Mapping of T helper cell epitopes by using peptides spanning the 19-kDa protein of Mycobacterium tuberculosis. Evidence for unique and shared epitopes in the stimulation of antibody and delayed-type hypersensitivity responses.

In vivo and in vitro T cell responses to overlapping 20-mer peptides that span the entire 19-kDa protein of Mycobacterium tuberculosis have been compared in three different strains of mice. Immunization of the mice with peptides and analysis of specific antibody production is an in vivo assay of Th cell activity. Peptides 1-20 and 61-80 elicited strong IgG1 responses in BALB/cJ, C57BL/10J, and B10.BR mice, indicating that these peptides could stimulate Th cells, possibly of a Th2 phenotype. T cells isolated from peptide-immunized mice were challenged in vitro with peptide, and their proliferative responses were analyzed. T cells from these three strains of mice immunized with peptides 1-20, 61-80, and 76-95 also responded to challenge with specific peptide in vitro. In addition, B10.BR mice and BALB/cJ mice showed antibody and T cell proliferative responses to peptides 136-155 and 145-159, respectively. Thus, in vitro proliferating T cells were found to possess specificities for peptide epitopes that were almost identical to those of the antibody-producing cells. Delayed-type hypersensitivity (DTH) responses to these peptides were also examined in the three strains. Interestingly, the T cells responding in the DTH assay had Ag specificities that were quite different from those identified in the antibody and proliferation assays. These results suggested that DTH Th cells form a separate population from antibody Th and proliferative T cells and these populations of cells were differentially activated, in an Ag-specific manner.     

Aqueous antigens induce in vivo tolerance selectively in IL-2- and IFN-gamma-producing (Th1) cells.

As a model for understanding in vivo immune responses, we have exposed mice to aqueous haptenated-protein Ag, and examined immune responses to subsequent immunization with Ag in adjuvant. Pretreating mice with soluble, TNP-conjugated Ag induces selective nonresponsiveness to Ag for both humoral and cell-mediated immune functions. Specific T cell proliferation in response to Ag is inhibited, and Ag-induced secretion of the lymphokines IL-2 and IFN-gamma, but not IL-4, is reduced. B cell responses after pretreatment are also affected. Although levels of TNP-specific IgG1 and IgE are similar in treated and untreated mice, soluble Ag pretreatment diminishes production of TNP-specific IgG2a and IgG2b. This is due to lack of T cell help and is not caused by tolerance in the B cell compartment. These results indicate that pretreatment of mice with aqueous Ag induces selective unresponsiveness in Th1-like Th cells, which secrete IL-2 and IFN-gamma, but not in Th2-like Th cells, which secrete IL-4.     

Abnormal regulation of IFN-gamma secretion in vitamin A deficiency

T lymphocytes from vitamin A-deficient (A-) mice show a decreased ability to stimulate B lymphocytes for Ag-specific secondary IgG1 responses in vivo and in vitro. Experiments reported here traced the molecular basis for this functional defect to an overproduction of IFN-gamma by A- CD4+ T cells compared with cells from A-sufficient (A+) mice. Secretion of IL-2 and IL-4 by cells from A- and A+ mice was equivalent. Retinoic acid supplementation in vitro decreased IFN-gamma secretion from A- T cells, indicating that IFN-gamma production is retinoid-responsive. Adding IFN-gamma neutralizing antibodies to cultures established with cells from immune A- mice substantially increased IgG1 production, whereas IL-4 addition moderately increased IgG1 production. Adding retinoic acid to the cultures either at initiation, or 48 h later, fully restored IgG1 production by A- cultures to the level of A+ control cultures. These results are consistent with a role for vitamin A in negatively regulating IFN-gamma secretion.     

Evidence for differential induction of helper T cell subsets during Trichinella spiralis infection

The H-2-compatible mouse strains, AKR and B10.BR, exhibit disparate responses to infection with the parasitic nematode Trichinella spiralis. The resistant AKR mice expel intestinal adult worms faster than susceptible B10.BR mice. We tested antibody and lymphokine responses in these strains. With respect to antibody responses, the B10.BR mice had 3- to 10-fold more serum IgE and T. spiralis-specific IgG1 and IgA than AKR mice. The B10.BR mice also had greater numbers of IgG and IgA plaque-forming cells than AKR mice. In contrast, AKR mice produced T. spiralis-specific IgG2a, whereas the B10.BR mice did not. The antibody response kinetics of these strains were similar. We also analyzed lymphokine secretion after restimulating lymphocytes in vitro with T. spiralis Ag. The AKR mesenteric lymph node cells produced more IFN-gamma and less IL-4 than the B10.BR mesenteric lymph node cells. The B10.BR splenocytes produced more IL-4 than the AKR splenocytes, although splenocyte IFN-gamma production was not different. The kinetics of IL-4 production also differed between the two strains. In summary, resistant AKR mice produced more IFN-gamma and T. spiralis-specific IgG2a than susceptible B10.BR mice, which produced more IL-4, IgE, and T. spiralis-specific IgG1. Our results are consistent with differential activation of Th cell subsets in T. spiralis-infected AKR and B10.BR mice.     

IgA responses in xid mice: oral antigen primes Peyer's patch cells for in vitro immune responses and secretory antibody production

Because the gut-associated lymphoreticular tissue (GALT), e.g., Peyer's patches (PP), of X-linked immunodeficient (xid) mice possesses a subpopulation of mature B cells, we have characterized the ability of xid mice to respond to the thymic-dependent antigen sheep erythrocytes (SRBC) given by the oral route. Gastric intubation of SRBC to xid (CBA/N X DBA/2) F1 male or CBA/N mice, followed by the in vitro culture of dissociated PP cells with SRBC, resulted in IgM, IgG1, IgG2, and high IgA anti-SRBC plaque-forming cell (PFC) responses. The addition of unprimed PP but not splenic T cells to splenic xid B cell cultures resulted in IgM anti-SRBC PFC responses, suggesting the importance of GALT T cells for support of the immune responses to SRBC by splenic B cells from xid mice. Furthermore, purified PP T cells from SRBC orally primed xid mice supported in vitro IgA anti-SRBC PFC responses in B cell cultures from either the PP or the spleens of nonprimed xid mice. Higher IgA responses, however, occurred in PP, when compared with splenic B cell cultures. Additional evidence that the GALT of xid mice contains functional IgA precursor cells was provided by the finding that cloned H-2k PP T helper cells (PP Th A) supported IgA responses in PP B cell cultures derived from (CBA/N X C3H/HeN) F1 male (xid) mice. On the other hand, splenic B cells from these xid mice, in the presence of PP Th A cells, did not support in vitro responses. These results suggest that unique subpopulations of T cells occur in the GALT of xid and normal mice; one T cell subpopulation may induce immature B cells to become precursor IgA cells in the PP. A separate GALT T cell subpopulation, e.g., isotype-specific helper T cells, effectively collaborates with mature IgA B cells for the induction of IgA responses to orally administered antigen. When xid mice were gastric intubated with SRBC, followed by i.p. injection of SRBC, good splenic IgA anti-SRBC PFC responses were seen. Salivary and serum IgA antibodies were also detected in these xid mice. Nevertheless, the magnitude of the anti-SRBC response in xid mice was lower than that seen in similarly treated normal mice. These studies indicate that the GALT of both xid and normal mice possess unique populations of T cells that support in vitro responses in xid B cell cultures from either the spleen or the PP, which direct the mature B cell populations present toward IgA isotype-specific responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

B cells are crucial for determinant spreading of T cell autoimmunity among beta cell antigens in diabetes-prone nonobese diabetic mice

The determinant spreading of T cell autoimmunity plays an important role in the pathogenesis of type 1 diabetes and in the protective mechanism of Ag-based immunotherapy in NOD mice. However, little is known about the role of APCs, particularly B cells, in the spreading of T cell autoimmunity. We studied determinant spreading in NOD/scid or Igmu(-/-) NOD mice reconstituted with NOD T and/or B cells and found that mice with mature B cells (TB NOD/scid and BMB Igmu(-/-) NOD), but not mice that lacked mature B cells (T NOD/scid and BM Igmu(-/-) NOD), spontaneously developed Th1 autoimmunity, which spread sequentially among different beta cell Ags. Immunization of T NOD/scid and BM Igmu(-/-) NOD mice with a beta cell Ag could prime Ag-specific Th1 or Th2 responses, but those T cell responses did not spread to other beta cell Ags. In contrast, immunization of TB NOD/scid and BMB Igmu(-/-) NOD mice with a beta cell Ag in IFA induced Th2 responses, which spread to other beta cell Ags. Furthermore, we found that while macrophages and dendritic cells could evoke memory and effector T cell responses in vitro, B cells significantly enhanced the detection of spontaneously primed and induced Th1 responses to beta cell Ags. Our data suggest that B cells, but not other APCs, mediate the spreading of T cell responses during the type 1 diabetes process and following Ag-based immunotherapy. Conceivably, the modulation of the capacity of B cells to present Ag may provide new interventions for enhancing Ag-based immunotherapy and controlling autoimmune diseases.     

Immune deficiency in chronic Trypanosoma cruzi infection. Recombinant IL-1 restores Th function for antibody production

Mice with chronic Trypanosoma cruzi infections are unable to mount primary responses to T-dependent Ag, such as SRBC. Responses to SRBC were restored in vitro and in vivo with rIL-1. The cellular basis of the immunodeficiency and the mechanism of IL-1 action were investigated. B cells from infected mice were capable of normal levels of PFC production when provided with the appropriate signals, IL-2 plus IL-1. T cells from infected mice were unable to provide Th function to normal B cells. However, Th activity was provided by these cells if IL-1 was added to the cultures. Furthermore, T-depleted spleen cells from infected mice did not make antibody in the presence of normal T cells unless IL-1 was added to the cultures. Neutralizing antibody against IL-2 greatly reduced the augmentation by IL-1 of the antibody response of cells from infected mice. Together these results indicate that splenic B cells from infected mice are capable of antibody production, but that Th function is lacking in the spleens of infected mice. These results suggest that the inability of mice with T. cruzi infection to mount primary antibody responses to T-dependent Ag may be due to a macrophage defect lending to impairment of Th function. These results document the potential of IL-1 in restoring immune competence in an infectious disease model.     

IgE class switching is critically dependent upon the nature of the B cell activator, in addition to the presence of IL-4.

Cross-linkage of membrane IgD on resting murine B cells, by anti-IgD mAb conjugated to dextran (alpha delta-dex), induces high levels of proliferation, and in the presence of IL-2 or IL-5, Ig secretion in vitro. The structural and functional similarities between alpha delta-dex and TNP-Ficoll for B cell responses led us to propose that alpha delta-dex could provide a model system for studying B cell activation induced by T cell-independent, type II Ag. In this report, we study the effects of Ig class switch and differentiation factors on Ig isotype production by murine B cells activated by alpha delta-dex, and directly compare these to responses obtained after activation by LPS. We show that an IL-4-containing CD4+ T cell supernatant (Th2 SN) stimulates large increases in IgG1 and IgE production by LPS-activated B cells, but fails to stimulate detectable levels of IgE by alpha delta-dex-activated cells, despite inducing high levels of secreted IgM and IgG1. This is correlated with undetectable steady state levels of both germ-line and rearranged (productive) IgE-specific RNA in B cells stimulated with alpha delta-dex + Th2 SN. Alpha delta-dex is selective in its failure to costimulate IgE production in that IFN-gamma-containing T cell supernatant (Th1 SN) and transforming growth factor-beta-supplemented Th2 SN selectively stimulate a large IgG2a and IgA secretory response, respectively. Anti-IgD conjugated to Sepharose beads, in distinct contrast to dextran, costimulates a strong IgE response. These findings underscore the importance of the specific B cell activator, in addition to IL-4, in the regulation of IgE production.     

GIF inhibits Th effector generation by acting on antigen-presenting B cells

Glycosylation-inhibiting factor (GIF) is a 13-kDa cytokine secreted from T cells. Administration of bioactive recombinant GIF inhibits IgG1 and IgE Ab responses in vivo. Treatment of B cells with the cytokine reduces the secretion of IgG1 and IgE induced by LPS and IL-4. To examine the effect on cognate T-B interaction, GIF was added to low-density B cells from MD4 transgenic (Tg) mice, which express B cell receptor specific for hen egg lysozyme (HEL). The B cells were subsequently pulsed with HEL-OVA conjugate and cultured with OVA-specific naive CD4 T cells from DO11.10 Tg mice. Treatment of Ag-presenting B cells with GIF reduced expansion and IL-2 secretion of naive T cells and rendered them hyporesponsive to antigenic restimulation, resulting in 50--95% reduction of IL-4 and IFN-gamma secretion upon restimulation with Ag. GIF dramatically inhibited Th effector generation when it was added to B cells before pulsing with HEL-OVA, whereas it showed little to no effect when added after B cells were pulsed with Ag. GIF was more effective when B cells from MD4 Tg mice were pulsed with HEL-OVA than when they were pulsed with OVA. This cytokine did not affect Th effector generation when B cells or irradiated splenocytes pulsed with OVA(323--339) peptide stimulated naive DO11.10 T cells. Confocal microscopy revealed that GIF inhibited internalization of HEL by B cells from MD4 Tg mice. Therefore, the cytokine may regulate early steps of Ag presentation involving B cell receptors to diminish Th effector generation from naive CD4 T cells.     

Antigen-specific sIalo B cells do not secrete IgG2a in response to TH1 cells and antigen: responsiveness can be restored by IL-4

The ability of Th cells, type 1 (TH1), to activate and induce differentiation of B cells into antibody-secreting cells is controversial because 1) some clones of TH1 cells provide help while others do not, and 2) by using the same TH1 clone, different laboratories disagree on whether they provide help to B cells. One possible explanation for the latter is the variability in the activation status of the B cells used in different laboratories. In the present studies, we have used Ag-specific B cells from athymic (nu/nu) mice, or sterilely housed nu/+ mice to study the TH1-mediated activation of B cells that had received little or no prior help from T cells and/or antigen in vivo. These B cells express low levels of surface Ia (sIa) Ag, and fail to secrete IgG2a in response to TH1 cells plus Ag; in contrast, responses to TH2 cells plus Ag are normal. To explore this observation further, we prepared "surface(s) Ia1o" B cells from conventionally housed BALB/c mice by sorting spleen cells on the fluorescence-activated cell sorter. This sIalo population also failed to produce IgG2a in response to TH1 cells plus Ag. In contrast, the sIahi, (presumably more mature) B cells, responded to both the TH1 and TH2 cells. The addition of LPS, TH2 cells or the lymphokine, IL-4, to cultures of sIalo B cells from normal or nu/nu mice (plus Ag and TH1 cells), restored IgG2a responses to control levels. Low sIa levels were not the sole cause of nonresponsiveness of the nu/nu B cells because a 24-h pulse with IL-4 restored sIa to control levels without restoring IgG2a production after activation with TH1 cells plus Ag. These data support the conclusion that sIalo B cells are immature and require an activation/maturation signal from IL-4 in vivo in order to respond to TH1 cells and Ag in vitro.     

The roles of CD28 and CD40 ligand in T cell activation and tolerance

Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28-/- T cells showed a greater reduction in proliferative responses to Ag than did CD40L-/- T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L-/- T cells were defective in Th1 development. In vivo, CD28-/- T cells failed to expand upon immunization, whereas CD40L-/- T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and CD40L:CD40 interactions has an additive effect in inhibiting T cell responses.     

Comparative study of the T cell response to two allelic forms of a malarial vaccine candidate protein.

T cell responses to two allelic forms of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum were mapped in mice using the rMSA2 proteins, Ag 1609 which has the sequence of the FCQ27/PNG strain and Ag 1615 which has the sequence of the Indochina 1 strain. Lymph node cells of BL/10 and B10.BR mice immunized with either Ag 1609 or Ag 1615 responded to both Ag in in vitro proliferation assays. Lymph node cells of BALB/c mice did not respond. The T cell determinants recognized by the responder strains were mapped to conserved and variant regions of these Ag using overlapping synthetic peptides. The determinants recognized by each mouse strain were distinct. Marked difference in sequence between the central regions of the two rMSA2 proteins did not affect antigenic processing of the conserved N and C terminal regions. Hence lymph node cells of BL/10 mice immunized with either Ag 1615 or Ag 1609 recognized an immunodominant T cell determinant at the highly conserved N terminal end within the sequence YSNTFINNAYNMSIR (peptide 3b) and B10.BR mice similarly immunized recognized an immunodominant determinant at the highly conserved C terminal within the sequence CTDGNKENCGAATSL (peptide 23). Several peptides identified as containing immunodominant T cell determinants specific to BL/10 mice induced peptide-specific T cells in both BL/10 and B10.BR mouse strains when used as immunogens. However, the ability of the peptide-primed T cells to proliferate in response to the rMSA2 proteins was confined to BL/10 mice. An example of this was observed with peptides 3b and N (KNESKYSNTFINNAYNMSIRRSMAN). Peptide N was able to prime B10.BR and BL/10 mice for an enhanced antibody response when these mice were subsequently immunized with Ag 1615 even though Ag 1615-specific T cell proliferation was not detected in B10.BR mice primed with N. The study concluded that 1) conserved sequences such as peptide N when used in vaccines may give rise to MSA2-specific memory Th cells amenable to boosting by subsequent exposure to all parasite strains and 2) peptide priming may be a useful pathway for inducing defined memory Th cells in a wider population and for preferentially inducing T dependent over T independent responses to some malarial Ag.     

Progressive depletion of peripheral B lymphocytes in 4-1BB (CD137) ligand/I-Ealpha)-transgenic mice

Interaction of 4-1BB (CD137) and its ligand (4-1BBL) is thought to positively regulate cell-mediated and humoral immune responses. We have prepared transgenic mouse strains that express 4-1BBL cDNA under the control of MHC class II I-Ealpha promoter. The 4-1BBL-transgenic mice show progressive splenomegaly and selective depletion of B220(+) B cells accompanied with low levels of circulating IgG and defective humoral responses to Ag challenge. In addition, splenocytes from the transgenic mice fail to provide stimulation for allogeneic T cells in both lymphoproliferative and CTL responses in vitro, whereas their T cells remain functionally normal. Our results reveal unexpected functions of 4-1BBL in the regulation of humoral immune responses and Ag presentation.     

ES-62, an immunomodulator secreted by filarial nematodes, suppresses clonal expansion and modifies effector function of heterologous antigen-specific T cells in vivo

ES-62 is a phosphorylcholine-containing glycoprotein secreted by filarial nematodes, which has previously been shown to possess a range of immunomodulatory capabilities. We now show, using a CD4+ transgenic TCR T cell adoptive transfer system, that ES-62 can modulate heterologous Ag (OVA)-specific responses in vivo. Thus, in contrast to the mixed IgG1-IgG2a response observed in control animals, ES-62-treated mice exhibited a Th2-biased IgG Ab response as evidenced by stable enhancement of anti-OVA IgG1 production and a profound inhibition of anti-OVA IgG2a. Consistent with this, Ag-specific IFN-gamma produced was suppressed by pre-exposure to ES-62 when T cells were rechallenged ex vivo. However, the response observed was not classical Th2, because although Ag-specific IL-5 production was enhanced by pre-exposure to ES-62, IL-13, and IL-4 were inhibited when T cells were rechallenged ex vivo. Moreover, such T cells produced lower levels of IL-2 and proliferated less upon Ag rechallenge ex vivo. Finally, pre-exposure to ES-62 inhibited the clonal expansion of the transferred Ag-specific CD4+ T cells and altered the functional response of such T cells in vivo, by modulating the kinetics and reducing the extent of their migration into B cell follicles.     

Cellular FLIP (long isoform) overexpression in T cells drives Th2 effector responses and promotes immunoregulation in experimental autoimmune encephalomyelitis

Cellular FLIP (c-FLIP) is an endogenous inhibitor of death receptor-induced apoptosis through the caspase 8 pathway. It is an NF-kappaB-inducible protein thought to promote the survival of T cells upon activation, and its down-regulation has been implicated in activation-induced cell death. We have generated transgenic mice overexpressing human c-FLIP long form (c-FLIP(L)) specifically in T cells using the CD2 promoter (TgFLIP(L)). TgFLIP(L) mice exhibit increased IgG1 production upon stimulation by a T cell-dependent Ag and a markedly enhanced contact hypersensitivity response to allergen. In addition to showing augmented Th2-type responses, TgFLIP(L) mice are resistant to the development of myelin oligodendrocyte glycoprotein 35-55 peptide-induced experimental autoimmune encephalomyelitis, a Th1-driven autoimmune disease. In vitro analyses revealed that T cells of TgFLIP(L) mice proliferate normally, but produce higher levels of IL-2 and show preferential maturation of Th2 cytokine-producing cells in response to antigenic stimulation. After adoptive transfer, these (Th2) cells protected wild-type recipient mice from experimental autoimmune encephalomyelitis induction. Our results show that the constitutive overexpression of c-FLIP(L) in T cells is sufficient to drive Th2 polarization of effector T cell responses and indicate that it might function as a key regulator of Th cell differentiation.     

Regulation of anthrax toxin-specific antibody titers by natural killer T cell-derived IL-4 and IFNγ

Activation of Natural Killer-like T cells (NKT) with the CD1d ligand α-GC leads to enhanced production of anthrax toxin protective Ag (PA)-neutralizing Abs, yet the underlying mechanism for this adjuvant effect is not known. In the current study we examined the role of Th1 and Th2 type responses in NKT-mediated enhancement of antibody responses to PA. First, the contribution of IL-4 and IFNγ to the production of PA-specific toxin-neutralizing Abs was examined. By immunizing C57Bl/6 controls IL-4(-/-) mice and IFNγ(-/-) mice and performing passive serum transfer experiments, it was observed that sera containing PA-specific IgG1, IgG2b and IgG2c neutralized toxin in vitro and conferred protection in vivo. Sera containing IgG2b and IgG2c neutralized toxin in vitro but were not sufficient for protection in vivo. Sera containing IgG1 and IgG2b neutralized toxin in vitro and conferred protection in vivo. IgG1 therefore emerged as a good correlate of protection. Next, C57Bl/6 mice were immunized with PA alone or PA plus a Th2-skewing α-GC derivative known as OCH. Neutralizing PA-specific IgG1 responses were modestly enhanced by OCH in C57Bl/6 mice. Conversely, IgG2b and IgG2c were considerably enhanced in PA/OCH-immunized IL-4(-/-) mice but did not confer protection. Finally, bone marrow chimeras were generated such that NKT cells were unable to express IL-4 or IFNγ. NKT-derived IL-4 was required for OCH-enhanced primary IgG1 responses but not recall responses. NKT-derived IL-4 and IFNγ also influenced primary and recall IgG2b and IgG2c titers. These data suggest targeted skewing of the Th2 response by α-GC derivatives can be exploited to optimize anthrax vaccination.     

A novel B cell-mediated transport of IgE-immune complexes to the follicle of the spleen

Ag administered i.v. to mice along with specific IgE or IgG2a induces higher Ab- and CD4(+) T cell responses than Ag administered alone. The IgE effect is completely dependent on the low-affinity receptor for IgE, CD23, whereas the IgG2a effect depends on activating FcgammaRs. In vitro studies suggest that IgE/Ag is presented more efficiently than Ag alone to CD4(+) T cells by CD23(+) B cells and that IgG2a/Ag is presented by FcgammaR(+) dendritic cells (DCs). In this study, we investigate in vivo the early events leading to IgE- and IgG2a-mediated enhancement of immune responses. OVA administered i.v. in PBS in combination with specific IgE binds circulating B cells after 5 min and is found in B cell follicles bound to follicular B cells (CD23(high)) after 30 min. This novel B cell-dependent route of entry is specific for IgE because IgG2a-Ag complexes were trapped in the marginal zone. OVA-specific CD4(+) T cells were found at the T-B border in the T cell zones 12 h after immunization both with IgE/OVA or IgG2a/OVA and proliferated vigorously after 3 days. The findings suggest that IgE- and IgG2a-immune complexes are efficient stimulators of early CD4(+) T cell responses and that Ag bound to IgE has a specific route for transportation into follicles.     

Ag85B of mycobacteria elicits effective CTL responses through activation of robust Th1 immunity as a novel adjuvant in DNA vaccine

CD4+ T cells play a crucial role in CTL generation in a DNA vaccination strategy. Several studies have demonstrated the requirement of CD4+ T cells for the induction of a sufficient immune response by coadministrating DNAs. In the present study we investigated the effectiveness of Ag85B of mycobacteria, which is known to be one of the immunogenic proteins for Th1 development, as an adjuvant of a DNA vaccine. HIV gp120 DNA vaccine mixed with Ag85B DNA as an adjuvant induced HIV gp120-specific Th1 responses, as shown by delayed-type hypersensitivity, cytokine secretion, and increasing HIV-specific CTL responses. Moreover, these responses were enhanced in mice primed with Mycobacterium bovis bacillus Calmette-Guérin before immunization of HIV DNA vaccine mixed with Ag85B DNA. Furthermore, these immunized mice showed substantial reduction of HIV gp120-expressing recombinant vaccinia virus titers compared with the titers in other experimental mice after recombinant vaccinia virus challenge. Because most humans have been sensitized by spontaneous infection or by vaccination with mycobacteria, these findings indicate that Ag85B is a promising adjuvant for enhancing CTL responses in a DNA vaccination strategy.     

Experimental murine schistosomiasis in the absence of B7 costimulatory molecules: reversal of elicited T cell cytokine profile and partial inhibition of egg granuloma formation

The granulomatous inflammation in infection with the helminth Schistosoma mansoni represents a cellular hypersensitivity reaction mediated by, and dependent upon, MHC class II-restricted CD4+ Th cells sensitized to parasite egg Ags. The current work examines the role and significance of the B7:CD28/CTLA-4 pathway in providing the costimulation necessary for the activation of these pathogenic T cells. In vitro T cell responses in B7-1-/- mice, 7-8 wk postinfection, were no different from wild-type controls, but the absence of B7-2 molecules resulted in a decrease in egg Ag-induced proliferation with increased IFN-gamma production. Both B7-1-/- and B7-2-/- mice exhibited intact granuloma formation. In contrast, CD4+ Th cells from B7-1/2 double-deficient mice displayed a dramatic loss of proliferative capacity upon stimulation with egg Ag. Most strikingly, these T cells secreted only IFN-gamma, but not IL-4 and IL-10, a pattern entirely opposite to that displayed by wild-type controls. Despite these major differences in T cell reactivity, B7-1/2-/- mice had only a limited reduction of granuloma size and fibrosis, without appreciable difference in cellular composition. These results show that substantial granuloma formation can occur under conditions of limited T cell expansion and restricted Th1-type cytokine production. They also support the notion that the combined effect of B7 signaling is not as critical for Th1 cell activation as it is for the development of the Th2 dominant environment characteristic of the evolving schistosome infection in H-2b mice.