The adaptor protein AP-3 is required for CD1d-mediated antigen presentation of glycosphingolipids and development of Valpha14i NKT cells

Relatively little is known about the pathway leading to the presentation of glycolipids by CD1 molecules. Here we show that the adaptor protein complex 3 (AP-3) is required for the efficient presentation of glycolipid antigens that require internalization and processing. AP-3 interacts with mouse CD1d, and cells from mice deficient for AP-3 have increased cell surface levels of CD1d and decreased expression in late endosomes. Spleen cells from AP-3-deficient mice have a reduced ability to present glycolipids to natural killer T (NKT) cells. Furthermore, AP-3-deficient mice have a significantly reduced NKT cell population, although this is not caused by self-tolerance that might result from increased CD1d surface levels. These data suggest that the generation of the endogenous ligand that selects NKT cells may also be AP-3 dependent. However, the function of MHC class II-reactive CD4+ T lymphocytes is not altered by AP-3 deficiency. Consistent with this divergence from the class II pathway, NKT cell development and antigen presentation by CD1d are not reduced by invariant chain deficiency. These data demonstrate that the AP-3 requirement is a particular attribute of the CD1d pathway in mice and that, although MHC class II molecules and CD1d are both found in late endosomes or lysosomes, different pathways mediate their intracellular trafficking.     

Gene transfer to liver cancer cells of B7-1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone

To investigate the cooperative effect of B7-1 and IL-12 in the induction of antitumor activity, we have developed retroviral vectors encoding human B7-1, murine IL-12, or both B7-1 and IL-12 coordinately. Murine transformed liver cells (BNL) were engineered to stably express B7-1, IL-12, or both by infection with corresponding retroviruses. No tumor was observed in 20, 75, and 95% of mice receiving, respectively, B7-1-, IL-12-, and B7-1/IL-12-modified tumor cells after 250 days of inoculation. In contrast, injection of parental BNL or BNL/Neo cells resulted in lethal tumor progression in all mice. Protection against rechallenge with parental tumor cells was observed only in mice who had rejected BNL/IL-12, but not in animals that rejected BNL/B7-1 or BNL/B7-1-IL-12. Growth of parental tumor cells was significantly delayed by simultaneous injection in a distant site of irradiated tumor cells engineered to express IL-12 or both B7-1 and IL-12 but not B7-1 alone. BNL/B7-1 and BNL/B7-1-IL-12 showed similar efficacy in these experiments. Antitumor immunity induced by B7, with or without IL-12, was found to depend mainly on CD4+ T cells with a minor contribution of a non-T cell mechanism; whereas the effect of IL-12 was dependent on CD8+ T cells and on non-T cell effectors. Immunization of mice with IL-12-modified BNL cells induced secretion of a Thl pattern of cytokines while immunization with cells expressing both IL-12 and B7-1 resulted in inhibition of IFN-gamma production. Immunization with BNL/B7-1-IL-12 cells in the presence of anti-human B7-1 MAb resulted in restoration of IFN-gamma production to the levels found in animals injected with BNL/IL-12 cells. To summarize, in our model coexpression of B7-1 and IL-12 in tumor cells does not result in improved antitumoral activity as compared with expression of IL-12 alone. This may be related to the fact that B7-1 changes the mechanisms of antitumor immunity and inhibits IFN-gamma production induced by IL-12 in vivo.     

Functional analysis of influenza-specific helper T cell clones in vivo. T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin

We compared the effects of adoptively transferred Th cell clones specific for the influenza hemagglutinin (HA), matrix (M), or nucleoprotein (NP) on the antibody response of nude mice infected with A/PR/8/34 influenza virus. We show that the production of antibodies to the HA absolutely requires the presence of virus-specific Th cells. Further, transfer of a Th clone specific for the internal proteins, M or NP, was as effective as was transfer of an HA-specific clone in supporting an antibody response to the HA. With each of the clones, the kinetics of the response were accelerated by approximately 3 d compared with the antibody response of normal BALB/c mice. The HA- and M-specific clones supported an isotype switch from IgM to IgG and IgA similar to that which occurs during a normal antibody response. Finally, as shown by coinfection experiments, the response required a cognate T-B interaction whether the determinants recognized by the Th and B cell are located on the same viral protein or on different viral proteins within the same virus particle. The implications of these findings for understanding the T-B interactions that occur during an effective antiviral antibody response are discussed.     

Induction of tolerance to 1-fluoro-2,4-dinitrobenzene contact sensitivity with hapten-modified lymphoid cells. II. Selective tolerance in F1 mice of T cell subsets recognizing 1-fluoro-2,4- dinitrobenzene associated with parental major histocompatibility complex antigens

F1 animals were tolerized to 1-fluoro-2,4-dinitrobenzene (DNFB) contact sensitivity with parentally derived, in vitro hapten-modified spleen cells. This tolerant state was found, upon adoptive transfer to naive parental strain recipients, to affect only that T cell subpopulation that recognized the parental haplotype of the cell used as the tolerogen, and did not inhibit the ability of the remaining T cell subset to confer immunity. This demonstrates that this tolerant state involves the inactivation of a cell required for the expression of contact sensitivity by recognizing DNFB in association with self major histocompatibility complex gene products.     

Genetic restrictions for the induction of suppressor T cells by hapten- modified lymphoid cells in tolerance to 1-fluoro-2,4-dinitrobenzene contact sensitivity. Role of the H-2D region of the major histocompatibility complex

Genetic restrictions governing the induction and expression of suppressor T cells (Ts) in tolerance to 1-fluoro-2,4-dinitrogenzene (DNFB) contract sensitivity were studied. Tolerance was induced by using 2,4-dinitrophenyl (DNP)-modified lymphoid cells (DNP-LC) as tolerogen. Two kinds of Ts were found-those produced by DNP-LC syngeneic to the donor of the Ts (syninduced Ts), and those produced by DNP-LC allogeneic to the donor of Ts (alloinduced Ts). Studies employing congenic resistant mouse strains indicated that recognition of DNP-modified-major histocompatibility region determinants on the tolerogenic DNP-LC was essential for the induction of both types of Ts. Non-H-2 genetic background was irrelevant to Ts induction. Mapping studies indicated that induction of both syninduced and alloinduced Ts was associated with recognition of DNP-modified-MHC region determinants which map to the right of the H-2G region (i.e., H-2D gene products). Tolerization of donor mice with DNP-LC which were H-2D region compatible, but not with H-2K or I region compatible DNP-LC, was both sufficient and required for the induction of hapten-specific syninduced Ts. Tolerization of donor mice with DNP-LC which were incompatible only at the H-2D region was sufficient for the induction of alloinduced Ts. These Ts were capable of suppressing recipient mice only if the recipients shared the H-2D region with the strain providing the DNP-LC tolerogen, and were not capable of suppressing recipients sharing all but the H-2D region with the tolerogen.     

Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein

The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.     

Evidence for extrathymic generation of intermediate T cell receptor cells in the liver revealed in thymectomized, irradiated mice subjected to bone marrow transplantation

In addition to the major intrathymic pathway of T cell differentiation, extrathymic pathways of such differentiation have been shown to exist in the liver and intestine. In particular, hepatic T cells of T cell receptors or CD3 of intermediate levels (i.e., intermediate T cell receptor cells) always contain self-reactive clones and sometimes appear at other sites, including the target tissues in autoimmune diseases and the tumor sites in malignancies. To prove their extrathymic origin and self reactivity, in this study we used thymectomized, irradiated (B6 x C3H/He) F1 mice subjected to transplantation of bone marrow cells of B6 mice. It was clearly demonstrated that all T cells generated under athymic conditions in the peripheral immune organs are intermediate CD3 cells. In the case of nonthymectomized irradiated mice, not only intermediate CD3 cells but also high CD3 cells were generated. Phenotypic characterization showed that newly generated intermediate CD3 cells were unique (e.g., interleukin 2 receptor alpha-/beta+ and CD44+ L-selectin-) and were, therefore, distinguishable from thymus-derived T cells. The precursor cells of intermediate CD3 cells in the bone marrow were Thy-1+ CD3-. The extrathymic generation of intermediate CD3 cells was confirmed in other combinations of bone marrow transplantation, C3H --> C3H and B10.Thy1.1 --> B6.Thy1.2. The generated intermediate CD3 cells in the liver contained high levels of self-reactive clones estimated by anti-V beta monoclonal antibodies in conjunction with the endogenous superantigen minor lymphocyte-stimulating system, especially the combination of B6 --> (B6 x C3H/He) (graft-versus-host- situation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen recognition. V. Requirement for histocompatibility between antigen-presenting cell and B cell in the response to a thymus- dependent antigen, and lack of allogeneic restriction between T and B cells

The restrictions imposed by the major histocompatibility complex on T-B- antigen-presenting cell (APC) interactions were studied with an in vivo adoptive transfer system, using mutually tolerant T and B cells taken from one-way fetal liver chimeras. It was found that the B cells and adoptive recipient (which provides APC function) have to share determinants encoded by the left-hand end of the H-2 complex for cooperation, whereas there is apparently no such requirement for T-B cell syngeneicity. Suppression arising from allogeneic effects between the host and the transferred T or B cells was excluded by the use of tolerant as well as normal adoptive recipients; both were functionally equivalent. We conclude that under experimental conditions, unrestricted helper T cell function and concurrent APC-B cell genetic restriction can be demonstrated in vivo.     

Human Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes home preferentially to and induce selective regressions of autologous EBV- induced B cell lymphoproliferations in xenografted C.B-17 scid/scid mice [published erratum appears in J Exp Med 1996 Sep 1;184(3):1199]

C.B-17 scid/scid (severe combined immunodeficiency [SCID]) mice inoculated with peripheral blood lymphocytes from Epstein-Barr virus (EBV)-seropositive donors, or with EBV-transformed lymphoblastoid B cell lines (EBV-LCL), develop lethal human EBV+ B cell lymphoproliferative disorders (EBV-LPD) with characteristics similar to those arising in immunodeficient patients. Using this model, we examined the capacity of human effector cells to control human EBV-LPD. SCID mice received rabbit anti-asialo GM1 antiserum to abrogate endogenous natural killer-cell function. Preliminary experiments showed that adoptive transfer of peripheral blood mononuclear cells (PBMC), purified T cells, interleukin (IL) 2-activated PBMC or anti-CD3- activated T cells derived from EBV-seropositive donors did not result in improved survival of treated mice (in vivo effector/target ratio 2:1 to 1:1). In contrast, EBV-specific cytotoxic T lymphocytes (CTL), derived from EBV-seropositive donors and expanded in vitro, exhibited strong EBV-specific and HLA-restricted activity both in vitro and in vivo. SCID mice inoculated intraperitoneally with autologous but not with HLA-mismatched EBV-LCL had significantly improved survival relative to untreated mice after inoculation of EBV-specific CTL either intraperitoneally (P<0.001) or intravenously (P<0.001) (in vivo effector/target ratio 1:1). SCID mice bearing large subcutaneous EBV+ tumors and treated intravenously with 10(7) EBV-specific CTL achieved complete tumor regression. Both CTL- and CTL-plus-IL-2-treated mice survived significantly longer than untreated animals or animals treated with IL-2 alone (P = 0.0004 and P<0.02, respectively). SCID mice bearing two subcutaneous EBV+ tumors, one autologous and the other HLA mismatched to the EBV-specific CTL donor, had regression of only the autologous tumor after intravenous infusion of 10(7) EBV-specific CTL. Moreover, we could demonstrate preferential homing of PKH26-labeled EBV- specific CTL to autologous but not to HLA-mismatched EBV+ tumors as early as 24 h after intravenous adoptive transfer. Immunophenotypic analyses also demonstrated preferential infiltration of T cells into the autologous EBV+ tumor in SCID mice bearing both the autologous and either fully HLA-mismatched or genotypically related haplotype-sharing EBV+ tumors. The human T cells infiltrating EBV+ tumors were CD3+ and, predominantly, CD8+CD4-. Our results indicate that EBV-specific CTL preferentially localize to and infiltrate EBV+ tumors bearing the appropriate HLA antigens and thereafter induce targeted regressions of disease.     

CC chemokine receptor (CCR)2 is required for langerhans cell migration and localization of T helper cell type 1 (Th1)-inducing dendritic cells. Absence of CCR2 shifts the Leishmania major-resistant phenotype to a susceptible state dominated by Th2 cytokines, b cell outgrowth, and sustained neutrophilic inflammation

There is growing evidence that chemokines and their receptors regulate the movement and interaction of antigen-presenting cells such as dendritic cells (DCs) and T cells. We tested the hypothesis that the CC chemokine receptor (CCR)2 and CCR5 and the chemokine macrophage inflammatory protein (MIP)-1alpha, a ligand for CCR5, influence DC migration and localization. We found that deficiency of CCR2 but not CCR5 or MIP-1alpha led to distinct defects in DC biology. Langerhans cell (skin DC) density in CCR2-null mice was normal, and their ability to migrate into the dermis was intact; however, their migration to the draining lymph nodes was markedly impaired. CCR2-null mice had lower numbers of DCs in the spleen, and this was primarily due to a reduction in the CD8alpha(1) T helper cell type 1 (Th1)-inducing subset of DCs. Additionally, there was a block in the Leishmania major infection-induced relocalization of splenic DCs from the marginal zone to the T cell areas. We propose that these DC defects, in conjunction with increased expression of B lymphocyte chemoattractant, a B cell-specific chemokine, may collectively contribute to the striking B cell outgrowth and Th2 cytokine-biased nonhealing phenotype that we observed in CCR2-deficient mice infected with L. major. This disease phenotype in mice with an L. major-resistant genetic background but lacking CCR2 is strikingly reminiscent of that observed typically in mice with an L. major-susceptible genetic background. Thus, CCR2 is an important determinant of not only DC migration and localization but also the development of protective cell-mediated immune responses to L. major.     

Restricted helper function of F1 hybrid T cells positively selected to heterologous erythrocytes in irradiated parental strain mice. II. Evidence for restrictions affecting helper cell induction and T-B collaboration, both mapping to the K-end of the H-2 complex

Studies with H-2-congenic and recombinant strains showed that when F1 hybrid T cells were activated to sheep erythrocytes in irradiated mice of parental strain or related strain, a population of helper cells was generated which collaborated only with B cells sharing the K-end of the H-2 complex with the strain used for activation. No evidence was found that the restriction in helper function (a) reflected a deficiency of appropriate macrophages during T-B collaboration, or (b) was influenced by the Ig allotype of the B cells. It was concluded that the results signified restrictions acting at both the level of helper cell induction (presumed to be a reflection of T-macrophage interactions in the irradiated intermediate hosts) and during T-B collaboration. With (CBA X C57BL/6)F1 cells, the restrictions at each level mapped to the same region i.e. to the left of the I-B subregion. Consequently, one gene (or set of genes) might control restriction at both levels. If so, T-cell recognition of major histocompatibility complex-associated antigen on macrophages and on specific B cells would be either identical or very similar. The fact that genes mapping to the K-end of the H-2 complex also control the restrictive interactions of homozygous T cells implies that F1 T cells behave functionally as a mixture of T cells derived from the two parental strains. Positive selection to antigen in parental strain mice appears simply to alter the ratio of these two populations.