Regulation of T15 idiotype dominance. I. Mice expressing the xid immune defect provide normal help to T15+ B cell precursors

The immune response to phosphocholine (PC) in many strains of mice is dominated by the T15 idiotype family of anti-PC antibodies. By introducing the CBA/N X-linked immune defect (xid gene) into these mice, one profoundly alters their ability to make a T15-predominant, IgM anti-PC response. This loss of T15 dominance in mice expressing the xid gene is not due to the presence of suppressor T cells or the lack of T15 idiotype-specific helper cells in these mice. Thus, one can reconstitute a T15 idiotype-dominant response in immune defective mice with B cells from normal mice, and in adoptive transfer assays the primed T helper cells from immune-defective mice provide qualitatively the same help to normal B cells as the T helper cells from normal mice. T15 idiotype dominance appears to be controlled by the expression and activation of Lyb-5+ PC-specific B cells. Thus, the majority of T15+ B cell precursors are restricted to this B cell subset, whereas the Lyb-5- B cell subset contains predominantly T15-, anti-PC B cell precursors, which produce mainly IgG antibodies after activation by PC-containing antigens.     

Polyclonal activation of xid B cells by auto-Ia-reactive T cell clones

The mechanism of T cell-dependent activation of xid B cells into Ig-producing cells was studied by employing H-2-restricted, antigen-specific T cell clones. Helper factors (B cell stimulatory factors, BSF) released from KLH-specific T cell lines could induce polyclonal Ig production in B cells from (CBA/N X BALB/c)F1 (NBF1) female mice but not from CBA/N or NBF1 male mice. Direct addition of helper T cell lines induced Ig production in xid B cells from CBA/N or NBF1 male mice. A T cell clone, MK6, which was derived from NBF1 male mice and specific against Iad determinant, could activate NBF1 male but not CBA/N B cells. Another clone, CK4, derived from CBA/N mice and having specificity against KLH plus I-Ak determinant could activate both CBA/N and NBF1 male B cells into IgM- and IgG-producing cells in the absence of KLH, and monoclonal anti-I-Ak antibody specifically blocked such activation. These results suggest that xid B cells are able to be activated by the signal provided by the recognition of Ia molecules on B cells by auto-Ia-reactive T cells. Xid B cells from CBA/N mice that had been co-cultured with a T cell line specific against I-Ak determinant for 24 hr became reactive to BSF and capable of differentiating into Ig-producing cells in the presence of BSF. The results showed that even xid B cells could be responsive to BSF if they were in a certain activation stage.     

Constitutive production by the WEHI-3 cell line of B cell growth and differentiation factor that co-purifies with interleukin 1

The myelomonocytic cell line WEHI-3 produces constitutively a factor that affects the growth and differentiation of murine B cells in culture. This cell line also secretes colony-stimulating factors (CSF), interleukin 1 (IL 1) but not interleukin 2. Sequential purification through AcA54 gel filtration, DEAE-Sephacel ion exchange chromatography, and buffer electrofocussing clearly resolved the B cell growth and differentiation factor (BGDF) from the CSF activities but failed to separate BGDF from IL 1. The WEHI-3-derived material responsible for BGDF/IL 1 activity, however, exhibited different behavior on DEAE chromatography (elution at 175 mM NaCl) to that reported for IL 1 from the P388D1 cell line (elution at 50 mM NaCl). B cell growth and differentiation could be induced by WEHI-3 BGDF/IL 1 in cultures of normal spleen cells depleted of T cells and adherent cells but not in cultures of spleen cells from B cell-deficient CBA/N mice, even though thymocytes from such mice displayed a normal response to IL 1. Significant B cell proliferation induced by BGDF/IL 1 was apparent only in the presence of submitogenic concentrations of anti-mouse IgM antibodies, but under these conditions few antibody-forming cells (AFC) were generated. In contrast, B cell differentiation to AFC occurred in the presence of the factor alone, and this response was inhibited by anti-IgM. Thus there appeared to be a reciprocal relationship between B cell proliferation and differentiation induced by BGDF/IL 1. The significance of these results is discussed in the light of other recent studies of BGDF.     

Detection of receptors for murine B cell stimulatory factor 1 (BSF1): presence of functional receptors on CBA/N splenic B cells

The presence of receptors specific for murine B cell stimulatory factor 1 (BSF1) was demonstrated by utilizing an internally radiolabeled recombinant BSF1. Radiolabeled BSF1 was efficiently produced in Xenopus laevis oocytes injected with a cloned mRNA for BSF1 and 35S-methionine. The labeled BSF1 specifically bound to splenic B cells. A Scatchard analysis indicated the existence of one class of receptor sites. BSF1 receptors were found to be distributed on a wide range of hematopoietic lineage cells, including B cells, T cells, macrophages, and mast cells. B cells from CBA/N mice with the xid gene defect had a similar level of BSF1 binding capacity compared with BALB/c strain B cells, and responded well to insoluble anti-Ig and BSF1 in proliferation assays, indicating that CBA/N B cells express functional BSF1 receptors at normal levels. Pre-B cell lines showed low levels of BSF1 binding, suggesting that cells in the B cell lineage acquire BSF1 responsiveness early in development.     

Transferred B cells from autoimmune NZB/N mice fail to activate T suppressor cells

T-cell-mediated suppression of the antibody response of autoimmune NZB/N mice to Type III pneumococcal polysaccharide (SSS-III) can readily be induced in situ by priming with a subimmunogenic dose of SSS-III; however, the transfer of either "young" (8 weeks old) or "old" (42 weeks old) SSS-III-primed B cells, which activates suppressor T cells in normal BALB/cByJ mice, fails to induce suppression of the antibody response in recipient NZB/N mice, regardless of the number of cells transferred or the time interval between transfer and immunization. Transfer of 51Cr-labeled B cells demonstrated that syngeneic primed B cells home to the spleens of NZB/N mice in somewhat lower numbers than in BALB/cByJ mice, although the differences observed may not be sufficient to explain the complete absence of activation of suppressor T cells. These findings suggest that B cells from autoimmune NZB/N mice are unable to activate T suppressor cells upon transfer; this disorder in a normal regulatory mechanism may be important in the pathogenesis of disease.     

Differential impact of T cell repertoire diversity in diabetes-prone or -resistant IL-10 transgenic mice

Expression of IL-10 transgene (tg) in pancreatic beta cells failed to induce autoimmune insulitis and diabetes in (BALB/c x NOD)F1 mice. However, IL-10-expressing tg littermates from backcrosses (N2 and N3) with NOD mice became diabetic at 5 to 10 weeks of age in an MHC-dependent manner. In this study, we tested the possibility that enhancement in frequency of islet antigen (Ag)-specific T cells overrides the protective effects of a diabetes-resistant genetic background and promotes diabetes in IL-10 tg (BALB/c x NOD)F1 mice. For this test, we introduced the IL-10 transgene into tg BDC2.5 mice expressing the islet Ag-specific Vbeta4 T cell repertoire by breeding Ins-IL-10+/BALB/c mice with BDC2.5 mice. The progeny (Ins-IL-10+/BALB/c x BDC2.5+)F1 mice doubly tg for IL-10 and Vbeta4 (BDC2.5) T cell repertoire, developed diabetes at 10 to 18 weeks of age with a much more aggressive T cell infiltrate in the pancreatic islets than in single tg mice. Surprisingly, these diabetic mice were free from acute pancreatitis but had apoptotic beta cells in the islet infiltrate. Conversely, mice tg for Vbeta4 (BDC2.5) T cell repertoire but not IL-10 had no diabetes and no apoptotic beta cells in the islet infiltrate. Therefore, an increase in the frequency of islet-specific T cells apparently overcomes the protection from diabetes by a resistant genetic background. Interestingly, N2 backcross mice doubly tg for Vbeta4 (BDC2.5) T cell repertoire and IL-10, compared to N2 backcross mice tg for IL-10 only, eventually became diabetic but with a delayed onset and reduced incidence of disease. These findings demonstrate that, along with IL-10, an increase in frequency of islet antigen-specific T cells (a) overrides the protective effect of genetic resistance to autoimmune diabetes in F1 mice and (b) delays the onset of an otherwise accelerated diabetes in (Ins-IL-10+/NOD)N2 backcross mice.     

Regulation of the antibody response to type III pneumococcal polysaccharide. V. Ontogeny of factors influencing the magnitude of the plaque-forming cell response.

Mice of different ages were evaluated with respect to their ability to give a plaque-forming cell (PFC) response to Type III pneumococcal polysaccharide (SSSIII), as well as the degree of amplifier and suppressor thymus-derived(T) cell activity present. Although the magnitude of the PFC response to an optimally immunogenic dose of SSS-III for 2-and 3-week old mice was only 7% and 14%, respectively, of that produced by adult (8-week old) mice, values comparable to those of adult animals were attained by 4 weeks of age; no significant changes in the ability to respond to SSS-III occurred thereafter. Amplifier T cell activity, which was minimal at 2 to 4 weeks of age, matured slowly and did not reach a maximum until 8 to 10 weeks of age. By contrast, suppressor T cell activity appeared to be fully developed at least as early as 2 weeks of age; here, the inhibitory effects produced could by abrogated by depletion of T cells, indicating that the unresponsiveneness induced by such cells does not result in the depletion ot irreversible inactivation of B cells capable of responding to SSS-III. These findings suggest that the inhibitory effects of suppressor T cells are predominant in young mice and that such cells may play an important role in determining the ease with which unresponsiveness is induced in neonates, and in the prevention of autoimmune disease. Also, studies conducted with adult-thymectomized mice showed that both amplifier and suppressor T cells, once seeded to the periphery, are stable and do not depend upon the presence of intact thymus for the expression or renewal of their activity.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

Modulated immune responsiveness associated with experimental Encephalitozoon cuniculi infection in BALB/c mice

Spleen cell blastogenesis to mitogens and antibody responses to sheep erythrocytes (sRBC) were tested in BALB/c mice with experimental E. cuniculi infections. Blastogenesis responses of spleen cells 1 week post-infection were significantly lower than normal to T-cell mitogens (Con A and PHA) and were unchanged in response to B-cell mitogens (LPS and PWM). After 2 weeks post-infection, the responses to T cell mitogens returned to normal. Mixing spleen cells from 1-week infected mice with cells from uninfected mice failed to reveal the presence of suppressor cells. Antibody responses to sRBC were significantly slower to develop in 1 week-infected mice compared with uninfected mice or mice infected 2 weeks earlier or at the same time as sRBC challenge. Infected mice displayed splenomegaly which was most pronounced 1 week post-infection and the differential spleen cell counts revealed the presence of lymphoblasts. Lymphohyperplasia appeared to cause the splenomegaly. No shifts in the proportion of Thy 1.2+ T cells, Ig+ B cells, or esterase-positive macrophages were detected. These results indicate that the immune system in BALB/c mice is depressed early during E. cuniculi infections.     

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.     

Contribution of Lyb 5+ and Lyb 5- B cells to the primary and secondary phosphocholine-specific antibody response

Due to a mutation on their X-chromosome, CBA/N mice lack the Lyb-5+ subset of B cells. The loss of this B cell subset results in a profound alteration in the immune response of these mice to the hapten phosphocholine (PC). Thus, when these mice are immunized with high doses of PC-KLH (200 micrograms) in CFA, they: 1) fail to produce IgM anti-PC antibodies; 2) produce little or no anti-PC antibody bearing the normally predominant T15-idiotype; and 3) produce IgG anti-PC antibodies only late in the primary response. In order to more fully delineate this defect in responsiveness to PC, the splenic focus assay was used to analyze Lyb-5- B cell precursors from both normal and immune defective mice. Lyb-5- cells were obtained from normal (CBA/N x DBA/2)F1 (CD) female spleens by treatment with anti-Lyb-5 serum and complement. These normal Lyb-5- cells and Lyb-5- cells from immune defective CD male mice were stimulated in vitro with either PC-Hy or TNP-Hy in the presence of Hy-primed T helper cells. The results demonstrate that primary Lyb-5- PC-specific B cells fail to respond in the splenic focus assay, while secondary Lyb-5- PC-specific precursors respond normally, and that both primary and secondary Lyb-5- TNP-specific precursors respond in the splenic focus assay. These data suggest that Lyb-5- PC-specific precursors must differentiate into memory cells before they can be activated to secrete antibody, and they also indicate that the Lyb-5- B cell subset may be composed of two subsets with different activation requirements.     

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)     

Immunoregulation in the Peyer's patch microenvironment. Cellular basis for the enhanced responses by the B cells of X-linked immunodeficient CBA/N mice

The Peyer's patches (PP) of X-linked immunodeficient (xid) CBA/N and hemizygous (CBA/N X DBA/2)F1 (CDF1) male mice contain a B cell subpopulation that expresses the Lyb-5 maturational marker and is responsive to type 2 and T cell-dependent antigens in vitro, a B cell phenotype which is absent from the spleens of xid mice. Experiments reported here show that xid spleen B cells co-cultured with B cell-depleted PP cells from xid mice differentiated into specific plaque-forming cells in response to trinitrophenyl-Ficoll (type 2) and sheep erythrocytes (T cell-dependent). Two cell types were involved in this normalization of xid B cell responses. An accessory cell activity present in the PP, but not the spleens, of both CDF1 male (xid) and CDF1 female (normal) mice was required for the response to either the type 2 or T cell-dependent antigens. In the presence of this PP accessory cell, T cells from the PP of either xid or normal mice supported responses to both classes of antigens. In contrast, T cells from the spleens of xid mice did not support the response to trinitrophenyl-Ficoll, although the splenic T cells from normal mice did synergize with PP accessory cells in allowing plaque-forming cell development by xid B cells to this type 2 antigen. The xid PP T cell activity required for the type 2 response by xid B cells was present in the Ly-1+, Lyt-2- subpopulation, and the xid PP accessory cell activity was provided by an enriched population of dendritic accessory cells. These results demonstrate the the lymphoreticular cells comprising the PP microenvironment provide effective support for the differentiation of xid B cells in response to type 2 and T cell-dependent antigens.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.     

The effects of age on the immune response to type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) in BALB/c, SJL/J, and C3H mice.

Type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) were used to evaluate B cell and T cell regulatory functions in BALB/c, SJL/J, and C3H mice of various ages. It was found that the BALB/c and C3H mice could mount high level plaque-forming cell (PFC) responses to SIII at various ages through 110 weeks whereas the levels of the SJL/J PFC responses had begun to decline by the age of 42 weeks through the age of 80 weeks. BALB/c mice were also capable of producing strong PFC responses to LPS at various ages through 110 weeks whereas the comparable SJL/J PFC responses to LPS had declined by 80 weeks of age. By using anti-lymphocyte serum (ALS) and low-dose paralysis to SIII, it was shown that suppressor T cell activity was apparently greater in young BALB/c mice than in older BALB/c mice. It was also found that paralysis to SIII in BALB/c mice was easier to achieve at an early age. SJL/J mice were found to have the necessary B cell activity to respond to SIII through 80 weeks of age and the PFC responses could be greatly enhanced by ALS. Implications of the roles of regulatory T cells in aging are discussed.     

RAGE ligation affects T cell activation and controls T cell differentiation

The pattern recognition receptor, RAGE, has been shown to be involved in adaptive immune responses but its role on the components of these responses is not well understood. We have studied the effects of a small molecule inhibitor of RAGE and the deletion of the receptor (RAGE-/- mice) on T cell responses involved in autoimmunity and allograft rejection. Syngeneic islet graft and islet allograft rejection was reduced in NOD and B6 mice treated with TTP488, a small molecule RAGE inhibitor (p < 0.001). RAGE-/- mice with streptozotocin-induced diabetes showed delayed rejection of islet allografts compared with wild type (WT) mice (p < 0.02). This response in vivo correlated with reduced proliferative responses of RAGE-/- T cells in MLRs and in WT T cells cultured with TTP488. Overall T cell proliferation following activation with anti-CD3 and anti-CD28 mAbs were similar in RAGE-/- and WT cells, but RAGE-/- T cells did not respond to costimulation with anti-CD28 mAb. Furthermore, culture supernatants from cultures with anti-CD3 and anti-CD28 mAbs showed higher levels of IL-10, IL-5, and TNF-alpha with RAGE-/- compared with WT T cells, and WT T cells showed reduced production of IFN-gamma in the presence of TTP488, suggesting that RAGE may be important in the differentiation of T cell subjects. Indeed, by real-time PCR, we found higher levels of RAGE mRNA expression on clonal T cells activated under Th1 differentiating conditions. We conclude that activation of RAGE on T cells is involved in early events that lead to differentiation of Th1(+) T cells.     

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.     

Regulatory mechanism of reagin production in mice at the T cell level. I. Suggestive evidence for the generation of class specific PPD-reactive helper T cell population in Mycobacterium-primed cells.

Helper T cell activities specific for purified protein derivative (PPD) generated by immunization with Mycobacterium tuberculosis (Tbc) or PPD were investigated concerning adoptive IgE and IgG antibody responses. It is interesting that preferential triggering activity of IgG antibody response was observed when PPD-reactive cells from mice immunized with Tbc were used as a helper cell source. The selective triggering of IgG B cells by Tbc-primed cells was consistently observed using DNP-primed B cell populations from mice immunized with DNP-carrier conjugate in either ICFA or alum. T cell dependency of helper activity was demonstrated by the fact that treatment of Tbc-primed cells with anti-Thy 1 antiserum plus complement abolished their helper activity. We also demonstrated that purified T cell populations selectively triggerred IgG B cells. Selective triggering of IgG B lymphocytes by Tbc-primed T cells may not be due to the influence of suppressor T cells supposedly present in Tbc-primed cells since this selectivity was not affected by X-irradiation of Tbc-primed T cell populations which may inactivate suppressor T cells. Furthermore, passive transfer of Tbc-primed cells into normal recipient mice, the condition which may detect the suppressor T cell effect much more sensitively in IgE production, or preimmunization with Tbc 2 weeks before, did not suppress primary anti-DNP IgE antibody response to DNP-PPD. Thus, the observations presented here are favorable to the concept of the presence of IgG class-specific helper T lymphocytes. Furthermore, PPD-reactive T cells from mice immunized with PPD itself exerted their helper function for triggering B cells of both IgE and IgG classes. This may also indicate that some of the components associated with Tbc other than PPD might negatively affect the development of PPD-reactive helper T cells specific to the IgE class. The generation of such IgG-specific T cell activity in the presence of Tbc will be discussed in the light of the T cell population involved in the regulation of antibody responses of different immunoglobulin classes.     

Characterization of a B cell defect in the NZB mouse manifested by an increased ratio of surface IgM to IgD.

In an effort to define the cellular basis of abnormalities in polyclonal B cell activation previously noted in NZB mice, the surface immunoglobulin (sIg) isotypes of spleen cells from NZB mice were examined. After lactoperoxidase-catalyzed radioiodination, the cell surface immunoglobulins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Spleen cells from 8- to 10-week-old NZB mice were found to have an increased ratio of cell surface IgM/IgD compared to cells from 11 control strains. The altered ratio of sIg isotypes was not a consequence of increased proteolytic activity present in NZB cell suspensions or of the presence of cytophilic antibody or autoantibody. Ontogenetic studies of the sIgM/sIgD (mu/delta) ration on splenocytes from NZB and BALB/c mice revealed that the former cells had higher mu/delta ratios as early as 2 weeks after birth. By 4 weeks of age the mu/delta ratios were equivalent. Between 4 weeks and 1 year of age, the mu/delta ratios on NZB splenocytes remained constant whereas those on BALB/c splenocytes decreased and reached adult levels at 6 weeks.