Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.     

Evidence for existence of two distinct TNP-Ficoll-responsive B cell subpopulations preferentially reactive either to a T cell-replacing factor (B151-TRF) or to a signal from accessory cells

The ability of B cells to respond to TNP-Ficoll has been shown to correlate with their ability to respond to T cell-replacing factor (TRF). The present study analyzed the relationship of TNP-Ficoll-responsive B cells to a TRF-responsive B cell subpopulation. The B cells from normal, unprimed mice responded to TNP-Ficoll in the presence of accessory cells. Such responses were notably augmented by the addition of TRF derived from a monoclonal T cell hybridoma, B151K12(B151-TRF). Interestingly, B cells of mutant X-linked immunodeficient DBA/2Ha which failed to respond to B151-TRF gave anti-TNP PFC responses to TNP-Ficoll comparable to those of normal mice, depending on the presence of accessory cells. However, under this condition, the addition of B151-TRF did not augment the TNP-Ficoll responses. One explanation of the augmentation of TNP-Ficoll response by TRF for the B cells from nondefective mice was that two distinct B cell subpopulations exist which differ in their respective activation requirement for TRF and accessory cells. To examine this possibility, syngeneic accessory cells were pulsed with TNP-Ficoll and were assayed for their ability to activate normal B cells in the presence or absence of B151-TRF. The results revealed that TNP-Ficoll-pulsed accessory cells were able to induce primary anti-TNP PFC responses in normal B cells to the same magnitude as soluble TNP-Ficoll. However, these B cell responses induced by the TNP-Ficoll-pulsed accessory cells were not augmented by the addition of B151-TRF to the culture. These results support the notion that two distinct TNP-Ficoll-responsive B cell subpopulations exist; one requires accessory cell-B cell interaction to be activated by TNP-Ficoll but fails to respond to TRF, and the other can be activated by TRF in a totally accessory cell-independent manner.     

Characteristics of amplifier T cells involved in the antibody response to the capsular polysaccharide of type III Streptococcus pneumoniae

Amplifier T cell activity can be transferred by spleen cells harvested 72 hr after priming with type III pneumococcal polysaccharide (SSS-III) and can be abolished by treating the transferred cells with monoclonal anti-Lyt-1, or anti-Thy-1 antibodies in the presence of complement; thus, amplifier cells represent a distinct subpopulation of T cells. Amplifier T cells were found to be sensitive to irradiation but not to treatment with cyclophosphamide. When amplifier cells were transferred to athymic nude (nu/nu) mice, the enhancement obtained was much greater than that produced in thymus-bearing (nu/+) mice; this is presumably due to the lack of suppressor T cell activity in nu/nu mice that enables amplifier T cell activity to be expressed more fully. Amplifier T cells also were found to be present in peripheral blood; these amplifier T cells were Lyt-2- in phenotype. Although the induction and activation of amplifier T cells appear to be antigen-specific, the product made by amplifier T cells may not be antigen specific in its mode of action. Because amplifier T cells can be induced and activated by exposure to immune B cells, specificity is presumably due in whole or in part to the ability of amplifier T cells to recognize the idiotypic determinants of B cell-associated antibody specific for SSS-III.     

Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurred between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the "effector phase" as well as in the "induction phase". The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the "bystander effect."     

Cutting edge: CD40 ligand is a limiting factor in the humoral response to T cell-dependent antigens.

CD40 ligand (CD40L) plays a crucial role in T cell-dependent B cell responses, but whether its abundance is a limiting factor in their development is unclear. This question was addressed in transgenic mice expressing the murine CD40L gene under the control of the IL-2-promoter (CD40Ltg+). The fraction of activated T cells from the CD40Ltg+ mice with detectable levels of surface CD40L was modestly greater (1.1- to 2-fold) than littermate controls and paralleled an approximately 1.8-fold increase in CD40L mRNA abundance. In response to trinitrophenol (TNP)-keyhole limpet hemocyanin and tetanus/diphtheria vaccine, CD40Ltg+ mice developed higher titers of high-affinity IgG and IgG1 Ab than wild-type mice. In contrast, the Ab response of CD40Ltg+ and control mice was similar in response to the T-independent Ag TNP-Ficoll. These results suggest that a modest increment in expression of CD40L accelerates the development of T-dependent responses, and that CD40L plays a limiting role in the induction of high-affinity Ab and Ab-class switching.     

T cell control of the antibody response to the T-independent antigen, DAGG-Ficoll

C57BL/6J nu/nu mice respond to the type 2 TI antigen DAGG-Ficoll, but not to the TD antigen SRC. A comparable difference can also be seen in vitro, but only at high spleen cell density and in the presence of selected batches of FBS. At low spleen cell density and in the absence of FBS, the DAGG-Ficoll-induced B cell response is strictly dependent on soluble helper factors or cloned specific helper T cells. The B cell response so induced requires that the T cell-depleted spleen cells be compatible in the I-A subregion of the H-2 complex. These helper factors, induced by antigen in an I-A-restricted T cell-macrophage interaction, provide helper for T cell-depleted spleen cells irrespective of their H-2 haplotype. Under conventional culture conditions, the stringent requirement for helper factors in the in vitro response to DAGG-Ficoll is obscured by FBS. In vitro culture of low numbers of spleen cells, in serum-free medium instead of FBS, provides a sensitive assay for helper factors. We have compared the helper activity for a B cell response to SRC or DAGG-Ficoll as provided by antigen-induced supernatants of various individual EA-specific T cell clones. There was a remarkable and consistent heterogeneity among individual T cell clones: their helper activity in the response to TI and TD antigens did not correlate, nor was there any correlation between helper activity and antigen-induced TCGF (interleukin 2) activity.     

Antigen-specific suppression of cytotoxic T cell responses: an idiotype-bearing factor regulates the cytotoxic T cell response to azobenzenearsonate-coupled cells

When A/J mice are injected subcutaneously with azobenzenearsonate- (ABA) coupled spleen cells, their splenocytes contain primed ABA-specific cytotoxic T lymphocyte (CTL) precursors. Animals that are not primed in vivo do not develop vigorous CTL activity when assessed after in vitro culture with ABA-coupled stimulators. Suppressor molecules derived from ligand-induced first-order ABA-specific suppressor T cells were evaluated for their ability to limit cytolytic T cell development. We have shown that an idiotype-bearing, hapten-specific suppressor factor suppresses priming for CTL in an H-2-unrestricted but allotype-restricted manner. The implication of these studies to regulatory networks is discussed.     

T cell regulation of polyclonal B cell responsiveness. I. Helper effects of T cells.

Polyclonal activation of murine splenic B lymphocytes to secrete immunoglobulin was shown to be subject to regulation by splenic T cells. By admixture of separated B and T cell populations it was demonstrated that normal fresh splenic T cells were able to augment polyclonal B cell responsiveness to LPS up to several-fold. Optimal collaboration between these two cell types ensued when they were co-cultured in equal numbers. T cell-mediated enhancement of polyclonal B cell responses was dependent upon the ability of T cells to divide and was manifested upon T cell interaction with B cells soon after culture initiation. Originally expounded as a one-signal phenomenon, polyclonal activation of lymphocytes by LPS is, under the circumstances described, attributable instead to two distinct, nonspecific signals acting in concert. The observation that T cells from LPS-nonresponder (C3H/HeJ) mice were deficient in the capacity to enhance polyclonal B cell responsiveness of B cells derived from responder (C3H/HeN) mice implied a direct action of LPS on the involved T cells as well as an active role for the T cell signal in this immunoregulatory event. The novel observation of a functional T cell defect in LPS responsiveness in the C3H/HeJ mouse is discussed in terms of its other cellular defects.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting 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.     

TIM-1 signaling in B cells regulates antibody production

Members of the T cell Ig and mucin (TIM) family have recently been implicated in the control of T cell-mediated immune responses. In this study, we found TIM-1 expression on anti-IgM- or anti-CD40-stimulated splenic B cells, which was further up-regulated by the combination of anti-IgM and anti-CD40 Abs. On the other hand, TIM-1 ligand was constitutively expressed on B cells and inducible on anti-CD3⁺ anti-CD28-stimulated CD4⁺ T cells. In vitro stimulation of activated B cells by anti-TIM-1 mAb enhanced proliferation and expression of a plasma cell marker syndecan-1 (CD138). We further examined the effect of TIM-1 signaling on antibody production in vitro and in vivo. Higher levels of IgG2b and IgG3 secretion were detected in the culture supernatants of the anti-TIM-1-stimulated B cells as compared with the control IgG-stimulated B cells. When immunized with T-independent antigen TNP-Ficoll, TNP-specific IgG1, IgG2b, and IgG3 Abs were slightly increased in the anti-TIM-1-treated mice. When immunized with T-dependent antigen OVA, serum levels of OVA-specific IgG2b, IgG3, and IgE Abs were significantly increased in the anti-TIM-1-treated mice as compared with the control IgG-treated mice. These results suggest that TIM-1 signaling in B cells augments antibody production by enhancing B cell proliferation and differentiation.     

The role of T cells in IgG production; thymus-dependent antigens induce B cell memory in the absence of T cells.

B cell memory was shown to develop in congenitally athymic (nu/nu) mice after injection with small amounts of thymus-dependent antigens, in particular heterologous serum proteins, such as fown gamma-globulin (FGG) or DNP-bovine-serum albumin (DNP-BSA). Large doses of proteins (10 mg) tended to produce a specific B cell unresponsiveness, although there was still some evidence of B cell priming. The antigen did not have to be in a multivalent form to interact with B cell so as to induce immunologic memory or tolerance. In contrast to the induction of B cell memory, the production of IgG antibody in this system was found to be strongly T cell dependent. Thymus-independent antigens like LPS or POL with pronounced adjuvant effects on IgG production in normal or surgically thymectomized mice, could not replace T cells in allowing an IgG response against thymus-dependent antigens in congenitally athymic mice. However, the action of T cells once activated is likely to be non-antigen-specific, since it was shown that supernatants of antigen-activated-syngeneic T cells stimulated IgG production in cultures of primed B cell populations non-antigen-specifically.     

IL-4 suppression of in vivo T cell activation and antibody production

Injection of mice with a foreign anti-IgD Ab stimulates B and T cell activation that results in large cytokine and Ab responses. Because most anti-IgD-activated B cells die before they can be stimulated by activated T cells, and because IL-4 prolongs the survival of B cells cultured with anti-Ig, we hypothesized that treatment with IL-4 at the time of anti-IgD Ab injection would decrease B cell death and enhance anti-IgD-induced Ab responses. Instead, IL-4 treatment before or along with anti-IgD Ab suppressed IgE and IgG1 responses, whereas IL-4 injected after anti-IgD enhanced IgE responses. The suppressive effect of early IL-4 treatment on the Ab response to anti-IgD was associated with a rapid, short-lived increase in IFN-gamma gene expression but decreased CD4+ T cell activation and decreased or delayed T cell production of other cytokines. We examined the possibilities that IL-4 stimulation of IFN-gamma production, suppression of IL-1 or IL-2 production, or induction of TNF-alpha or Fas-mediated apoptosis could account for IL-4's suppressive effect. The suppressive effect of IL-4 was not reversed by IL-1, IL-2, or anti-TNF-alpha or anti-IFN-gamma mAb treatment, or mimicked by treatment with anti-IL-2Ralpha (CD25) and anti-IL-2Rbeta (CD122) mAbs. Early IL-4 treatment failed to inhibit anti-IgD-induced Ab production in Fas-defective lpr mice; however, the poor responsiveness of lpr mice to anti-IgD made this result difficult to interpret. These observations indicate that exposure to IL-4, while T cells are first being activated by Ag presentation, can inhibit T cells activation or promote deletion of responding CD4+ T cells.     

Modulation of Immune Response by Bacterial Lipopolysaccharide (LPS): Roles of Macrophages and T Cells in In Vitro Adjuvant Effect of LPS on Antibody Response to T Cell-Dependent and T Cell-Independent Antigens

The roles of macrophages and T cells in the adjuvant effect of lipopolysaccharide (LPS) were studied. In vitro anti-trinitrophenyl (anti-TNP) antibody responses to TNP-Ficoll and TNP-keyhole limpet hemocyanine (TNP-KLH) in spleen cells of C57BL/6 mice showed the most enhancement, when LPS was added to cultures at 1 μg/ml 48 hr after culture was started. The responses to these antigens were enhanced markedly by LPS in whole and macrophage-depleted spleen cells. The enhancement was greater in the latter group than in the former. The adjuvant effect among whole, T cell-depleted, macrophage-depleted and both macrophage- and T cell-depleted spleen cells was compared. The response to TNP-Ficoll was enhanced markedly by LPS in all groups. The enhancement was greater in the latter two groups than in the first two groups. The response to TNP-KLH was enhanced by LPS strongly in macrophage-depleted spleen cells, moderately in whole and both macrophage- and T cell-depleted spleen cells, and only slightly in T cell-depleted spleen cells. Enhancement was restored to T cell-depleted spleen cells by adding T cells. The response to TNP-KLH of macrophage-depleted spleen cells of LPS-responsive C3H/HeN mice which was enhanced by LPS was suppressed by adding splenic macrophages of C3H/HeN mice, but not of LPS-nonresponsive C3H/HeJ mice. The response to TNP-KLH of macrophage-depleted spleen cells of C3H/HeJ mice was not enhanced by LPS, irrespective of the addition of macrophages of C3H/HeN mice. The results indicate that B cells are activated directly by LPS, and T cells enhance and macrophages suppress the adjuvant effect of LPS.     

Follicular helper NKT cells induce limited B cell responses and germinal center formation in the absence of CD4(+) T cell help

B cells require MHC class II (MHC II)-restricted cognate help and CD40 engagement by CD4(+) T follicular helper (T(FH)) cells to form germinal centers and long-lasting Ab responses. Invariant NKT (iNKT) cells are innate-like lymphocytes that jumpstart the adaptive immune response when activated by the CD1d-restricted lipid α-galactosylceramide (αGalCer). We previously observed that immunization of mice lacking CD4(+) T cells (MHC II(-/-)) elicits specific IgG responses only when protein Ags are mixed with αGalCer. In this study, we investigated the mechanisms underpinning this observation. We find that induction of Ag-specific Ab responses in MHC II(-/-) mice upon immunization with protein Ags mixed with αGalCer requires CD1d expression and CD40 engagement on B cells, suggesting that iNKT cells provide CD1d-restricted cognate help for B cells. Remarkably, splenic iNKT cells from immunized MHC II(-/-) mice display a typical CXCR5(hi)programmed death-1(hi)ICOS(hi)Bcl-6(hi) T(FH) phenotype and induce germinal centers. The specific IgG response induced in MHC II(-/-) mice has shorter duration than that developing in CD4-competent animals, suggesting that iNKT(FH) cells preferentially induce transient rather than long-lived Ab responses. Together, these results suggest that iNKT cells can be co-opted into the follicular helper function, yet iNKT(FH) and CD4(+) T(FH) cells display distinct helper features, consistent with the notion that these two cell subsets play nonredundant functions throughout immune responses.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

IgE enhances antibody and T cell responses in vivo via CD23+ B cells

IgE Abs, passively administered together with their specific Ag, can enhance the production of Abs recognizing this Ag by >100-fold. IgE-mediated feedback enhancement requires the low affinity receptor for IgE, CD23. One possible mechanism is that B cells take up IgE-Ag via CD23 and efficiently present Ag to Th cells, resulting in better Ab responses. To test whether IgE Abs have an effect on Th cells in vivo, mice were adoptively transferred with CD4+ T cells expressing a transgenic OVA-specific TCR, before immunization with IgE anti-TNP (2,4,6-trinitrophenyl) plus OVA-TNP or with OVA-TNP alone. IgE induced a 6- to 21-fold increase in the number of OVA-specific T cells. These cells acquired an activated phenotype and were visible in splenic T cell zones. The T cell response peaked 3 days after immunization and preceded the OVA-specific Ab response by a few days. Transfer of CD23+ B cells to CD23-deficient mice rescued their ability to respond to IgE-Ag. Interestingly, in this situation also CD23-negative B cells produce enhanced levels of OVA-specific Abs. The data are compatible with the Ag presentation model and suggest that B cells can take up Ag via "unspecific" receptors and activate naive T cells in vivo.     

The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses

Polyomavirus (PyV) infection elicits protective T cell-independent (TI) IgG responses in T cell-deficient mice. The question addressed in this report is whether CD40 signaling plays a role in this TI antiviral IgG response. Because CD40 ligand (CD40L) can be expressed on numerous cell types in addition to activated T cells, it is possible that cells other than T cells provide CD40L to signal through CD40 on B cells and hence positively influence the antiviral TI IgG responses. In this study we show, by blocking CD40-CD40L interactions in vivo with anti-CD40L Ab treatment in TCR betaxdelta-/- mice and by using SCID mice reconstituted with CD40-/- B cells, that the lack of CD40 signaling in B cells results in a 50% decrease in TI IgG secreted in response to PyV. SCID mice reconstituted with CD40L-/- B cells also responded to PyV infection with diminished IgG secretion compared with that of SCID mice reconstituted with wild-type B cells. This finding suggests that B cells may provide the CD40L for CD40 signaling in the absence of T cell help during acute virus infection. Our studies demonstrate that, although about half of the TI IgG responses to PyV are independent of CD40-CD40L interactions, these interactions occur in T cell-deficient mice and enhance antiviral TI Ab responses.     

In vivo treatment with monoclonal anti-I-A antibodies: disappearance of splenic antigen-presenting cell function concomitant with modulation of splenic cell surface I-A and I-E antigens

A single injection of anti-I-Ak antibody (AB) into H-2k mice resulted in abrogation of splenic antigen-presenting cell (APC) function for protein antigen-primed T cells or alloantigen-specific T cells. Spleen cells from anti-I-A-treated mice are not inhibitory in cell mixing experiments when using cloned antigen-specific T cells as indicator cells, thus excluding a role for suppressor cells in the observed defect. Also, nonspecific toxic effects and carry-over of blocking Ab were excluded as causes for the defect. Experiments with anti-I-Ak Ab in (H-2b X H-2k)F1 mice showed abrogation of APC function for T cells specific for both parental I-A haplotypes. In homozygous H-2k mice, anti-I-Ak treatment not only abrogated APC function for I-Ak-restricted cloned T cells but also for I-AekE alpha k-restricted cloned T cells. FACS analysis of spleen cells from anti-I-Ak-treated (H-2b X H-2k)F1 mice revealed the disappearance of all Ia antigens (both I-A and I-E determined), whereas the number of IgM-bearing cells was unaffected. The reappearance of APC function with time after injection was correlated with the reappearance of I-A and I-E antigen expression. In vitro incubation of spleen cells from anti-I-A-treated mice led to the reappearance of Ia antigen expression and APC function within 8 hr. Thus, it appears that B cells (as determined by FACS analysis) and APC (as determined by functional analysis) behave similarly in response to in vivo anti-I-A Ab treatment. We interpret these findings as suggesting that in vivo anti-I-A treatment temporarily reduces the expression of Ia molecules through co-modulation on all Ia-bearing spleen cells, thereby rendering them incompetent as APC. Such modulation of Ia molecules does not occur when spleen cells are incubated in vitro with anti-I-A antibodies. These results imply that a primary defect purely at the level of APC in anti-I-A-treated mice may be responsible for the observed T cell nonresponsiveness when such mice are subsequently primed with antigen.     

Functional analysis of T lymphocyte subsets in antiviral host defense

The role of different T cell subsets in antiviral host defense was investigated by treating thymectomized C57BL/6 and CBA/J mice with monoclonal rat anti-Lyt-2 or anti-L3/T4 IgG 2b antibodies 14 and 10 days before infection. This treatment depleted the respective T cell subsets to undetectable levels in peripheral blood when assayed by immunofluorescence. In mice treated with anti-Lyt-2, induction of cytotoxic T cells was reduced to less than 1 to 2% after intravenous infection with Armstrong strain of lymphocytic choriomeningitis virus (LCMV). In addition, no primary swelling of the footpad could be detected following local inoculation of the virus. In animals treated with anti-L3/T4, antiviral cytotoxic T lymphocyte responses were reduced by a factor of 10. These L3/T4+ cell-depleted mice showed delayed footpad swelling after local injection of LCMV Armstrong. After intracerebral infection with LCMV, anti-Lyt-2-treated mice were resistant and those injected with anti-L3/T4 were totally susceptible to LCMV Armstrong-triggered immunopathologic disease. Virus could be detected in the blood of antibody-treated mice 7 days after inoculation; however, no virus could be measured in the blood of surviving anti-Lyt-2-treated animals 15 days after intracerebral infection. Serum titers of interferon-alpha,beta induced by viral infection remained unaffected by depletion of T cell subsets. Anti-L3/T4 antibody-treated C57BL/6 mice failed to generate IgG antibodies against the New Jersey strain of vesicular stomatitis virus, whereas Lyt-2+ cell-depleted mice had normal antivesicular stomatitis virus (New Jersey strain) IgG antibody titers.