Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo and in vitro expression of an interleukin 2 receptor by murine B and T lymphocytes

Interleukin 2 (IL 2), which is well established to be a T cell growth factor, has more recently been shown to stimulate B lymphocyte growth and differentiation in vitro. Responsiveness of B and T cells to IL 2 has been associated with expression of a cell membrane IL 2 receptor (IL 2R). To investigate the role of IL 2 in B cell growth and differentiation in vivo, a system was used in which the injection of mice with a goat antibody to mouse IgD (GaM delta) induces polyclonal T-independent B cell proliferation first, and later induces polyclonal T-dependent B cell proliferation and IgG secretion. IL 2R expression by splenic B and T lymphocytes from GaM delta injected mice was studied by a dual label immunofluorescence technique. Although GaM delta was found to be a strong inducer of B cell IL 2R expression in vitro, even in serum-free medium, and stimulated up to 50% of splenic T cells to express considerable quantities of IL 2R in vivo, it failed to induce more than minimal B cell IL 2R expression in vivo. Concanavalin A and bacterial lipid A also induced B cells to express IL 2R to a much greater extent in vitro than in vivo. Although these agents and GaM delta acted synergistically to stimulate B cell IL 2R expression both in vitro and in vivo, a single agent induced B cell IL 2R expression to a considerably greater extent in vitro than did all three agents acting together in vivo. In vitro GaM delta-induced B cell IL 2R expression was not suppressed by inclusion of IL 2 in the culture medium but was suppressed by the presence of 10% normal mouse serum or plasma. These observations suggest that polyclonal T-dependent B cell proliferation and antibody secretion may not require an interaction between B cells and IL 2; the in vivo environment may downregulate IL 2R expression by B cells: and in vivo B cell IL 2R expression and consequently, induction of B cell responsiveness to IL 2, may require stimuli beyond those sufficient to induce B cell IL 2R expression and IL 2 responsiveness in vitro.     

Polyclonal activation of the murine immune system by an antibody to IgD. VII. Demonstration of the role of nonantigen-specific T help in in vivo B cell activation

Previous studies from this laboratory have shown that the injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) induces T-independent polyclonal increases in: 1) B cell DNA synthesis, and 2) expression of surface Ia antigen and receptors for T helper factors, 1 to 2 days after injection. In addition, T-independent polyclonal increases in B cell number and IgG1 secretion are observed 6 to 7 days after injection. The administration of normal goat IgG (GIgG) along with GaM delta has been shown to augment GaM delta-induced polyclonal IgG1 secretion. To obtain information about the characteristics of the T help that is required for polyclonal antibody production in this model system, we investigated: 1) the length of the period during which GaM delta must be present to induce day 7 polyclonal antibody production, and 2) the kinetics of the induction of splenic T cell DNA synthesis. We found that GaM delta can be neutralized 3 days after injection by the administration of IgD without decreasing day 7 polyclonal IgG1 secretion, as long as mice are given GIgG at the time that GaM delta is neutralized. In contrast, polyclonal IgG1 secretion is greatly inhibited if GaM delta is neutralized 1 to 2 days after injection or if GaM delta is neutralized 3 days after injection, but GIgG is not administered at this time. Because GIgG can stimulate activated GIgG-specific T cells to secrete helper factors, but, unlike GaM delta cannot focus GIgG-specific T help polyclonally onto B cells, these findings suggest that nonspecific T help, rather than antigen-specific T help, is required in this system after day 3 for the induction of polyclonal IgG1 secretion. Determination of the kinetics of the induction of T cell DNA synthesis in this system by in vivo [3H] thymidine incorporation studies, as well as dual laser fluorescence-activated cell sorter analysis of T and B cell DNA content, indicate that T cells are induced to synthesize DNA 2 days after GaM delta injection and reach plateau rates of DNA synthesis 3 days after injection. Taken together, the GaM delta neutralization experiments and DNA synthesis studies suggest that one reason that GaM delta is required for 3 days in this system is to allow maximal activation of GIgG-specific T cells, which when stimulated later by GIgG secrete nonantigen-specific helper factors that induce GaM delta-activated B cells to secrete IgG1.     

Polyclonal activation of the murine immune system by an antibody to IgD. X. Evidence that the precursors of IgG1-secreting cells are newly generated membrane IgD+B cells rather than the B cells that are initially activated by anti-IgD antibody

Injection of BALB/c mice with an affinity-purified goat antibody to mouse IgD (GaM delta) stimulates T cell-independent B cell activation as well as later T cell activation. Activated T cells then induce polyclonal differentiation of B cells into IgG1-secreting cells, which results in an approximately 100-fold increase in serum IgG1 level. It is not known whether the same B cells that are initially activated by GaM delta are the progenitors of the IgG1-secreting cells. To investigate this issue a system was developed in which CB20 mice, which are congenic to BALB/c mice but express Ig of the beta allotype rather than the BALB/c alpha allotype, were injected with GaM delta and simultaneously or subsequently also received BALB/c B cells. The IgG1 response generated by the donor BALB/c B cells was quantitated by an assay specific for IgG1 of the alpha allotype. Our experiments with this system indicate that: 1) BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta generate a much larger IgG1 response than do BALB/c B cells transferred simultaneously with GaM delta antibody; 2) B cells that express membrane IgD generate the great majority of this response; 3) differences in the magnitudes of the responses of BALB/c B cells transferred at different times after CB20 mice were injected with GaM delta antibody cannot be explained by differences in homing of the donor B cells to the host spleen or by short survival of donor BALB/c B cells after their transfer; and 4) the response made by donor BALB/c B cells transferred 2 days after CB20 mice were injected with GaM delta is proportionate to donor cell representation in the host spleen 1 day after their transfer, whereas the response made by donor cells transferred simultaneously with GaM delta is disproportionately small. These observations suggest that most of the IgG1 antibody made by GaM delta-injected mice is generated by newly produced, mIgD+ B cells that appear approximately 2 days after GaM delta injection, rather than by those B cells that are present in the spleen at the time of GaM delta injection, and support the view that signals that induce B cell secretion of Ig require an interaction with at least partially activated Th cells.     

Polyclonal activation of the murine immune system by a goat antibody to mouse IgD. IX. Induction of a polyclonal IgE response

The possibility that injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) that stimulates polyclonal IgG1 secretion might also stimulate differentiation of B cells into IgE-secreting cells was suggested by the observation that such treatment induces T cells from those mice to secrete a lymphokine, B cell stimulatory factor 1 (BSF-1), that can stimulate both IgG1 and IgE secretion in vitro. Studies described in this paper show that injection of BALB/c mice with 200 to 3200 micrograms of GaM delta greatly increased the quantity of splenic epsilon chain-encoding mRNA, the number of spleen cells with cytoplasmic IgE, and the concentration of serum IgE 7 days after injection. Serum IgE levels obtained in these mice were approximately 100 times baseline levels and were comparable with those found in mice infected with the nematode parasite Nippostrongylus brasiliensis, but were approximately 2000-fold less than the peak serum IgG1 levels induced by GaM delta injection. Both IgE and IgG1 secretion in GaM delta injected mice were T dependent (blocked by anti-L3T4 antibody). These observations are consistent with the hypothesis that BSF-1 may play a role in the in vivo stimulation of IgE secretion and provide an easy to apply model for the investigation of in vivo regulation of IgE responses.     

Murine B cells expressing Thy-1 after in vivo immunization selectively secrete IgE

IL-4 induces Thy-1 expression and IgE secretion by LPS--and T cell-stimulated murine B cells in vitro. IFN-gamma inhibits both of these IL-4-mediated effects. IL-4 and IFN-gamma are often exclusively produced by different CD4+ T cell subsets. Injection of mice with a polyclonal goat anti-mouse IgD antibody (GaM delta) stimulates large increases in the serum concentration of IgE through the production of IL-4. Neutralization of endogenous IFN-gamma production in GaM delta-injected mice leads to further increases in serum IgE levels. We show that IL-4 and IFN-gamma, produced after GaM delta injection, respectively, stimulate and inhibit the number of splenic B cells expressing Thy-1 in vivo. Increased numbers of Thy-1-expressing B cells are observed concomitantly with the onset of enhanced IgE secretion. These Thy-1-expressing B cells are highly and selectively enriched for IgE-secreting cells.     

Polyclonal activation of the murine immune system by an antibody to IgD. VIII. Stimulation of IgD secretion

The low levels of serum IgD found in mice and the lack of a typical DNA switch sequence between C delta and C mu raise the possibility that the generation of murine IgD-secreting cells results from a chance "mistake" rather than a controlled process. The recent observation that injection of mice with purified IgD upregulates IgD receptor expression on helper T cells and enhances the ability of these T cells to induce B cells to differentiate into antibody secreting cells led us to look for evidence of controlled differentiation of B cells into IgD-secreting cells. To do this, we injected mice with a goat antibody to IgD (GaM delta), because this antibody stimulates large increases in IgM, IgG1, IgG2a, and IgE secretion. Mice injected with GaM delta demonstrated a large increase in splenic content of mRNA specific for the secreted form of delta-chain, as well as a greater than 100-fold increase in the percentage of splenic IgD-containing plasmablasts. The secretory IgD response was totally T-dependent. Production of the secretory form of IgD was not seen until 7 days after GaM delta injection, and peaked sharply on day 8, whereas by day 6 IgM secretion had already peaked and IgG1 and IgG2 secretion had attained substantial levels. This observation suggests that: 1) either cells that synthesize large quantities of the secretory form of delta-chain, unlike cells that synthesize large quantities of the secretory forms of gamma-, epsilon-, or alpha-chains, do this without deleting C mu, or, despite the absence of a typical DNA switch sequence between C mu and C delta, controls must exist to effect the C mu deletion and VDJ-C delta joining; and 2) if secreted IgD has a role in the regulation of a humoral immune response it most likely is involved in later processes, such as memory cell generation or response termination, rather than in relatively early processes, such as helper T cell activation.     

Comparison of phorbol ester/calcium ionophore and phytohemagglutinin-induced signaling in human T lymphocytes. Demonstration of interleukin 2-independent transferrin receptor gene expression

The proliferation of human T lymphocytes is regulated, in part, by the coordinated expression of genes encoding T cell growth factor (interleukin 2 (IL-2), IL-2 receptors, and transferrin receptors (TFR). We examined the time course of accumulation of mRNA for these genes in T cells stimulated with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore, ionomycin, and compared their expression to T cells stimulated with phytohemagglutinin. In cells treated with PDB/ionomycin, maximum expression was observed at 3 hr for IL-2 mRNA and at 6 hr for TFR mRNA, whereas the level of IL-2 receptor mRNA reached a peak 24 to 48 hr after stimulation. In phytohemagglutinin-stimulated T cells IL-2 mRNA was detectable within 3 hr but peaked later at 12 hr; the level of IL-2 receptor mRNA similarly peaked 24 to 48 hr later. Accumulation of TFR mRNA in phytohemagglutinin-stimulated T cells, however, was not detectable at 6 hr and reached a peak only between 12 to 24 hr. The early accumulation of TFR mRNA in PDB/ionomycin-stimulated T cells seemed, in part, independent of the interaction of IL-2 with its own receptor, because TFR mRNA was detectable as early as 1 hr after stimulation and addition of cycloheximide before addition of PDB/ionomycin did not abolish the PDB/ionomycin-induced accumulation of TFR mRNA. In addition, either PDB or ionomycin used alone induced the expression of TFR mRNA but not IL-2 mRNA. These results indicated that the combination of PDB/ionomycin accelerated the expression of IL-2 and TFR genes in T cells compared to phytohemagglutinin and triggered an IL-2-independent pathway for the induction of TFR mRNA.     

Role of antigen-specific T cell help in the generation of in vivo antibody responses. I. Antigen-specific T cell help is required to generate a polyclonal IgG1 response in anti-IgD antibody-injected mice.

A system in which injection of mice with an antibody to mouse IgD that they recognize as foreign stimulates a large, T cell-dependent IgG response was used to study whether Ag-specific T cell help is required to stimulate polyclonal (non-Ag-specific) IgG production in vivo. Igha x Ighb allotype heterozygous mice were injected with a conjugate of a foreign Ag coupled to a mAb specific for one of the two IgD allotypes expressed in these mice. This conjugate cross-links mIgD on B cells that express the recognized allotype. These cells process the conjugate and present the foreign Ag to Ag-specific T lymphocytes, which become activated. Thus, B cells of the recognized allotype can be stimulated by cross-linking of their mIgD, Ag-specific T cell help, non-Ag-specific cytokines, and non-Ag-specific contact with activated T cells. In contrast, B cells that express the Igh allotype not recognized by the Ag-anti-IgD antibody conjugate (bystander B cells) can be stimulated in this system only by non-Ag-specific cytokines and non-Ag-specific contact with activated T cells. Although both recognized and bystander B cells in conjugate-injected mice demonstrated substantial increases in size and Ia expression, only the recognized B cells were induced to synthesize DNA and to make a substantial polyclonal Ig response. Bystander B cells still failed to secrete IgG when mice were injected with an anti-IgD-Ag conjugate specific for the other Igh allotype as well as a mAb that cross-linked IgD of the bystander B cell allotype. These observations demonstrate that although non-Ag-specific cytokine and contact-mediated T cell help are sufficient to induce B cells to increase in size and Ia expression in anti-IgD antibody-injected mice, Ag-specific T cell help is required to stimulate the generation of an IgG response in these mice.     

Expression of Lyt-1 by a subset of B lymphocytes

Using two-color flow cytometry and multiparameter data analysis, we have shown that the IgM bright, large subset of mouse splenic B lymphocytes express Lyt-1. This is not due to B cell uptake of immune complexes of Lyt-1 and antibody from T cells. The IgM bright cells of autoimmune NZB mice express more Lyt-1 than normal controls. This is because IgM containing plasmablasts, which are greatly increased in NZB spleens, are Lyt-1+. NZB spleen also contains more cells that are Lyt-1+ (but perhaps Lyt-1.2-), Thy-1.2 dull, and smaller in size than cells in normal mice. Thus, Lyt-1 is common to the T and B cell precursor or is induced independently during the ontogeny of T and at least one subset of B cells. We suggest that it be called Lyt-1.     

Sequential expression of immunoglobulin on developing mouse B lymphocytes: a systematic survey that suggests a model for the generation of immunoglobulin isotype diversity.

Paired immunofluorescent staining with antibodies specific for the major isotypes of mouse immunoglobulin was used to study the ontogenetic expression of diversity of cell surface immunoglobulin. The first B lymphocytes to emerge, derived from cytoplasmic IgM+ precursors, express sIgM exclusively. Between birth and 3 days of age separate populations of sIgM+ B lymphocyte acquire a second isotype: sIgD, one of the subclasses of sIgG, or sIgA. At 3 days, all splenic B lymphocytes that bear sIg or sIgA also express sIgM, but virtually none stain for sIgD. By 7 days, a substantial porportion of sIgG+ or sIgA+ lymphocytes in spleen and most of those in lymph node express both sIgM ans sIgD. Anti-mu antibody treatment from birth prevented development of B lymphocytes expressing any isotype. These observations suggest that the immature sIgM+ B lymphocyte is the pivotal cell in the generation of the different sublines of B cells and that sIgD ig or IgA. The frequency of lymphocytes bearing only sIgG or sIgA is higher in old than in young mice, suggesting that sIgD and sIgM may be lost after stimulation by antigens. The occurrence of a nearly identical distribution of sIg isotypes on B lymphocytes from athymic, pathogen-free mice suggests that primary expression of isotype diversity does not require T cells.     

Regulation of the anti-alpha(1----3) dextran IgG antibody response of BALB/c mice by idiotype-specific T suppressor lymphocytes.

The immune response of BALB/c mice against the so-called thymus-independent bacterial Ag alpha(1----3) dextran (Dex) is restricted to the expression of few major idiotypes (Id). It is furthermore under the control of T lymphocytes which regulate the isotype expression in such a way that they prevent anti-Dex IgG antibody production upon immunization. At the same time these T cells are part of a regulatory system for Dex-specific B cell memory formation. The underlying Ts cell activity has previously been analyzed by using euthymic and athymic congenic animals. Now we have isolated CD4-positive Id-specific T cell lines and clones which by several criteria are representatives of the above Ts cells. They inhibit in vitro proliferation and antibody secretion of Dex-specific hybridoma B cells. They prevent Id-restricted in vivo IgG anti-Dex antibody formation in T cell-reconstituted BALB/c nu/nu mice. At the same time they enforce, again Id-specific, accumulation of Dex-specific B memory cells. As has been shown previously under the influence of splenic Ts cells, these B memory cells are arrested in the original host but can be expanded and activated for anti-Dex IgG antibody formation upon adoptive transfer into X-irradiated allotype congenic nonresponder BALB.Ighb mice. The data show that the regulatory influence of T cells on the anti-Dex response is Id specific. It can now be studied by means of cloned Ts cells.     

Delineation and characterization of human B cell subpopulations at various stages of activation by using a B cell-specific monoclonal antibody

Human tonsillar B cells were separated into three distinct subpopulations, Ba-/IgD+, Ba+/IgD+, and Ba+/IgD-, by using a B cell-specific monoclonal antibody (anti-Ba) that recognizes only activated B cells, and anti-IgD antibody. Stimulation of Ba-/IgD+ cells with anti-mu plus PHA-conditioned culture supernatant (PHA-sup) or TPA induced Ba+/IgD+ cells, which reverted to Ba-/IgD+ phenotype in the absence of continuous stimulation. Further stimulation of Ba+/IgD+ cells with several B cell activators, such as TPA plus anti-mu or PWM plus T cells, resulted in the loss of IgD expression. Three-color FACS analysis showed that the expression of transferrin receptor (TFR) was at its maximum in Ba+/IgD- cells, and the intensity of this expression was proportional to that of Ba expression in Ba+/IgD+ cells. PHA-sup induced maximum proliferation in Ba+/IgD- cells, and the degree of response was a function of the intensity of Ba expression in Ba+/IgD+ cells. PHA-sup or purified BCDF (BSF-2) induced Ig secretion preferentially in Ba+/IgD- cells. Taken together, these results show that resting B cells (Ba-/IgD+) are activated into Ba+/IgD+ cells, and then into Ba+/IgD- cells, under mitogenic stimulation, and BCDF induces the final maturation of Ba+/IgD- cells into Ig-secreting cells. Ba+/IgD- cells, which maximally expressed TFR as well as Ba and displayed maximum proliferative response to PHA-sup, did not express any Tac antigen. On the other hand, in vitro activated B cells expressed Ba and TFR as well as Tac antigen.     

Differential effect of cyclosporin A on the expression of T and B lymphocyte activation antigens

We have used a monoclonal antibody (Mab) to the interleukin 2 (IL 2) receptor as well as a Mab to the transferrin receptor to analyze the effects of cyclosporin A (CsA) on the induction and expression of these activation antigens on mitogen-stimulated murine T and B lymphocytes. The same concentration (0.25 microgram/ml) of CsA that produced optimal inhibition of the T cell proliferative response to concanavalin A (Con A) was also very effective at inhibiting IL 2 production and the induction of IL 2 responsiveness, as well as the expression of the IL 2 and transferrin receptors when measured 72 hr after mitogen activation. Surprisingly, CsA only minimally inhibited expression of these receptors 24 hr after the addition of mitogen; however, T cell blasts recovered from these cultures failed to respond to IL 2 even though IL 2 receptor expression was only modestly decreased. These results suggest that inhibition of the maturation of receptor expression is secondary to an early effect of CsA in blocking the induction of IL 2 responsiveness or to an arrest in the sequence of events required for maturation of T cells that bear high densities of these receptors. In contrast to the results observed with T lymphocytes, CsA had no effect on the B cell proliferative response to lipopolysaccharide (LPS) or on the induction of the IL 2 and transferrin receptors on activated B lymphocytes.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

小鼠乳腺表达抗PD-1抗体对其脾脏T细胞的影响

为从细胞水平研究小鼠乳腺表达抗PD-1抗体对转基因鼠脾脏T细胞表面抗原蛋白、细胞因子表达、脾脏CD4⁺T细胞增殖以及增殖相关通路的影响,将8周龄未经历过怀孕的和18周龄经历过哺乳的表达抗人PD-1抗体的转基因小鼠分成两组,每组以转基因阴性鼠为对照,提取脾脏淋巴细胞,检测脾脏淋巴细胞的变化。与转基因阴性小鼠相比,乳腺表达抗PD-1抗体的转基因小鼠的免疫系统中的脾脏T细胞的效应T细胞比例上升,Treg细胞比例下降,CD4⁺T细胞表达的IFN-γ、IL-17以及IL-2有不同程度的增加。IL-4、IL-10以及TGF-β都没有发生变化。与T细胞刺激相关的一些细胞表面的蛋白分子也没有引起变化。转基因阳性鼠和转基因阴性鼠中T细胞增殖没有显著性差异,转基因阳性鼠中PI3K/Akt/mTOR和Ras/MEK/ERK这两条通路上的磷酸化蛋白只有部分表达上调,整个通路没有完全上调。结果表明,转基因小鼠作为表达抗PD-1抗体这类免疫系统相关单克隆抗体的宿主是可行的。     

Interleukin-15 expression affects homeostasis and function of B cells through NK cell-derived interferon-γ

Interleukin-15 (IL-15) is a cytokine important for the development, maturation, and function of many cells of the immune system including NK, NKT, γδT, and CD8⁺ T cells. The relationship between IL-15 and B lymphocytes however, is not well characterized and is the focus of our study. Previous in vitro reports have shown that IL-15 increases proliferation of B lymphocytes and increases antibody secretion however, this relationship remains inadequately defined in vivo. The focus of this study was to examine the role of IL-15 in B cell homeostasis and function in vivo using mice that either over express IL-15 (IL-15tg mice) or are deficient in IL-15 (IL-15^−/− mice) production. Here we report significant differences between the B cell populations of IL-15^−/−, C57BL/6, and IL-15tg mice. In fact, increased expression of IL-15 resulted in a significant decrease in the percentage and absolute number of CD19⁺ cells. In vitro B cell co-cultures implicate interferon-gamma (IFN-γ) as the factor responsible for inhibiting B cell proliferation. We also show that IL-15 expression affects B cell function, as B cells from IL-15 transgenic mice produce greater amounts of IgG and IgA than IL-15 knockout mice in vitro. Interestingly, despite significant differences in B cell numbers in these strains, there were no significant differences in total antibody titers in serum and vaginal washes of these mice. Results from our in vivo and in vitro experiments suggest that altered expression of IL-15 affects B cell homeostasis through the induction of NK cell-derived IFN-γ.     

CD28 costimulation induces delta opioid receptor expression during anti-CD3 activation of T cells

Previous studies have demonstrated that naive splenic mouse T cells express no or only very low levels of the delta-type opioid receptor (delta OR), but stimulation of mouse splenocytes with Con A results in induction of delta OR mRNA and protein. In this report we have shown that stimulation of highly purified populations of naive mouse T cells with anti-CD3 mAb alone results in T cell activation, as evidenced by sustained IL-2 secretion and cell proliferation, but fails to elicit delta OR expression. However, delta OR expression is induced by costimulation of these very pure T cells with anti-CD3 and anti-CD28 mAbs. The delta OR induction by anti-CD3 and anti-CD28 costimulation was completely blocked by inhibition of phosphatidylinositol 3-kinase with wortmannin. Because phosphatidylinositol 3-kinase activation in T cells is linked to costimulation, these results suggest that induction of delta OR expression during T cell activation is strictly dependent on costimulation. It also appears that costimulatory receptors other than CD28 can provide the signaling required for delta OR expression because delta OR mRNA was induced by Con A stimulation of splenocytes from CD28-deficient mice.     

Utility of antiPax5 in the diagnosis of lymphoproliferative disorders and neoplasia in mice

CD45R/B220 antigen (B220) is a common mouse panB-cell marker used for paraffin-embedded tissues. However, antiB220 has limited specificity in diagnostic pathology because the B220 antigen is expressed on subsets of cytotoxic T lymphocytes and natural killer cells, on plasmacytic dendritic cells, and on T lymphocytes of mice with the lymphoproliferative disorder associated with Fas (lymphoproliferative mutant mouse, B6.MRL-Fas(lpr/J)) or Fas ligand (generalized lymphoproliferative disease mutant mouse, C3H/ HeJ-Fasl(gld/J) or B6Smn.C3-Fasl(gld/J)). In addition, mouse B lymphocytes vary in the amount of B220 expressed, and some subsets of mouse B lymphocytes do not express B220 at all. In comparison, Pax5 expression (detected by immunohistochemistry using antiPax5) offers greater specificity and sensitivity because of its earlier expression during B-cell differentiation, its ability to detect all committed B cells, and its restriction to the B-cell lineage. Here we describe the use of an antibody to human Pax5 in diagnostic pathology with formalin-fixed, paraffin-embedded mouse tissue.     

Murine B cell development and antibody responses to model antigens are not impaired in the absence of the TNF receptor GITR

The Glucocorticoid-Induced Tumor necrosis factor Receptor GITR, a member of the tumor necrosis factor receptor superfamily, has been shown to be important in modulating immune responses in the context of T cell immunity. B lymphocytes also express GITR, but a role of GITR in humoral immunity has not been fully explored. To address this question, we performed studies to determine the kinetics of GITR expression on naïve and stimulated B cells and the capacity of B cells to develop and mount antibody responses in GITR^−/− mice. Results of our studies indicate that all mature B cells express GITR on the cell surface, albeit at different levels. Expression of GITR on naïve mature B cells is upregulated by BCR signaling, but is counteracted by helper T cell-related factors and other inflammatory signals in vitro. In line with these findings, expression of GITR on germinal center and memory B cells is lower than that on naïve B cells. However, the expression of GITR is strongly upregulated in plasma cells. Despite these differences in GITR expression, the absence of GITR has no effect on T cell-dependent and T cell-independent antibody responses to model antigens in GITR^−/− mice, or on B cell activation and proliferation in vitro. GITR deficiency manifests only with a slight reduction of mature B cell numbers and increased turnover of naïve B cells, suggesting that GITR slightly contributes to mature B cell homeostasis. Overall, our data indicate that GITR does not play a significant role in B cell development and antibody responses to T-dependent and independent model antigens within the context of a GITR-deficient genetic background.