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PubMed1998

Dose-dependent priming or desensitization induced by chemotactic agents in chemiluminescence experiments with canine and human neutrophils.

We explored whether receptor-specific desensitization in repeated stimulation offers an opportunity to identify chemotactic factors particularly in species, e.g. the dog, for which immunochemical methods to determine chemotactic factors are not commercially available. Complement fragment 5a and interleukin-8 act via distinct receptors. They were used as test agents for neutrophils in luminol-dependent chemiluminescence. These experiments led to the observation that exposure of human and canine neutrophils to low concentrations of a chemotactic agent shows an increased response when stimulated again with the same agent (priming). In the heterologous system even cross-priming could be observed after a second stimulation with another agent. The concentration at which priming rather than desensitization occurred were lower for homologous factor-cell combinations (<10-20 nM) than for heterologous combinations (<45-60 nM). The mechanism underlying this phenomenon is unknown. However, it raises the possibility that it subserves an important function in the recruitment and activation of cells by low agonist concentration. This study shows that despite the phenomenon of priming receptor-specific desensitization may be used to determine chemotactic agents in the homologous system.

Animals↗
PubMed1998

Induction of MHC class I-restricted CTL response by DNA immunization with ubiquitin-influenza virus nucleoprotein fusion antigens.

DNA vaccines have been shown to be an effective means of inducing cytotoxic T-lymphocyte (CTL) responses in both young and aged mice. Better understanding of the pathways by which antigens encoded by DNA vaccines are processed and presented to CTL may allow for improvements in CTL responses in older animals. Since CTL recognize short peptides presented by MHC class I molecules, and since ubiquitin-dependent proteolysis is widely believed to be responsible for degradation of endogenously synthesized antigens and generation of these peptide ligands, we sought to use ubiquitin (Ub) conjugation to target influenza virus nucleoprotein (NP) antigen into the Ub-proteasome degradation pathway for MHC class I-restricted antigen processing and presentation. However, the addition of the Ub moiety did not affect the half-life of Ub-NP protein in transiently transfected human rhabdomyosarcoma (RD) cells. Moreover, the modifications of NP DNA vaccine with Ub conjugation did not affect their ability to induce a CTL response specific for the H-2Kd-restricted NP147-155 epitope, as assessed by both percent cytolysis in bulk CTL culture and by CTL precursor (CTLp) frequency in limiting dilution analysis (LDA). In contrast, the anti-NP antibody (Ab) responses were dramatically reduced in mice immunized with low doses (1 microgram) of Ub-NP constructs, compared with mice immunized with wild-type NP DNA. These results demonstrate that Ub conjugation alone does not guarantee targeting of endogenously synthesized antigens for rapid degradation by proteasomes. Furthermore, the ability of ubiquintination to reduce Ab responses to NP without affecting CTL responses suggests that the Ub modifications result in a lower availability of full-length NP from transfected cells in vivo. The implications of these data on antigen presentation and cross-priming are discussed.

Animals↗
PubMed1996

CD4 T cell tolerance to nuclear proteins induced by medullary thymic epithelium.

Thymic epithelium is involved in negative selection, but its precise role in selecting the CD4 T cell repertoire remains elusive. By using two transgenic mice, we have investigated how medullary thymic epithelium (mTE) and bone marrow (BM)-derived cells contribute to tolerance of CD4 T cells to nuclear beta-galactosidase (beta-gal). CD4 T cells were not tolerant when beta-gal was expressed in thymic BM-derived cells. In contrast, CD4 T cells of mice expressing beta-gal in mTE were tolerized. Tolerance resulted from presentation of endogenous beta-gal by mTE cells but not from cross-priming. mTE cells presented nuclear beta-gal to a Th clone in vitro, while thymic dendritic cells did not. The data indicate that mTE but not thymic BM-derived cells can use a MHC class II endogenous presentation pathway to induce tolerance to nuclear proteins.

Animals↗
PubMed1998

Help for cytotoxic-T-cell responses is mediated by CD40 signalling.

Cytotoxic T lymphocytes (CTLs) which carry the CD8 antigen recognize antigens that are presented on target cells by the class I major histocompatibility complex. CTLs are responsible for the killing of antigen-bearing target cells, such as virus-infected cells. Although CTL effectors can act alone when killing target cells, their differentiation from naive CD8-positive T cells is often dependent on 'help' from CD4-positive helper T (TH) cells. Furthermore, for effective CTL priming, this help must be provided in a cognate manner, such that both the TH cell and the CTL recognize antigen on the same antigen-presenting cell. One explanation for this requirement is that TH cells are needed to convert the antigen-presenting cell into a cell that is fully competent to prime CTL. Here we show that signalling through CD40 on the antigen-presenting cells can replace the requirement for TH cells, indicating that T-cell 'help', at least for generation of CTLs by cross-priming, is mediated by signalling through CD40 on the antigen-presenting cell.

Animals↗
PubMed1998

Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs.

CD8+ cytotoxic T lymphocytes (CTLs) mediate resistance to infectious agents and tumours. Classically, CTLs recognize antigens that are localized in the cytoplasm of target cells, processed and presented as peptide complexes with class I molecules of the major histocompatibility complex (MHC). However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. The most dramatic example is the in vivo phenomenon of cross-priming: antigens from donor cells are acquired by bone-marrow-derived host antigen-presenting cells (APCs) and presented on MHC class I molecules. Two unanswered questions concern the identity of this bone-marrow-derived cell and how such antigens are acquired. Here we show that human dendritic cells, but not macrophages, efficiently present antigen derived from apoptotic cells, stimulating class I-restricted CD8+ CTLs. Our findings suggest a mechanism by which potent APCs acquire antigens from tumours, transplants, infected cells, or even self-tissue, for stimulation or tolerization of CTLs.

Antigen Presentation↗
PubMed1998

CTLA-4 blockade synergizes with tumor-derived granulocyte-macrophage colony-stimulating factor for treatment of an experimental mammary carcinoma.

Generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. CTLA-4 is a second B7 receptor expressed by T cells upon activation that, unlike CD28, appears to deliver an inhibitory signal to T cells. Recently, we and others demonstrated that administration of an anti-CTLA-4 antibody was sufficient to promote regression of several murine tumors. However, certain tumors, such as the SM1 mammary carcinoma, remain refractory to this type of immunotherapy. In the present study, we report that the combination of both CTLA-4 blockade and a vaccine consisting of granulocyte-macrophage colony-stimulating factor-expressing SM1 cells resulted in regression of parental SM1 tumors, despite the ineffectiveness of either treatment alone. This synergistic therapy resulted in long-lasting immunity to SM1 and depended on both CD4(+) and CD8(+) T cells. Interestingly, synergy was not observed between CTLA-4 and a B7-expressing SM1 vaccine. Given that granulocyte-macrophage colony-stimulating factor promotes differentiation and activation of dendritic cells as well as enhances cross-priming of T cells to tumor-derived antigens and that SM1 is major histocompatibility complex class II-negative, our findings suggest that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell. In addition, these results also support the idea that the most effective and synergistic vaccine strategy targets treatments that enhance T cell priming at the level of host-derived antigen-presenting cells.

Abatacept↗
PubMed1978

T-cell populations specifically depleted of alloreactive potential cannot be induced to lyse H-2-different virus-infected target cells.

Mouse lymphocyte populations of one parental H-2 type (A) were specificially depleted of alloreactive potential by filtration through irradiated A X B F1 recipients, and thoracic duct cells were then stimulated with virus in an A X B F1 environment. Experiments using T cells that had previously been exposed to influenza virus in the context of A established that cross-priming for recognition of viral components expressed on H-2-different (B) target cells does not occur. Furthermore, immunologically naive T cells stimulated with vaccinia virus, subsequent to negative selection for reactivity to B, could not be shown to interact with virus-infected cells of type B. Either there is no significant T-cell repertoire for recognition of virus associated with an H-2 determinant not encountered during ontogeny, or such T cells are also alloreactive and are removed during filtration.

Animals↗
PubMed1978

Cytotoxic T-cell responses in mice infected with influenza and vaccinia viruses vary in magnitude with H-2 genotype.

Secondary effector T-cell populations generated by cross-priming with heterologous influenza A viruses operate only in H-2K or H-2D compatible situations, when assayed on SV40-transformed target cells infected with a range of influenza A viruses. The H2-Kb allele is associated with a total failure in the generation of influenza-immune cytotoxic T cells, though this is not seen for the primary response to vaccinia virus. In both influenza and vaccinia development of effector T cells operating at H-2Db is greatly depressed in B10.A(2R) (kkkddb) and B10.A(4R) (kkbbbb), but not in B10 (bbbbbb), mice. However, there is no defect in viral antigen expression at either H-2Kk or H-2Db in B10.A(2R) target cells. This apparently reflects some inadequacy in the stimulator environment, as (A/J X B6) F1 T cells can be induced to respond at H-2Db when exposed to vaccinia virus in an irradiated B6 but not in a B10.A(4R) recipient. The present report, together with the accompanying paper by Zinkernagel and colleagues, records the first rigorous demonstration of both a nonresponder situation and a probable Ir-gene effect for conventional infectious viruses. Possible implications for the evolution of H-2 polymorphism and mechanisms of Ir gene function are discussed.

Animals↗
PubMed1979

T cells recognize minor histocompatibility antigens on H-2 allogeneic cells.

B10.A animals were rendered tolerant to B10.M spleen cells by injection of (B10.A X B10.M)F1 cells into neonates. Adult animals accepted B10.M skin grafts and failed to generate cytotoxic effector cells in vitro against B10.M H-2 antigens. In vivo inoculation of tolerant animals with A.CA spleen cells, followed by in vitro challenge with similar cells, resulted in the generation of cytotoxic effector cells that had specificity for the A strain minor histocompatibility (H)-antigens in the context of the H-2f haplotype. If these animals were boosted in vitro with A strain spleen cells, cross-priming could be demonstrated, whereby the cytotoxic effect was restricted by the H-2a haplotype. These data indicate that at least two sets of T cells co-exist in tolerant animals, one capable of recognizing antigens in the context of the host H-2 haplotype, and the other able to recognize antigens in the context of the tolerated H-2-allogeneic haplotype. Because tolerant animals inoculated with A-strain spleen cells in vivo and boosted in vitro with A.CA spleen cells failed to generate a cytotoxic effect against A.CA, it is unlikely that minor H-antigens need to be processed by host lymphoreticular cells.

Animals↗
PubMed1981

Special features of the priming process for a secretory IgA response. B cell priming with cholera toxin.

Administration of cholera toxin/toxoid by either intraduodenal or parenteral routes increases the frequency of antigen-sensitive B cells in Peyer's patches (PP) and in distant lymphoid tissues greater than 50-fold. The special feature of mucosal priming with toxin is its unique effectiveness at generating secondary B cells, whose progeny express IgA exclusively, and such cells appear in highest frequency in PP and in appreciable numbers in spleen. Thus, this deliberate intraduodenal immunization seems to mimic the natural priming process induced by enteric bacterial colonization, which we have postulated to account for the high frequencies of IgA-committed cells specific for bacterial determinants in the PP of conventionally reared mice. furthermore, as a result of intraduodenal immunization, antigen-specific memory B cells are disseminated to sites distant form that of antigen application, including the lymphoid follicles associated with the respiratory mucosa. Direct antigenic stimulation of cells in the PP therefore results in effective cross-priming among mucosal and systemic sites through division, differentiation, and disemination of antigen-sensitive secondary B cells.

Animals↗
PubMed1999

DNA vaccination: transfection and activation of dendritic cells as key events for immunity.

The mechanisms underlying initiation and maintenance of CD4 T cell responses after DNA vaccination were studied using a construct coding for nonsecreted fifth component of complement (C5) protein, thus restricting the availability of antigen. The only cell types to express C5 were keratinocytes at the site of DNA application and a small number of dendritic cells present in the draining lymph nodes. Antigen expression persisted for up to 12 wk in keratinocytes, but dendritic cells did not express C5 beyond 2 wk after vaccination. Cross-priming of dendritic cells by C5 expressed in keratinocytes did not occur unless keratinocyte death was induced by irradiation in vitro. CD4 T cells were activated in the draining lymph nodes only and subsequently migrated to the spleen, where memory T cells persisted for longer than 40 wk despite the absence of a source of persistent antigen. While DNA vaccination resulted in transfection of a small proportion of dendritic cells only, it led to general activation of all dendritic cells, thus providing optimal conditions for effective T cell activation and maintenance of memory.

Animals↗
PubMed1985

Graft rejection in a congenic panel of rats with defined immune response genes for MHC class I antigens. I. Rejection of and priming to the RT1Aa antigen.

Allograft rejection in the rat has been shown to be under stringent immune response (Ir) gene control using major histocompatibility complex recombinant animals as donors. Presentation of an isolated class I antigenic difference to high responder recipients results in rapid graft rejection, but low responders fail to reject. This striking qualitative difference is also seen in some liver grafting experiments in which the donor presents a full MHC haplotype and minor antigen mismatch to the responders. Grafts of other organs, however, do not discriminate qualitatively between high and low responders when a full haplotype mismatch exists. We have used the canonical high and low-responder animals, (PVG X PVG-RT1u)F1 and PVG to examine whether any qualitative difference in responsiveness can be detected against the a haplotype using a variety of organ grafts. We have confirmed a qualitative difference between high and low responders using PVG.R1 donors presenting an isolated class I (Aa) difference. Rapid rejection by high responders contrasted with complete failure to reject by the low responders. No difference in rejection tempo was found when a full a haplotype mismatch was introduced. This could have reflected vigorous responses to I and C region differences, because rapid rejection through these regions was demonstrated using the PVG.r1 (AaIcCc) and PVG.r8 (AaIuCu) recombinants. The feeble immunogenicity of the Aa antigen for PVG animals was revealed by priming and cross-priming experiments showing not only that r1 failed to prime for subsequent r1 graft rejection, but that the Aa antigen presented in concert with Ia and Ca also failed to prime. An unexpected result was that the Aa antigen of r1 actually suppressed responsiveness, especially when delivered by a heart graft. This suppression not only extended to subsequent r1 grafts (for example, skin rafts) but also to subsequent grafts of a tissue. The mechanism of this suppression remains unclear but preliminary experiments argue in favor of enhancement rather than active suppression.

Animals↗
PubMed1993

Genetic control of the cellular in vitro response to the H-Y antigen in the rat.

The immune response against the male-specific H-Y antigen has been studied in RT1 congenic rat strains by assaying the cytotoxic and proliferative response in vitro after in vivo priming. The capacity to respond is associated with the RT1a and RT1n major histocompatibility haplotypes, and nonresponsiveness with the RT1u haplotype. Both, genes of the RT1.A region, which encodes class I antigens, and of the RT1.B/D region, which includes class II and transporter genes, are involved in the genetic control. In F1 hybrids haplotype preference of H-Y restriction occurs in favor of RT1a. Cross-priming can be induced in F1 hybrids by parental cells for RT1a-restricted cytotoxic and proliferative T cells. Allopriming is successful only in the RT1a-carrying strain, whereas xenopriming with mouse cells could not be elicited. The results are discussed in the context of current views on processing and presentation of antigens, and their relevance for transplantation is pointed out.

Animals↗
PubMed1998

Microchimerism after liver transplantation: prevalence and methodological aspects of detection.

BACKGROUND: Microchimerism after liver transplantation is a readily observed phenomenon. The immunological implications, however, remain unclear. Moreover, methodological approaches and their detection limits in the study of allogeneic microchimerism have not been studied in detail. METHODS: Therefore, the aim of this study was to evaluate the single-step and nested formats of the polymerase chain reaction/sequence-specific priming (PCR-SSP) approach under standardized conditions. For that purpose, a panel of recombinant plasmid clones was generated by PCR cloning. The panel contained the allelic sequences of the second exon of DRB1 covering all DR specificities on a low-resolution level. Using this panel, limiting dilution assays for various DR sequences in the presence and absence of competitor DNA were carried out to determine the minimal number of copies required for detection by single-step and nested PCR-SSP. Subsequently, 22 liver transplant recipients were analyzed in a retrospective study for the presence of allogeneic microchimerism by nested PCR-SSP. RESULTS: Although at least 10 copies of template DNA could be detected by nested PCR-SSP overall, single-step PCR-SSP was on average 10(2) to 10(3) times less sensitive. Upon the addition of human competitor DNA, the detection limits decreased on average by a factor of 10. In addition, sequence-specific differences in amplification efficiency could be appreciated. Using nested PCR-SSP, peripheral blood allogeneic microchimerism could be observed in 17 of 22 HLA-DR-mismatched liver recipients. Recombinants representing recipient DRB1 specificities were used to exclude false-positive results by lack of cross-reactivities of the donor-specific primers and to evaluate negative results due to sample-related reduced amplification efficiencies in microchimerism-negative recipients. In donor/recipient combinations that differed by at least one DR specificity, allogeneic microchimerism was seen in 87.5% of the cases. In five chimerism-negative cases, sample-related problems were detected in two cases. CONCLUSION: The optimization and standardization of the detection of genomic HLA sequences at low copy number may be greatly facilitated using a clonal reference system. Furthermore, a clonal reference system may be used to conduct cross-priming experiments to exclude false-positive results and may allow the determination of sample-specific detection limits for donor-derived HLA-DR specificities in chimerism-negative patients. Our evaluation of the PCR-SSP approach for the study of allogeneic microchimerism indicated that nested PCR-SSP provides the most sensitive format when HLA sequences are targeted. Yet, the detection sensitivity may vary between individual alleles and specificities. Allogeneic microchimerism in liver recipients can be observed in the majority of patients. However, the detection may be subject to the degree of mismatching, the HLA-DR alleles involved, and sample-related impaired PCR amplification efficiency.

Alleles↗
PubMed1995

A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides.

Endogenously synthesized antigenic determinants are generally presented on major histocompatibility complex (MHC) class I molecules, whereas exogenous determinants are presented by MHC class II molecules. Here, it is shown that exogenous antigens chaperoned by a heat shock protein can be channeled into the endogenous pathway, presented by MHC class I molecules, and recognized by CD8+ T lymphocytes. This pathway is functional only in a subset of macrophages among the cell types tested. These observations provide a basis for the tumor-specific and virus-specific immunogenicity of cognate heat shock protein preparations and offer a mechanism for the classical phenomenon of cross-priming.

Amino Acid Sequence↗
PubMed1996

Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys.

We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasite-purified or recombinant merozoite surface protein MSP1 of Plasmodium falciparum. Cross-priming studies also suggested that a conserved T-helper epitope(s) is efficient in inducing the anti-MSP1 antibody response. In this study, we determined whether a similar profile of immune responses was induced during live P. falciparum infections. Naive Aotus monkeys were infected by blood-stage challenge with either one of the two dimorphic MSP1 alleles represented by the FUP and FVO parasites. Sera collected after parasite clearance were analyzed by enzyme-linked immunosorbent assays (ELISAs). Monkeys infected with parasites carrying one allelic form of MSP1 had antibodies that were equally reactive with homologous or heterologous MSP1s. This preferential recognition of conserved epitopes of MSP1 was confirmed by competitive binding ELISAs. Studies with Plasmodium yoelii and P. falciparum show that the C-terminal 19-kDa fragment of MSP1, MSP1(19), is the target of protective immunity. Thus, monkey sera were assayed for recognition with recombinant MSP1(19)s expressing variant and conserved B epitopes. Results of direct and competitive binding ELISAs showed that the anti-MSP1(19) antibodies were also directed primarily against conserved determinants. The similarities between vaccine- or infection-induced antibody responses suggest a possible reciprocal enhancement of the two populations of anti-MSP1 antibodies when a subunit MSP1 vaccine is introduced into populations living in areas where malaria is endemic. This together with previous observations that conserved determinants are important in MSP1-mediated immunity provides an optimistic outlook that a subunit MSP1 vaccine may be effective and practical for field applications in malaria-exposed populations.

Alleles↗
PubMed1991

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.

Animals↗
PubMed1999

Host B7-1 and B7-2 costimulatory molecules contribute to the eradication of B7-1-transfected P815 tumor cells via a CD8+ T cell-dependent mechanism.

B7-1 (CD80)-transfected P815 tumor cells were previously shown to elicit tumor-eradicating immunity that leads to the regression of B7-1+ P815 tumors after transient growth in normal syngeneic (DBA/2) mice. Here, we show that not only the B7-1 molecule but also the B7-2 (CD86) molecule contributed to the eradication of B7-1+ P815 tumors. The B7-1 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed not only on the tumor cells but also on host APCs, including MAC-1+ cells. The B7-2 molecule that contributed to the eradication of B7-1+ P815 tumors was expressed only on host APCs, such as B220+ cells, and not on the tumor cells. In spite of the fact that B7-expressing host APCs contributed to the eradication of B7-1+ P815 tumors, only CD8+ T cells without help from CD4+ T cells were important for tumor eradication. Taken together, these findings indicate that in addition to the ability of B7-1-transfected tumor cells to stimulate CD8+ T cell-mediated tumor-eradicating immunity directly, such tumor cells can also stimulate CD8+ T cell-mediated tumor-eradicating immunity indirectly as a result of cross-priming through B7-expressing host APCs.

Animals↗