Critical role of CD81 in cognate T-B cell interactions leading to Th2 responses

We previously demonstrated that CD81-/- mice fail to develop Th2-biased immune responses and allergen-induced airway hyper-reactivity. Because CD81 is expressed on both activated T and on B cells, we examined the role of CD81 expression by each cell type. We established an in vitro system by backcrossing the CD81 deletion to TCR transgenic (Tg) mice and to BCR Tg mice. Here we demonstrate that CD81 expression by T cells is critical for their induction of IL-4 synthesis by B cells. CD81-/- TCR Tg T cells were impaired in IL-4 production compared to CD81+/+ TCR Tg T cells, whereas CD81-/- and CD81+/+ BCR Tg B cells induced equivalent amounts of IL-4 in CD81+/+ TCR Tg T cells. CD81-/- TCR Tg T cells expressed reduced levels of ICOS, GATA-3, STAT6 and phosphorylated STAT6 when activated by antigen-presenting B cells. Taken together, these results indicate that CD81 expression by T cells greatly enhances cognate T-B cell interactions and greatly augments intracellular activation pathways leading to Th2 polarization.     

IL-4 and IL-2 upregulate the expression of antigen B7, the B cell counterstructure to T cell CD28: an amplification mechanism for T-B cell interactions.

We recently generated mAb 104 which is specific for the B cell activation antigen Ag B7. With this we studied the regulation of Ag B7 expression on normal tonsillar B lymphocytes as well as the activities of B7+ and B7- activated B cells. SAC and to a lesser extent anti-IgM antibody upregulated Ag B7 and this was further enhanced by IL-2 and most notably IL-4. Ag B7 was expressed on virtually all sIgG+ and sIgA+ B cells and approximately half of the sIgD+ and sIgM+ B cells. SAC-stimulated B7+ B cells proliferated and produced IgM, IgG and IgA in response to IL-2 and IgM and IgG in response to IL-4. SAC-stimulated B7- B cells proliferated and produced only IgM in response to IL-2 and IL-4. Considering that Ag B7 has recently been shown to be the counterstructure of the T cell CD28 and that CD28 triggering strongly enhances cytokine production by T cells, it is likely that the CD28/B7 interaction represents an important amplification phenomenon in T-B cell interaction leading to humoral immune responses. The preferential expression of Ag B7 on IgG and IgA committed cells suggests that CD28/B7 interaction may be more specific to secondary antibody responses provided by memory T and B cells.     

Mechanism of antigen binding by T cells: H-2 (I-A)-restricted binding of antigen plus Ia by helper cells

The binding of a complex containing self Ia and antigen (IAC) to T cells was investigated. Poly-L (Tyr, Glu)-poly-DLAla—poly-LLys [(T, G)-A—L], bovine serum albumin, ovalbumin or fowl gamma-globulin were used as antigen. The effect of this complex was investigated in three experimental systems: autoradiographic antigen binding, in vivo immunization, and in vitro radioactive suicide of helper T cells. Autoradiographic experiments have shown that antigen binding to T cell-enriched spleen cells is a slow process with a half-life period of 20 min. It was found that during this time, antigen which can bind to Lyt-1⁺ T cells with much faster kinetics (half-life period = 2 min), is liberated from adherent cells. This “processed” antigen was retained by anti-Ia or lentil lectin affinity columns, and its binding to T cells was restricted by the I-A subregion of H-2. IAC was 100 to 1000-fold more immunogenic in vivo than the same amount of untreated antigen. This immunogenicity could be removed on anti-Ia columns, and was found to be under similar H-2 restriction as was its binding to T cells. The functional T cells, to which IAC binds, were identified by radioactive antigen suicide. It was found that the IAC killed syngeneic but not allogeneic, helper T cells. For the binding of processed antigen, helper cells required metabolic energy and a nonspecific soluble factor of adherent cells. These data are interpreted to suggest that H-2 restriction is directly determined by the interaction of the helper cell receptor with self Ia and foreign antigen. It also appears that the Ia-containing antigen may be a potent, cell type-directed immunogen.     

Peripheral blood-derived bovine dendritic cells promote IgG1-restricted B cell responses in vitro

Regulation of humoral responses involves multiple cell types including the requirements for cognate interactions between T and B cells to drive CD40-dependent responses to T-dependent antigens. A third cell type has also been shown to play an essential role, the dendritic cell (DC). We demonstrate that bovine peripheral blood-derived (PB)-DC are similar in function to features described for human interstitial DC including the production of signature type 2 cytokines [interleukin (IL)-13, IL-10]. PB-DC express moderate-to-high costimulatory molecule expression, and major histocompatibility complex class II is negative for CD14 expression and has low or no expression of CD11c. Consistent with the interstitial phenotype is the ability of PB-DC to influence B cell activation and differentiation via direct expression of CD40L and type 2 cytokines. Collectively, these results suggest that direct B cell-DC interactions may promote an immunoglobulin-isotype expression pattern consistent with type 2 responses, independent of direct T cell involvement.     

Immunoglobulin-specific T-B cell interaction. III. B cell activation by immunoglobulin-recognizing T cell clones

Immunoglobulin (Ig)-specific T-B cell interactions were studied in the model of T cell recognition of Ig kappa chain Ig kappa-1b allotype in Ig kappa-1-congenic rats. Using Ig kappa-1b-recognizing major histocompatibility complex (MHC)-restricted T helper clones from August rats we have shown that Ig kappa-1b+ B cells from congenic August.1b rats presented Ig kappa-1b epitope of the processed self-synthesized Ig to T clones. This interaction was found to be a bidirectional regulatory event inducing specific MHC-dependent proliferation of both interacting T cell and B cell as well as Ig(Ig kappa-1b) synthesis. Small Ig kappa-1b+ B cells were capable of inducing T clone proliferation and becoming activated in response to the same T clone. Limiting dilution analysis suggested that every tenth cell in Ig kappa-1b+ B cell population is involved in this interaction. The bystander activation of Ig kappa-1a+ B cells by T clones in the presence of irradiated Ig kappa-1b+ spleen cells, if observed, was less than the level of specific Ig kappa-1b+ B cell proliferation. In contrast to a 20-fold increase of Ig(Ig kappa-1b) levels upon stimulation of Ig kappa-1b+/1a+ B cell population from heterozygous (August x August.1b)F1 rats by T clones, a "nonspecific" increase of Ig(Ig kappa-1a) was not observed. This result demonstrates the requirement for direct T-B contact for B cell activation to occur. The data suggest a great functional potency of T-B interactions mediated by T cell recognition of Ig-derived peptide/MHC class II complexes on the B cell surface. The implication of the data for idiotypic regulation enables us to propose the existence of putative idiopeptidic network T-B cell interactions.     

Inhibition of T cell-dependent human B cell proliferation and B cell differentiation by polyspecific monomeric IgG.

A commercially available polyspecific, monomeric IgG preparation suitable for intravenous administration (IgSRK; Sandoglobulin) can inhibit pokeweed mitogen (PWM)-induced proliferation of peripheral blood mononuclear cells (PBMC) by a small, but statistically significant, amount compared to control cultures. Such inhibition could not be demonstrated when PBMC were stimulated with the T cell mitogen phytohaemagglutinin. Surface phenotype analysis of the PWM-stimulated cells indicated that in IgSRK-containing cultures, the proportion of B cells was decreased and the proportion of T cells was increased compared to control cultures. This alteration in T:B ratio was not due to antigenic modulation of B or T cell markers from their surfaces. In addition, IgSRK inhibited the proliferation of T cell-depleted PBMC cultures stimulated by B cell proliferation factors (BCPF) but not by fixed protein A-bearing Staphylococcus aureus strain Cowan I. The capacity to inhibit B cell proliferation was independent of and distinct from its capacity to inhibit B cell differentiation, since IgSRK inhibited the differentiation of a B cell differentiation factor (BCDF)-sensitive line by BCDF (which contains no BCPF activity). IgSRK inhibited PWM-induced generation of cytoplasmic Ig+ cells but had no effect on Ig secretion from mature Ig-secreting cells. Taken together, these findings suggest that IgSRK (which contains the IgG fraction from pooled plasma from 2,000 healthy donors) can inhibit T cell-dependent or T cell factor-dependent B cell proliferation and B cell differentiation.     

Selective role of PKCbeta enzymatic function in regulating cell survival mediated by B cell antigen receptor cross-linking

Cross-linking of the B cell antigen receptor (BCR) results in the activation of several protein tyrosine kinases leading to phospholipase C-gamma2-dependent phospholipid hydrolysis and Ca2+ mobilization, followed by activation of the protein kinase C (PKC) family members. Sustained Ca2+ release in B lymphocytes is dependent on the membrane localization and activation of the protein tyrosine kinase BTK. Ca2+ release is a tightly regulated process involving BTK membrane localization through its phosphorylation by PKCbeta. A selective role of PKCbeta in B cell signaling was first revealed by the characterization of PKCbeta knockout mice, which displayed decreased B cell proliferation in response to various mitogenic stimuli. However, it is not clear whether the B cell defects displayed by the PKCbeta knockout mice are due a B cell developmental defect or the scaffolding function of PKCbeta, resulting in a defect in the recruitment or formation of signal transducing complex molecules. Thus, in this report we investigated the effects of pharmacologic inhibition of the catalytic function of PKCbeta on B cell survival and growth. Treatment of Daudi B lymphoma cell line with a selective PKCbeta inhibitor, LY333531, inhibited anti-IgM-induced phosphorylation of BTK on Ser180 in a concentration-dependent manner, which was concomitant with an increase in BTK activation, and Ca2+ mobilization. In primary splenic B cells, LY333531 inhibited BCR-induced B cell proliferation, but did not affect basal or LPS-induced proliferation. Finally, LY333531 treatment resulted in the induction of apoptosis of anti-IgM-activated B cells, which corroborated with their inability to up-regulate pro-survival factors, Bcl-X(L) and Bcl-2. These results support the important and selective role of the PKCbeta enzymatic function in controlling Ca2+ release during BCR signaling leading to B lymphocyte survival and growth.     

Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery

Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freund's adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.     

Induction of Th2 responses to soluble proteins is independent of B cell tolerance status

Injection of high doses of monomeric human gamma globulins (dHGG)In naive, adult mice causes antigen-specific tolerance of Bcell and T_h1 lymphocytes, while inducing the selective expansionof antigen-specific T^h2 cells. Several parameters of toleranceinduction were analyzed in this work, In order to establishwhether B cell tolerance and T_h1 unresponsiveness were functionallyrelated in this in vivo model. By varying the antigen form andsite of injection, we demonstrate in this work that T_h1 unresponsivenessto HGG is not a consequence of peripheral B cell tolerance.In particular, mice pretreated with heat-aggregated antigen(HAHGG) or F(ab')² HGG were found to develop a strong humoralresponse while displaying a defective T_h1 response. In fact,these animals developed a strong T^h2 response in vivo, demonstratingthat selective expansion of antigen-specific T_h2 cells in thismodel is not a consequence of B cell tolerance or antigen captureby Fc receptor-expressing cells. We conclude that while B celltolerance in this model is only observed in response to deaggregatedantigen, Injection of all forms of adjuvant-free, protein antigensinduces T helper precursor cells to differentiate into T_h2-typehelper cells in vivo Irrespectively of the B cell tolerancestatus     

Recognition of influenza A virus nucleoprotein by an H-2-restricted cytotoxic T-cell clone

Cytotoxic T-cell clones raised against X-31 (H3N2) influenza virus in C57BL/6 mice can be directed against an influenza A virus subtype specific determinant (1). A representative T-cell clone (A3.1) has been used in combination with a set of genetically typed recombinant viruses, to show that the A/PR/8/34 nucleoprotein can be responsible for cytotoxic T-lymphocyte recognition of infected target cells.     

Molecular definition of retrovirus-induced antigens recognized by tumour-specific H-2-restricted cytolytic T lymphocytes

The antigenic specificities of H-2-restricted, tumour-specific cytolytic T lymphocytes (CTL) were studied at the molecular level using CTL from BALB.B and C57BL/6 (H-2b) mice sensitized to an H-2b Gross murine leukaemia virus (MuLV)-induced tumour. Target cells were produced by the double transfection of mouse L cells (H-2k) with the cloned H-2Kb or H-2Db gene and retroviral DNA derived from a molecular clone of Akv MuLV (closely related to Gross MuLV). Doubly transfected L cells which express either H-2Kb or H-2Db antigen and retroviral antigens are lysed in a virus-specific manner by Gross MuLV-immune CTL. The existence of two independent Gross MuLV-immune CTL subpopulations, one restricted by H-2Kb and the other by H-2Db, is thus confirmed. Gross MuLV-immune CTL from both BALB.B and C57BL/6 mice killed L cells that express Akv MuLV gag gene products and H-2Kb or H-2Db antigen. In contrast, only CTL from C57BL/6 mice killed L cells that express Akv MuLV env gene products and H-2Kb or H-2Db. This indicates that specific recognition of MuLV-induced antigens by CTL can be selective and varies according to the origin of the CTL.     

Role of glycosylation in the H-2-restricted cytolysis of virus-infected cells

The role of the oligosaccharide portions of cell surface glycoproteins in the susceptibility of virus-infected cells to H-2-restricted cytolysis was investigated by using the antibiotic tunicamycin (TM). TM inhibits the addition of sugars to the polypeptides of glycoproteins. TM treatment of P 815 cells before and during infection with vesicular stomatitis virus (VSV) inhibited glycosylation of proteins and reduced by about 50% the lysis of infected P 815 cells by VSV-immune, H-2-identical killer cells. In contrast, TM treatment had a modest inhibitory effect on cytolysis of P 815 cells by alloimmune effector cells. TM treatment did not inhibit the surface expression of either H-2 or VSV glycoprotein. Thus, glycosylation of H-2 and/or viral glycoprotein is a prerequisite for the lysis of infected cells by H-2-identical, VSV-immune cytotoxic cells.     

T-T cell interactions during in vitro cytotoxic T lymphocyte responses. III. Antigen-specific T helper cells release nonspecific mediator(s) able to help induction of H-2-restricted cytotoxic T lymphocyte responses across cell-impermeable membranes

T helper cell induction and the specificity of T cell-mediated help as generated during alloreactive and H-2-restricted, virus- or hapten-specific cytotoxic T lymphocyte (CTL) responses have been compared. With the use of a double-chamber culture system, it was possible to dissect and separately analyze the induction phase of T helper cells from the T helper cell effector function. The data obtained revealed that during alloreactive as well as H-2-restricted T cell responses, antigen-specific T helper cells are induced. Upon specific restimulation of T helper cells, helper cell function is mediated across a cell-impermeable membrane via soluble products in an apparently nonspecific and nonrestricted manner. The data suggest that similar rules govern T-T cell interactions in alloreactive and H-2-restricted CTL responses.     

Modulation of human B cell responses by a monoclonal antibody to an activation antigen 4F2.

Antigen specific human antibody responses can be modulated in vitro by the addition of 4F2 antibody, a monoclonal antibody (MoAb) which recognizes an antigen on activated T cells and B cells. Specific antibody responses induced with the antigen are suppressed by the addition of 4F2. However, specific antibody responses induced with the polyclonal activator, pokeweed mitogen (PWM), are significantly enhanced by the addition of 4F2. Proliferative responses to both antigen and PWM are suppressed by the addition of 4F2. The enhancement of PWM stimulated responses by 4F2 is mediated by T cells. However, in the absence of T cells, 4F2 can directly inhibit antigen specific B cells. Polyclonal Ig production stimulated by PWM was also enhanced by 4F2. Thus, the immunomodulating effects of the 4F2 MoAb are the result of a balance of enhancement and suppression mediated at the T cell and the B cell level, respectively.     

Control of primary IgM antibody responses to H-2 alloantigens by antigen-bearing live B lymphocytes

Alloantigen-bearing (H-2d+) peripheral red blood cells, but not red cell-depleted H-2d+ spleen cells, induce primary IgM anti-H-2d plaque-forming cell responses. In this study it is reported that the primary antibody responses to H-2d+ peripheral red blood cells can be markedly suppressed by a subpopulation of H-2d+ spleen cells when they are injected simultaneously or a few days before injection of red blood cells. This suppression was antigen (H-2d)-specific, did not depend on T cells of either the donor or the recipient, and strictly required live donor cells. An energy-dependent action of the donor cell cortex and some proliferation of donor cells in the recipient seemed to be involved in the mechanism of suppression. The donor-suppressor cell type was largely present in the spleen but not in the bone marrow and thymus, and was present in the spleen of athymic nude mice. The suppressor cells displayed the properties of B lymphocytes: they adhered to the nylon wool but not to glass, were of relatively low density (rho less than 1.09), and were surface Ig+, Ia+, Fc receptor-positive but Thy-1-. H-2d+ suppressor-donor B lymphocytes might directly signal to antigen-specific recipient B cells competing with the signal provided by H-2d+ red blood cells for the B cell activation.     

CD40-CD40 ligand interactions stimulate B cell antigen processing

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II E^k molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.     

Recognition of shared melanoma antigen by HLA-A2-restricted cytolytic T cell clones derived from human tumor-infiltrating lymphocytes

Three melanoma-specific cytotoxic T lymphocytes (CTL) clones were derived from the tumor-infiltrating lymphocyte (TIL) of human melanoma M17, and were used to study the expression of immunogenic melanoma peptides on allogeneic tumors. Antibody inhibition studies showed that two of these TIL clones were restricted by an HLA-A2 molecule which was identified as A2.1 by gene sequencing. The third CTL clone was not restricted by HLA-A2, but by a B or C HLA antigen. HLA-A2-restricted CTL clones M17-1 and M17-2 lysed 5 and 12 out of 15 HLA-A2⁺ allogeneic melanomas, respectively. Since they did not lyse autologous Epstein-Barr virus B cells, HLA-A2.1-transfected P815 cells,13 HLA-A2⁺ non-melanoma tumor cell lines and 10 HLA-A2^? melanomas, these clones appeared specific for melanoma-restricted epitopes presented by the HLA-A2.1 molecule. We then tried to determine why a few HLA-A2⁺ melanomas were refractory to TIL lysis. By using a combination of flow cytometry analysis, partial cloning and sequencing of their HLA-A2 genes, we show that failure to lyse did not result from low expression or polymoprhism of the HLA-A2 molecule, or from deficient expression of the adhesion molecules ICAM-1 and LFA-3 by these melanomas. Taken together, our data confirm at the clonal level the existence of shared melanoma antigens recognized by TIL in the HLA-A2.1 context. They further show that individual peptides derived from these antigens are expressed by a large majority of HLA-A2⁺ melanomas. Identification of such peptides appears crucial for the future of vaccination therapies.     

Antigen-dependent,H-2-restricted cytolytic and noncytolytic T cell lines with specificity for minor histocompatibility antigens

Several cloned T cell lines were isolated from primed mixed lymphocyte cultures immunized against minor histocompatibility antigens. These lines were selected with irradiated stimulator cells as antigen and require restimulation at intervals to keep growing. They are responsive, as measured by proliferation, to interleukin 2 (T cell growth factor) but cannot be grown in it continuously. These T cell lines have either the H-2^d or H-2^k haplotype. They all show exquisite H-2 restriction and minor histocompatibility antigen specificity. We did not observe any alloreactivity on 8 different H-2 haplotypes. For the H-2^k T cell lines, the restriction element could be mapped to either the K or D end of the H-2 complex. No I-A-restricted cell line was found. It is of interest that all these T cell lines need the presence of T cells in the irradiated stimulator cell population. This suggests a more complex interaction between irradiated stimulators and responder T cells than just H-2K- or D-restricted antigen interaction. This recognition, though necessary, does not seem sufficient to induce the T cell clones to proliferate.     

A comment on H-2-restricted T cell recognition of Mls determinants: a question of perspective

Data presented here addressed the question of whether T lymphocyte recognition of Mls determinants is MHC restricted. Using isolated T cell blasts from primary MLCs involving H-2-identical, Mls-disparate strain combinations as responding cell populations in secondary MLCs, and measuring their responses against fresh stimulating cells from different strains, has permitted the study of reactions towards Mls determinants in the presence of MHC antigenic differences. Results strongly suggest that recognition of Mlsc (Mls-2 locus) determinants is H-2 restricted. In contrast, there is still little evidence that the recognition of Mlsa (Mls-1 locus) determinants is H-2 restricted. Furthermore, data are also presented which refute the notion that different H-2 haplotypes possess different abilities to present Mlsa determinants to responding T cells.