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.     

Genetic differences in BCG-induced resistance to Schistosoma mansoni are not controlled by genes within the major histocompatibility complex of the mouse.

Induction of nonspecific resistance to Schistosoma mansoni infection after the i.v. injection of viable BCG was investigated in outbred mice and a panel of inbred and H-2 congenic strains. Significant protection was induced in CF1, A/J, C57BL/6, C57BL/10, DBA/2, C57BR, and SJL mice. BALB/c mice were not protected whereas CBA and C3H mice expressed intermediate degrees of protection. Expression of the protective phenomenon is not controlled by genes within the MHC as shown by the marked differences in response between BALB/c and DBA/2 (H-2d) as well as between C57BR and C3H (H-2k) mice. H-2 congenic strains with C57BL/10 background (B10.A and B10.D2) were high responders. BALB.B10 mice carrying the high responder (B10) MHC on the nonresponder (BALB/c) background were not protected. The degree of splenic hypertrophy did not correlate with the expression of nonspecific resistance. These results demonstrate that, in addition to controlling specific immune responses, genetic differences influence the nonspecific protective phenomena related to BCG administration as well.     

CD3- bone marrow cells augment the generation of cytotoxic T lymphocytes showing a preference for the X-chromosome linked gene product of stimulator cells

Responder cells, composed of both a limited number of nylon wool-passed lymph node (NW-LN) cells and an excess number of CD3+ cell-depleted bone marrow (CD3- BM) cells from the same strain of mice, were stimulated with allogeneic spleen cells in vitro. The CD3- BM cells augmented the generation of allogeneic major histocompatibility complex (MHC) class I specific cytotoxic T lymphocytes (CTL) from NW-LN cells. C3H/He (H-2k, C3H background) responder cells were stimulated with either B10.D2 (H-2d, B10 background) or BALB/c (H-2d, BALB background) spleen cells. In the former stimulation, the CTL induced lysed B10.D2 target cells more efficiently than the BALB/c cells. Furthermore, these CTL lysed more (B10.D2 x BALB/c) F1 male target cells than (BALB/c x B10.D2) F1 male. In the latter stimulation, the CTL lysed more BALB/c than B10.D2 cells, and more (BALB/c) x B10.D2) F1 male than (B10.D2 x BALB/c) F1 male. The reciprocal mixed lymphocyte cultures (MLC) were carried out, in which BALB/c responder cells were stimulated with either C3H/He or B10.BR (H-2k, B10 background) spleen cells. In the former stimulation, the CTL induced lysed more C3H/He or (C3H/He x B10.BR) F1 male target cells than B10.BR or (B10.BR x C3H/He) F1 male, and in the latter, the reciprocal results were obtained. These results suggested that the CTL induced had a preference for the X-chromosome linked gene products (Xlgp), besides the specificity for the allogeneic MHC class I, of the mice used as stimulator.     

Anti-MHC class I antibody activation of proliferation and survival signaling in murine cardiac allografts

Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2K(b)/D(b)) received a fully MHC-incompatible BALB/c (H-2K(d)/D(d)) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2K(d) Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.     

Induction of robust diabetes resistance and prevention of recurrent type 1 diabetes following islet transplantation by gene therapy

We have previously shown that the development of type 1 diabetes (T1D) can be prevented in nonobese diabetic (NOD) mice by reconstitution with autologous hemopoietic stem cells retrovirally transduced with viruses encoding MHC class II I-A beta-chain molecules associated with protection from the disease. In this study we examined whether a blockade of the programmed death-1 (PD-1)-programmed death ligand-1 (PD-L1) pathway, a major pathway known to control diabetes occurrence, could precipitate T1D in young NOD mice following reconstitution with autologous bone marrow retrovirally transduced with viruses encoding protective MHC class II I-A beta-chain molecules. In addition, we examined whether the expression of protective MHC class II alleles in hemopoietic cells could be used to prevent the recurrence of diabetes in mice with pre-existing disease following islet transplantation. Protection from the occurrence of T1D diabetes in young NOD mice by the expression of protective MHC class II I-A beta-chain molecules in bone marrow-derived hemopoietic cells was resistant to induction by PD-1-PD-L1 blockade. Moreover, reconstitution of NOD mice with pre-existing T1D autologous hemopoietic stem cells transduced with viruses encoding protective MHC class II I-A beta-chains allowed for the successful transplantation of syngeneic islets, resulting in the long-term reversal of T1D. Reversal of diabetes was resistant to induction by PD-1-PDL-1 blockade and depletion of CD25(+) T cells. These data suggest that expression of protective MHC class II alleles in bone marrow-derived cells establishes robust self-tolerance to islet autoantigens and is sufficient to prevent the recurrence of autoimmune diabetes following islet transplantation.     

Macrophage-mediated suppression of immune responses in Toxoplasma-infected mice. II. Both H-2-linked and -nonlinked control of induction of suppressor macrophages

The suppressive effect of Toxoplasma infection on initiation of memory cells to dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) was drastically different among inbred strains of mice. C57BL/6 (B6), C57BL/10 (B10), and SJL mice showed markedly suppressed secondary anti-DNP responses when infected. In contrast, the suppression did not occur in BALB/c mice. The infected DBA/2 and C3H/He mice produced moderately suppressed responses. In B6 mice, an injection with 1 X 10(2) organisms of T. gondii induced a suppressed elicitation of the memory cells to DNP-KLH. However, in BALB/c mice, the responses were not affected even by inoculation with 1 X 10(4) organisms. The difference in the suppressive effect of infection between B6 and BALB/c mice was also observed in the primary anti-DNP antibody responses to DNP-KLH. Both H-2-linked and -nonlinked genes appeared to be responsible for the regulation of the immunosuppression, since the suppressive effect of infection in B10.D2 mice, which have the B10 background and the same H-2 haplotype as BALB/c, was weaker than that of B10 mice, but stronger than in BALB/c mice. In vitro studies using a primary anti-sheep erythrocytes (SRBC) antibody response system demonstrated that the activation of plastic-adherent suppressor cells by Toxoplasma infection, in which suppressor macrophages have been proved to be the responsible cells for the suppressive activity, was controlled by both H-2-linked and -nonlinked genes.     

Comparison of antigen specificity, class II major histocompatibility complex restriction, and in vivo behavior of myelin basic protein-specific T cell lines and clones derived from (BALB/c x SJL/J) mice

Immunization with myelin basic protein (BP) causes experimental allergic encephalomyelitis (EAE) in certain strains of mice. SJL/J (H-2s) is the prototype sensitive strain. Although BALB/c (H-2d) is resistant to EAE through use of an identical immunization protocol, (BALB/c x SJL/J)F1 hybrid mice develop EAE after immunization with BP. T cell clones specific for BP have been isolated from a highly encephalitogenic line of (BALB/c x SJL/J)F1 hybrid T cells raised against bovine BP. The clones were examined for their H-2 restriction and specificity for heterologous forms of BP (mouse, rat, and bovine BP). The results revealed the clones cross-reacting with mouse (self) BP were almost always restricted to F1 hybrid class II major histocompatibility complex (MHC) elements. In contrast, mouse cross-reactive clones derived from a nonencephalitogenic (BALB/c x SJL/J) T cell line raised against rat BP were largely restricted to H-2d elements. These clones did not cross-react with bovine BP. Four additional lines were generated by carrying the original rat and bovine F1 T cell lines on parental antigen-presenting cells thus generating lines biased toward homozygous (SJL/J, H-2s, or BALB/c, H-2d) restriction elements. These "parentally restricted" T cell lines did not induce EAE when injected in vivo. These results suggest that in this F1 strain sensitivity to T cell-induced EAE is associated with epitopes on murine BP that associate with F1 class II MHC restricting elements. In contrast, nonencephalitogenic T cell lines contain a high proportion of murine cross-reactive clones restricted to H-2d, the haplotype of the classically resistant BALB/c mouse. This work illustrates the use of T cell lines and clones in a model system to further analyze the role of MHC restriction elements in autoimmune disease occurring in heterozygous individuals.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to Mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.     

Low CD86 expression in the nonobese diabetic mouse results in the impairment of both T cell activation and CTLA-4 up-regulation

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune insulin-dependent diabetes mellitus and serves as a model for human type I diabetes. NOD spleen cells proliferate to a lesser extent than those from C57BL/6 and BALB/c mice in response to anti-CD3. To investigate the cause of this reduced T cell proliferation, costimulatory molecule expression was investigated. It was found that NOD macrophages, dendritic cells, and T cells, but not B cells, expressed lower basal levels of CD86, but not CD80, CD28, or CD40, compared with C57BL/6 and BALB/c. This low CD86 expression was not dependent on the MHC haplotype or on diabetes development since the NOD-related, diabetes-free mouse strains NON (H-2nb1) and NOR (H-2g7) exhibited similar low levels of CD86 expression and proliferation. Furthermore, following activation, the relative up-regulation of CTLA-4, as compared with CD28, was more pronounced on C57BL/6 and BALB/c T cells as shown by an increased CTLA-4/CD28 ratio. This activation-induced increase in the CTLA-4/CD28 ratio was markedly reduced on NOD T cells compared with the other two strains. The low CD86 expression in NOD mice may account for the reduced increase in both proliferation and the CTLA-4/CD28 ratio, since reducing CD86 expression in C57BL/6 and BALB/c cultures to NOD levels significantly reduces the proliferation and the CTLA-4/CD28 ratio. Therefore, we propose that a low level of CD86 expression in the NOD mouse contributes to a defective regulation of autoreactive T cells by preventing the full activation of T cells and therefore the up-regulation of CTLA-4.     

MHC class I allele dosage alters CD8 expression by intestinal intraepithelial lymphocytes

The development of TCR alphabeta(+), CD8alphabeta(+) intestinal intraepithelial lymphocytes (IEL) is dependent on MHC class I molecules expressed in the thymus, while some CD8alphaalpha(+) IEL may arise independently of MHC class I. We examined the influence of MHC I allele dosage on the development CD8(+) T cells in RAG 2(-/-) mice expressing the H-2D(b)-restricted transgenic TCR specific for the male, Smcy-derived H-Y Ag (H-Y TCR). IEL in male mice heterozygous for the restricting (H-2D(b)) and nonrestricting (H-2D(d)) MHC class I alleles (MHC F(1)) were composed of a mixture of CD8alphabeta(+) and CD8alphaalpha(+) T cells, while T cells in the spleen were mostly CD8alphabeta(+). This was unlike IEL in male mice homozygous for H-2D(b), which had predominantly CD8alphaalpha(+) IEL and few mostly CD8(-) T cells in the spleen. Our results demonstrate that deletion of CD8alphabeta(+) cells in H-Y TCR male mice is dependent on two copies of H-2D(b), whereas the generation of CD8alphaalpha(+) IEL requires only one copy. The existence of CD8alphabeta(+) and CD8alphaalpha(+) IEL in MHC F(1) mice suggests that their generation is not mutually exclusive in cells with identical TCR. Furthermore, our data imply that the level of the restricting MHC class I allele determines a threshold for conventional CD8alphabeta(+) T cell selection in the thymus of H-Y TCR-transgenic mice, whereas the development of CD8alphaalpha(+) IEL is dependent on, but less sensitive to, this MHC class I allele.     

Strain variations in anti-sperm antibody responses and anti-fertility effects in inbred mice

This study provides evidence for polygenic controls of antisperm antibody levels in inbred male mice immunized with syngenic testis and epididymis. H2-linked and non-H2-linked genes were involved. Mice of H-2d haplotype were high responders, whereas those with H-2k haplotype were nonresponders; however, B10.D2/nSnJ mice (H-2d) were also nonresponders. In vitro fertilization inhibition by antisera correlated positively with the serum antisperm antibody levels, particularly with antibody of the immunoglobulin (Ig) G class. Inheritance of antibody response that inhibited in vitro fertilization (IVF) was an autosomal dominant trait, but this was not apparent for the control of antibody levels per se. Since IVF was inhibited by both IgG and fragment antigen-binding (Fab) isolated from immune sera, but not by immune IgG previously absorbed by sperm or testis, the biologic effect is antigen-specific and probably involved blockade of functional antigenic epitopes. Antisera to testis, caput sperm or cauda sperm were found to inhibit IVF to a similar degree. Inbred strains of mice that produced the highest levels of serum antisperm antibodies that inhibited IVF were A/J, SJL/J, DBA/1J and BALB/cByJ mice, and their antisera immunoprecipitated a common sperm antigen molecule of 35,000 to 40,000 Mr. In contrast, C57BL/6 and C57BL/10 mice produced significant antibody levels that had no effect on IVF, and their sera did not react with the 35,000- to 40,000-Mr peak. Moreover, among BALB/c H-2 congenic mice, only antiserum of responder BALB/cByJ (H-2d) mice immunoprecipitated the 35,000- to 40,000 Mr peak. Thus the 35,000- to 40,000-Mr protein may be of functional significance in the fertilization process.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. II. Significance of MHC antigens in anti-Mls tolerance

Specificity of anti-Mlsa tolerance induced in BALB/c (H-2d, Mlsb) neonates was investigated by a popliteal lymph node (PLN)-swelling assay for the local graft-versus-host (GVH) reaction by injecting tolerant thymus cells into the footpads of several types of F1 hybrid mice. When thymus cells were obtained from 1-week-old normal BALB/c, they evoked enlargement of PLNs of (BALB/c X DBA/2)F1 (H-2d, Mlsb/a) [CDF1] recipients and of other hybrid recipients, heterozygous in Mlsa,c,d alleles, irrespective of the major histocompatibility complex (MHC) haplotypes. The same thymus cells did not cause the response in MHC-heterozygous F1 hybrids when the hybrids were homozygous in Mlsb, identical with BALB/c mice. Therefore, the PLN response to Mls antigens, known to be closely associated with MHC-class II antigens, was not directed to the class II antigens themselves. This enabled us to examine the effects of MHC on tolerance induction to the Mls antigens. When BALB/c neonates were injected with CDF1 bone marrow cells, complete tolerance to Mlsa-H-2d antigens of CDF1 cells was induced in the thymus, while responsiveness to Mlsa antigens in the context of H-2k and H-2b antigens, was not affected. This indicates MHC-restriction of neonatal tolerance to Mls antigens. Furthermore, when Mls and H-2-heterozygous (BALB/c X AKR)F1 (H-2d/k, Mlsb/a) bone marrow cells served as the tolerogen, thymus cells of BALB/c neonates were also tolerized to Mlsa-H-2k antigens as well as to Mlsa-H-2d antigens, which suggests the involvement of MHC, probably class II antigens of tolerance-inducing cells.     

MHC class II molecules play a role in the selection of autoreactive class I-restricted CD8 T cells that are essential contributors to type 1 diabetes development in nonobese diabetic mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2(g7) molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2(b)) or CD8 expression (H2(q)). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2(nb1) MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.     

Specificity of the mouse cytotoxic T lymphocyte response to adenovirus 5. E1A is immunodominant in H-2b, but not in H-2d or H-2k mice

The Ag specificity and MHC restriction of the CTL response to adenovirus 5 (Ad5) in three strains of mice, C57BL/10 (H-2b), BALB/c (H-2d), and C3H/HeJ (H-2k), were tested. Polyclonal Ad5-specific CTL were prepared by priming mice in vivo with live Ad5 virus followed by secondary in vitro stimulation of the spleen cells with virus-infected syngeneic cells. The Ad5-specific CTL were Db restricted in C57BL/10 and Kk restricted in C3H/HeJ. In BALB/c mice both Kd- and Dd/Ld-restricted CTL were detected. The polyclonal Ad5-specific CTL response in C57BL/10 mice is directed exclusively against the products of the E1A region, which comprises only 5% of the Ad5 genome. In BALB/c mice E1A is at best a very minor target Ag and in C3H/HeJ mice E1A is not recognized at all. Using the H-2 congenic mouse strains B10.BR (H-2k) and C3H.SW (H-2b) it was shown that the immunodominance of E1A is H-2 dependent. The 19-kDa glycoprotein encoded in the E3 region of Ad5, which binds to class I MHC in the endoplasmic reticulum and prevents its translocation to the cell surface, does not affect the specificity of the CTL response in C57BL/10 mice toward E1A. However, it affects the MHC restriction of the Ad5-specific response in BALB/c mice, selectively inhibiting generation of Kd-restricted CTL.     

Target antigens determine graft-versus-host disease phenotype

Chronic graft-vs-host disease (cGVHD) is an increasingly frequent complication of allogeneic stem cell transplantation. Phenotypically, cGVHD differs from patient to patient; in particular, a subset of patients develops extensive cutaneous fibrosis. Similarly, graft-vs-host disease (GVHD) is distinct in inbred murine donor:recipient pairings, indicating a genetic component to disease phenotype. The B10.D2 -->BALB/c (H-2d) strain pairing uniquely recapitulates key pathologic features of fibrotic human cutaneous cGVHD. To distinguish whether this genetic component is due to differences in genes that modulate immune responses or to the specific Ags targeted, we asked whether skin-dominant cGVHD also develops in the B10 -->BALB.B (H-2b) and B10.BR -->BALB.K (H-2k) MHC-congenic pairings. Because each MHC haplotype presents different peptides and selects different T cell repertoires, GVHD in each donor:recipient pair undoubtedly targets different Ags. We found that, in contrast to BALB/c recipients, BALB.B mice never manifested skin disease while BALB.K mice developed a modified form of skin disease. Instead, BALB.B and BALB.K recipients developed systemic GVHD which was absent in BALB/c mice. Moreover, in (B10 x B10.D2)F1 -->(BALB.B x BALB/c)F1 H-2b/d transplants, recipients developed both cutaneous and systemic disease. Thus, the selection of immunodominant Ags determines the target and character of GVHD, providing insight into the genetic basis for different forms of GVHD.     

Enhanced priming of multispecific,murine CD8+ T cell responses by DNA vaccines expressing stress protein-binding polytope peptides

A polytope DNA vaccine (pCI/pt10) was used that encodes within a 106-residue sequence 10-well characterized epitopes binding MHC class I molecules encoded by the K, D, or L locus (of H-2(d), H-2(b), and H-2(k) haplotype mice). The pCI/pt10 DNA vaccine efficiently primed all four K(b)/D(b)-restricted CD8(+) T cell responses in H-2(b) mice, but was deficient in stimulating most CD8(+) T cell responses in H-2(d) mice. Comparing CD8(+) T cell responses elicited with the pCI/pt10 DNA vaccine in L(d+) BALB/c and L(d-) BALB/c(dm2) (dm2) mice revealed that L(d)-restricted CD8(+) T cell responses down-regulated copriming of CD8(+) T cell responses to other epitopes regardless of their restriction or epitope specificity. Although the pt10 vaccine could thus efficiently co prime multispecific CD8(+) T cell responses, this priming was impaired by copriming L(d)-restricted CD8(+) T cell responses. When the pt10 sequence was fused to a 77-residue DnaJ-homologous, heat shock protein 73-binding domain (to generate a 183-residue cT(77)-pt10 fusion protein), expression and immunogenicity (for CD8(+) T cells) of the chimeric Ag were greatly enhanced. Furthermore, priming of multispecific CD8(+) T cell responses was readily elicited even under conditions in which the suppressive, L(d)-dependent immunodominance operated. The expression of polytope vaccines as chimeric peptides that endogenously capture stress proteins during in situ production thus facilitates copriming of CD8(+) T cell populations with a diverse repertoire.     

NOD bone marrow-derived dendritic cells are modulated by analogs of 1,25-dihydroxyvitamin D3

The immune effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) are mainly mediated through dendritic cells (DCs). In vitro, 1,25(OH)(2)D(3) treatment renders murine bone marrow (BM)-derived DCs more tolerogenic, indirectly altering behavior and fate of T lymphocytes. In vivo, treatment with 1,25(OH)(2)D(3) or its analogs prevents diabetes in NOD mice. The aim of this study was to investigate the effects of the 1,25(OH)(2)D(3)-analog TX527 on the expression of antigen-presenting and costimulatory/migratory molecules on BM-derived DCs from NOD mice. After culture with 20 ng/ml GM-CSF + 20 ng/ml IL-4 (8 days) followed by 1000 ng/ml LPS + 100 U/ml IFN-gamma (2 days), with or without 10(-8)M TX527, cells were counted and analyzed by FACS for MHC II, CD86, CD40 and CD54 expression within the CD11c(+) DC population. Upon TX527 treatment, cell recovery was significantly reduced whereas the CD11c(+) DC fraction remained constant. On CD11c(+) DCs, MHC II, CD86 and CD54 were significantly down-regulated and CD40 was twofold upregulated. Globally, BM-derived DCs from NOD mice become more tolerogenic upon TX527 treatment, confirming the effects of 1,25(OH)(2)D(3) on murine DCs and possibly explaining the protective effects of 1,25(OH)(2)D(3) and its analogs from diabetes in NOD mice.     

Development of diabetogenic T cells from NOD/Lt marrow is blocked when an allo-H-2 haplotype is expressed on cells of hemopoietic origin, but not on thymic epithelium

Diabetes is a T cell-mediated process in NOD/Lt mice, with a major genetically recessive component of susceptibility linked to homozygous expression of the unique H-2g7 MHC haplotype. Heterozygous expression of the H-2nb1 haplotype derived from the NON/Lt strain confers diabetes resistance both in (NOD x NON)F1 hybrids and in NOD mice congenic for the H-2nb1 haplotype. However, diabetes resistance is abrogated in F1 hybrids by NOD/Lt bone marrow reconstitution. To establish whether the generation of beta cell autoreactive T cells from NOD/Lt bone marrow-derived precursors required at least heterozygous expression of the H-2g7 haplotype on thymic epithelium, adolescent thymectomized (NOD x NON)F1 mice were implanted with neonatal NON/Lt thymus grafts before lethal radiation and reconstitution with NOD/Lt bone marrow. Peripheral T cells maturing through this ectopic thymic implant exclusively expressed the NOD H-2g7 haplotype and were tolerant to H-2nb1 skin grafts. Nevertheless, diabetes developed in 32% of the NON/Lt thymus-grafted chimeras vs 38% of the sham-thymectomized NOD bone marrow chimeras. Thus, homozygous expression of the diabetes-resistant H-2nb1 haplotype on thymic epithelium failed to block development of a diabetogenic T cell repertoire. To examine if expression of H-2nb1 on hemopoietically derived APC could alter the diabetogenic potential of NOD/Lt marrow, diabetes-resistant NOD.NON-H-2nb1 congenic mice were mated with NOD/Lt mice to produce NOD-H-2g7/H-2nb1 heterozygous recipients. These were lethally irradiated and reconstituted with either NOD/Lt marrow alone, NOD.H-2nb1 homozygous congenic marrow alone, or a 1:1 mixture of the two marrow populations. By 25 wk of age, all of the MHC heterozygous recipients of NOD.NON-H-2nb1 marrow remained diabetes-free whereas 75% of the MHC heterozygous recipients of NOD/Lt marrow developed diabetes. A striking decrease in diabetes was observed when T cell precursors derived from NOD/Lt marrow interacted with H-2nb1 gene products on hemopoietically derived APC, inasmuch as only 7% of the MHC heterozygous recipients reconstituted with a 1:1 mixture of NOD/Lt and NOD.NON-H-2nb1 marrow developed diabetes. Peripheral leukocytes in all reconstitution classes expressed the MHC phenotype(s) of the marrow donor(s). Skin grafting confirmed that all reconstitution classes of MHC heterozygous recipients were tolerant to the H-2nb1 haplotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Broad recognition of cytotoxic T cell epitopes from the HIV-1 envelope protein with multiple class I histocompatibility molecules.

A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.     

Transgene number-dependent, gene expression rate-independent rejection of Dd -, Kd-, or Dd Kd -transgened mouse skin or tumor cells from C57BL/6 Db Kb mice

Recipient cells migrating into the transplantation site of an allograft recognize histocompatibility antigens on the grafts and are cytotoxic against the grafts. Although the alloreactive immune response is predominantly directed at the major histocompatibility complex (major histocompatibility complex [MHC]; H-2 in mice) class I molecules, the basic mechanisms of allograft rejection (e.g., ligand-receptor interaction) remain unclear, because of the polymorphism and complexity of the MHC. To examine the role of MHC class I molecules in allograft rejection, D(d) , K(d) or D(d) K(d) -transgenic skin or tumor cells we established on a C57BL/6 (D(b) K(b) ) background and transplanted into C57BL/6 mice. Skin grafts from allogeneic (i.e., BALB/c, B10.D2, and BDF1) strains of mice were rejected from C57BL/6 mice on days 12-14 after grafting, whereas isografts were tolerated by these mice. Unexpectedly, skin grafts from D(d) -, K(d) -, and D(d) K(d) -transgenic C57BL/6 mice were rejected on days 12-14 in a transgene expression rate-independent manner from 9/19 (47%), 20/39 (51%), and 12/17 (71%) of C57BL/6 mice, respectively. Similarly, intradermally transplanted allogeneic (i.e., Meth A), but not syngeneic (i.e., EL-4), tumor cells were rejected from C57BL/6 mice; the growth of D(d) - or K(d) -transfected EL-4 cells was delayed by 10-13 days; and 4/10 (40%) of D(d) K(d) -transfected tumor cells were rejected from C57BL/6 mice. These results indicate that D(d) and K(d) genes are equivalent as allogeneic MHC class I genes and that C57BL/6 (D(b) K(b) ) mice reject D(d) -, K(d) -, or D(d) K(d) -transgened skin or tumor cells in a transgene number-dependent, gene expression rate-independent manner.