumococcal polysaccharide (S3), i.e. S3 coupled to erythrocytes (S3-RBC) produces S3-specific IgG antibody after secondary challenge with S3-RBC. When mice are depleted of T cells by treatment with anti-lymphocyte serum (ALS) at the time of priming, no IgG antibody is produced after secondary challenge. In order to determine the cellular basis for this phenomenon, various combinations of T and/or B cells from ALS-treated or normal primed mice were transferred to irradiated recipients prior to secondary challenge with S3-RBC. The results indicated that T cells were required at the time of priming with S3-RBC in order to (a) prevent the induction of tolerance in S3-specific B cells in mice primed with high doses of S3-RBC, and (b) induced differentiation of IgG-producing B cell precursors to Bgamma memory cells in mice primed with low doses of antigen.     

A combination of soluble helper factors bypasses the requirement for stimulator cells and induces nonspecific cytotoxic T cell responses

The specificity of cytotoxic T lymphocyte (CTL) responses generated in the presence of lymphokines was studied. Thymic responder cells were activated in the presence of stimulator cells that differed in their metabolic activity. After 5 days of culture, the cytotoxic response was estimated in a 4-h 51Cr-release test. Coculture of thymic responders with irradiated splenic stimulator cells in the presence of interleukin 2(IL 2) led to preferential cytolysis of target cells that expressed the same histocompatibility antigens as the cells used for sensitization. Addition of T cell cytotoxicity-inducing factor 1 (TCF1), however, to those cultures made the presence of stimulator cells unnecessary and induced cytotoxic responses against all target cells tested, including target cells syngeneic to the responder cells. This activation was neither due to contaminating mitogen nor to the effect of heterologous serum in the assay system. The conclusion of these findings was that either polyclonal activation of CTL was induced by TCF1 or that some specific CTL clones differentiated into unrestricted killer cells under the influence of TCF1.     

Genetics of human T cell-monocyte interaction in helper cell induction.

The generation of human antigen-specific helper cells from unprimed peripheral blood lymphoid cells in tissue culture requires the presence of the appropriate number of blood monocytes. The role of the HLA complex in this cell interaction was investigated, by using HLA-type donors, and it was found that the monocytes had to share at least one HLA-DR specificity with the T cell donor. These experiments suggested that the genes controlling the macrophage-T cell interaction are closely associated or in linkage disequilibrium with the HLA-DR region.     

Evaluation of accessory cell heterogeneity. I. Differential accessory cell requirement for T helper cell activation and for T-B cooperation

Several Ia+ tumor cell lines and peritoneal exudate macrophages were tested as accessory cells (AC) for the activation of antigen-specific T cells and for T-B cooperation. The macrophages and all the Ia+ tumor lines tested induced the release of lymphokines from T cells in a major histocompatibility complex (MHC)-restricted fashion and reconstituted the antibody responses of AC-depleted spleen cells or of purified T and B cells. However, only the normal macrophages but none of the tumor lines induced carrier-specific T helper (Th) cells which help B cells for specific antihapten antibody responses by linked recognition. For T-B cooperation accessory cells were also required, but in contrast to Th cell activation any type of Ia+ AC (e.g. macrophage or tumor line) was effective. Strong MHC-restriction between the lymphocytes and the AC was seen if antigen-pulsed AC were added into the AC-depleted T-B cooperation cultures. If the AC and antigen were concomitantly added to the AC-depleted T-B cultures, MHC-restriction was less obvious. Concanavalin A supernatant reconstituted the response of AC-depleted T-B cultures provided antigen-specific Th cells and the hapten-carrier conjugate were present. If, however, tumor line-activated T cells were added instead of macrophage-induced Th cells, no cooperation with B cells took place even in the presence of Con A supernatant. The results obtained demonstrate a differential AC requirement for the induction of Th cells depending on the differentiation stage of the Th cells.     

The role of macrophages in the generation of T helper cells. III. Influence of macrophage-derived factors in helper cell induction

Helper cell induction to soluble or particulate antigens in vitro requires the cooperation of T cells and macrophages. A direct contact between macrophages and T cells is not obligatory for this cooperation and factors released from macrophages are as effective in activating T cells as the cells themselves. Two different types of macrophage-derived factors where found. The supernatant obtained from purified macrophages incubated with antigen for several days generates helper cells in absence of macrophages or additional antigen, but only if obtained from macrophages which were identical at the I-A subregion of the H-2 complex as the T cells. This factor was called genetically related macrophage factor (GRF). The other factor(s), which is present in the supernatant obtained from macrophages incubated for several days without antigen, replaces macrophages only if the antigen is particulate. This factor(s), called nonspecific macrophage factor (NMF) is not restricted genetically and is also obtained from allogeneic macrophages. The importance of both these factors in helper cell induction is discussed.     

Stimulation with dendritic cells decreases or obviates the CD4+ helper cell requirement in cytotoxic T lymphocyte responses

We investigated the need for CD4+ helper T (Th) cells in the induction of murine cytotoxic T lymphocyte (Tc) responses across minor or major histocompatibility (MHC) antigenic differences with either normal spleen cells (NSC) or purified dendritic cells (DC) as antigen-presenting cells (APC). Generation of a secondary in vitro class II MHC-specific Tc response was totally CD4+ Th cell-dependent with both types of APC. Likewise, male antigen (H-Y)-primed class II mutant bm12 T cells, which do not respond to H-Y presented on NSC, do respond to H-Y presented on DC in a completely CD4+ Th cell-dependent fashion. All other Tc responses, including primary anti-class I MHC, primary anti-class I + II MHC plus anti-minor H, and secondary C57BL/6 (B6) anti-H-Y, although not completely CD4+ Th cell dependent, were greatly augmented in the presence of CD4+ Th cells, but only with NSC as APC. In contrast, with DC as APC these responses were entirely or largely CD4+ Th cell independent. Similarly, H-Y primed class I MHC mutant bm14 T cells, which do not respond to H-Y presented on NSC, do respond to H-Y presented on DC in a completely CD4+ Th cell-independent fashion. The combined results indicate that DC can directly present class I MHC alloantigen or class I MHC plus nominal antigen (e.g. minor H) to CD8+ cells and generate a Tc response by these cells without the requirement for CD4+ Th cells.     

Impairment of T helper and T regulatory cell responses at birth

Background: There is strong evidence that reduced exposures to microbial compounds triggering innate immune responses early in life are critical for the development of allergic illnesses. The underlying mechanisms remain unknown, but will include T‐cell responses either along T helper type 1 (Th1)/Th2 pathways or via T regulatory and Th17 cells. Yet, little is known about innate immune responses and the function of T regulatory/Th17 cells at birth. The aim of this study was to investigate T‐cell responses to innate (Lipid A/LpA, peptidoglycan/Ppg) and adaptive (phytohemagglutinin) stimuli at birth and to compare these findings with adult immune responses. Methods: Cord and peripheral blood mononuclear cells including T regulatory and Th17 cells from 25 neonates and 25 adults were examined for proliferation, cytokine secretion, surface, mRNA expression and functional suppression assays. Results: Proliferation and cytokine responses to innate stimuli were less mature at birth than in adulthood. T regulatory and Th17 cells were less expressed in cord than in adult blood (Ppg‐induced Foxp3, P = 0.001, LpA‐induced CD4⁺ CD25⁺ high, P = 0.02; Th17 : P < 0.0001). Mitogen‐induced suppression of T‐regulatory cells on T‐effector cell function was less efficient in cord than in adult blood (P = 0.01). At both ages, Th17 cells were correlated with Th1/Th2 cells (P < 0.01), but not with interleukin‐10 secretion following innate‐stimulation. Conclusion: Innate immune responses are immature at birth. Furthermore, the function of T regulatory and Th17 cells is impaired. Th17 cells in association with Th1/Th2 cells may be involved in early immuno‐modulation. Potent innate immune stimulation early in life can potentially contribute to protection from allergic diseases.     

T-T cell interactions during in vitro cytotoxic T cell responses. VI. The role of T cell-derived colony-stimulating factor in helper T cell activation

A limiting dilution culture system has been used to analyze the role of T cell-derived colony stimulating factor (CSF) during the activation of IL 2-producing helper T lymphocytes (HTL). EL4 thymoma-derived supernatant (EL4-SN) increased about 4-fold the frequency of HTL precursors responding to metabolically active allogeneic stimulator cells. Upon biochemical separation this biological activity within the EL4-SN segregated from interleukin 2 (IL 2) and gamma interferon but was associated with or identical to CSF. Further, a comparable rise in HTL precursor frequencies was observed when semipurified interleukin 1 (IL 1) derived from the P388 D1 cell line was added to limiting dilution cultures. In contrast to IL 1, semipurified CSF failed to facilitate the activation of HTL precursors when heat-treated allogeneic spleen cells were used as stimulator cells. Because EL4-derived CSF was found to induce IL 1 production by macrophages we conclude that T cell-derived CSF amplifies, via stimulation of antigen-presenting cells the number of inducible HTL precursors. Therefore, the CSF/IL 1-dependent increase in HTL precursor frequencies reported here may reflect the differential activation threshold of HTL-precursors, most of which will not be activated by antigen per se but only in presence of additional cytokines.     

Requirement for interactions between two subpopulations of T cells for helper cell induction in vitro.

The evidence for the interaction of 2 subpopulations of T cells, short-lived cells sensitive to adult thymectomy (T1 cells), and long-lived recirculating cells, sensitive to the action of antilymphocyte serum (T2 cells) in the induction of helper cells is presented. This T-T interaction occurred across a cell-impermeable nucleopore membrane, indicating that it did not depend on cell contact, but was mediated by subcellular factors. There was no genetic restriction on this T-T interaction, if it was performed across a nucleopore membrane. The implications of these results on our concepts of the mechanism of help are discussed.     

The induction and migration of antigen-specific helper cells for IgA responses in the intestine.

The distribution of keyhole limpet haemocyanin (KLH)-specific helper cells for antibody responses of IgA, IgM and IgG isotypes in Peyer's patch (PP), mesenteric lymph node (MLN) and peripheral lymph node (PLN) was examined following oral, intraduodenal (ID), intraperitoneal (IP), intra-Peyer's patch (IPP) or subcutaneous (SC) immunization with KLH. Oral or ID immunization gave little or no response in any tissue studied. IP immunization with or without a subsequent ID challenge gave rise to a modest IgA and IgM helper response in MLN but a small IgA and IgM helper response in PP and PLN. IP immunization alone did not stimulate IgG-specific help in any tissues studied, but a small IgG helper response occurred in MLN and PLN after subsequent ID challenge. IPP was the most effective route of immunization, giving rise to a large helper response for IgA, IgM and IgG isotypes in PP, a smaller response in MLN and no response in PLN. The helper response following IPP immunization was not augmented by subsequent ID challenge. SC immunization gave a small but significant helper response for all isotypes in PLN but no response in PP or MLN. The kinetics of the helper response were examined in PP, MLN, PLN and thoracic duct lymph (TDL) following IPP immunization. The helper response for all isotypes in PP was maximal at 2 weeks and then declined. Similar kinetics but of lower magnitude were observed in MLN and TDL. The presence of IgA-specific helper cells in TDL demonstrates that these cells migrate, presumably from GALT, and may constitute an important component of mucosal responses at extraintestinal sites.     

Protein kinase C isotype expression and regulation of lymphoid cell motility

Lymphocyte migration into inflammatory sites involves a change from a spherical, non-motile phenotype to an irregular, constantly shape-changing, motile phenotype. We have previously shown that lymphocytes are maintained in the non-motile state by the constitutive activity of protein kinase C (PKC). In this paper we have attempted to identify the PKC isotype which regulates these morphological changes by three different approaches. (a) Motile and non-motile T-cell lines were compared for expression of the α, βI, βII, γ, δ, ε, η, ζ and θ isotypes by Western blotting. There was no obvious correlation of isotype expression with motility. (b) Two different PKC inhibitors, one specific for classical isotypes, Go6976 and the other GF109203X, which inhibits both classical and non-classical isotypes were compared for induction of motility in non-motile lymphocytes. Only GF109203X induced motility implying that a non-classical isotype is involved. (c) Non-motile lymphocytes were chronically treated with the PKC activator bryostatin and the time courses of induction of motility and downregulation of PKC isotypes were compared. Induction of motility correlated better with downregulation of ε, η and θ than with α or β. It is concluded that the data fit best with the involvement of a non-classical PKC isotype in regulating lymphocyte motility although no association with a particular isotype was found.     

The regulation of T cell responses by spontaneously active suppressor cells.

In studying T cell regulation, peripheral blood mononuclear cells from normal subjects were examined for 'spontaneous', rather than mitogen-induced, suppressor cell activity. Normal blood leucocytes from 30 subjects included a subpopulation of cells capable of suppressing the response of lymphocytes to the T cell mitogen phytohaemagglutinin by 21-35%. The indicator system for these studies consisted of fresh normal lymphocytes stimulated by three concentrations of PHA in the presence or absence of normal but mitomycin C treated peripheral blood lymphocytes. To measure accurately the spontaneous suppressor cell activity, additional cultures were needed to control for the suppressive effects of crowding and metabolic competition. Allogeneic, cryopreserved 'B' cell enriched populations, supplied satisfactory control cells for this purpose. While allogeneic culture systems could induce significant suppressor cell activity after 7 days of co-culture, they could not induce this activity in the 3 days required to assay spontaneous suppressor cell effects. In developing this assay we noted that (a) crowding became a factor in the cellular response to mitogens with concentrations higher than 2 X 10(4) cells/well, (b) spontaneous suppressor cell activity decreased rapidly once cells were placed in culture and (c) both spontaneous and concanavalin A (Con A) activated suppressor cells could significantly reduce the response to PHA even when added to cultures established with mitogens 72 hr earlier. The ability to measure spontaneous suppressor cell activity in vitro will allow more physiological studies of the membrane markers and functional characteristics of these cells than is possible in conventional studies utilizing Con A. In addition, this assay allows the detection of enhanced in vivo activity of suppressor cells not easily detected in assays relying on mitogen induction of suppression. Such increased activity is thought to be an important factor in the pathogenesis of a number of human diseases.