Gene therapy for tolerance: high-level expression of donor major histocompatibility complex in the liver overcomes naive and memory alloresponses to skin grafts.

BACKGROUND: The liver has long been recognized as having tolerogenic properties. We investigated whether recombinant adenoassociated virus (rAAV)-mediated expression of donor major histocompatibility complex in recipient livers could induce tolerance to donor-strain grafts. METHODS: Naive B10.BR (H-2) or B10.BR recipients primed with a H-2K-expressing (K) skin graft were injected with rAAV-expressing H-2K (rAAV-K) to induce K expression on hepatocytes 7 days before challenge with a K skin graft. K-specific responses were measured by interferon (IFN)-γ ELISpot and flow cytometric assessment of directly H-2K reactive cells. Fully allogeneic grafts from C57BL/6 (H-2) donors were transplanted onto longstanding B10.BR recipients of K skin to test for linked epitope suppression. RESULTS: rAAV-K-treated B10.BR mice accepted K skin grafts with increased median survival time (MST) more than 169 days compared to uninoculated (MST=18.5 days) and rAAV-K-treated controls (MST=19 days). rAAV-K-treated B10.BR animals primed with K skin grafts also accepted secondary K skin grafts in the long term (MST>100 days) compared to accelerated rejection in primed, uninoculated mice (MST=12 days). Treatments did not induce liver pathology, assessed by serum alanine aminotransferase levels and histology. IFN-γ ELISpot analysis of splenocytes from rAAV-K-treated mice indicated reduced responses to donor K antigen, but protection was not extended to fully allogeneic C57BL/6 skin or heart grafts, even in recipients that had accepted K skin grafts in the long term. CONCLUSIONS: High-level expression of donor major histocompatibility complex in recipient livers promotes tolerance to skin allografts, even in animals primed to produce a memory response. This provides proof of concept for an approach using liver-targeted gene delivery for tolerance induction to donor antigen.

TAP expression reduces IL-10 expressing tumor infiltrating lymphocytes and restores immunosurveillance against melanoma.

Many immune therapeutic strategies are under development for melanoma to treat metastatic disease and prevent disease reoccurrence. However, human melanoma cells are often deficient in antigen processing and this appears to play a role in their expansion and escape from immunosurveillance. For example, expression of the transporters associated with antigen processing (TAP1 and TAP2) is down-regulated in the mouse melanoma cell line B16F10. This results in a lack of tumor-associated antigen processing, low surface expression of MHC Class I molecules and low immunogenicity. We observe that restoration of TAP1 expression by transfection resurrects the processing and presentation of viral antigens, and the melanoma-associated antigen, TRP-2. Immunization with irradiated B16F10/rTAP1 transfected cells generates CTLs that are capable of killing B16F10/rTAP1 transfected targets and B16F10 targets deficient in TAP1. Furthermore, B16F10/rTAP1 transfectants grow at a significantly slower rate in mice than B16F10 cells. In an experimental model that closely recapitulates the clinical situation, treatment of B16F10 tumors in mice with a vaccinia virus vector expressing TAP1 also significantly decreases tumor growth in vivo. Furthermore, tumors treated with vaccinia TAP1 had significantly reduced numbers of immunosuppressive, CD3(+)/IL-10 positive, tumor infiltrating lymphocytes. Therefore, TAP1 expression restores both antigen presentation and immunogenicity in B16F10 melanoma cells and concomitantly reduces immunosuppressive IL-10 production at the local tumor site, thereby increasing immunosurveillance mechanisms against tumors.

New strategy for the identification of squamous carcinoma antigens that induce therapeutic immune responses in tumor-bearing mice.

This study describes a new strategy for the identification of squamous carcinoma antigens tumor-associated antigens (TAA). The antigens were discovered by comparing microarrays of squamous carcinoma vaccines highly enriched for immunotherapeutic cells with non-enriched vaccines. The vaccines were prepared by transferring sheared genomic DNA fragments (25 kb) from KLN205 cells, a squamous carcinoma cell line (DBA/2 mouse origin (H-2(d)) into LM fibroblasts (C3H/He origin, H-2(k)). The transferred tumor DNA segments integrate spontaneously into the genome of the recipient cells, replicate as the cells divide and are expressed. As only a small proportion of the transfected cell population was expected to have incorporated DNA segments that included genes specifying TAA (the vast majority specify normal cellular constituents), a novel strategy was employed to enrich the vaccine for TAA-positive cells. Microarrays were used to compare genes expressed by enriched and non-enriched vaccines. Seventy-five genes were overexpressed in cells from the enriched vaccine. One, the gene for Cytochrome P450 (family 2, subfamily e, polypeptide 1) (Cyp2e1), was overexpressed in the enriched but not the non-enriched vaccine. A vaccine for squamous carcinoma was prepared by transfer of a 357 bp fragment of the gene for Cyp2e1 into the fibroblast cell line. Robust immunity, sufficient to result in indefinite survival, was induced in tumor-bearing mice immunized with cells transfected with this gene fragment.

The replacement of graft endothelium by recipient-type cells conditions allograft rejection mediated by indirect pathway CD4(+) T cells.

BACKGROUND: Whereas the participation of alloreactive T cells sensitized by indirect allorecognition in graft rejection is well documented, the nature of recipient antigen presenting cells recognized by indirect pathway CD4(+) T cells within the graft has yet to be identified. The purpose of this study was to determine the role played by graft endothelium replacement in the immune recognition of cardiac allografts rejected by indirect pathway CD4(+) T cells. METHODS: Transgenic RAG2(-/-) mice expressing I-A(b)-restricted male antigen H-Y-specific TcR were studied for their capacity to reject H-2(k) male cardiac allografts. Chronic vascular rejection in this model was due to the indirect recognition of H-Y antigen shed from H-2(k) male allograft and presented by the recipient's own I-A(b) APC to transgenic T cells. RESULTS: Immunohistochemical analysis of rejected grafts revealed the presence of numerous microvascular endothelial cells (EC) that expressed recipient's I-A MHC class II molecules. This observation suggested that graft endothelium replacement by I-A(b)-positive cells of recipient origin could stimulate the rejection of male H-2(k) graft by I-A(b)--restricted H-Y--specific T cells. To investigate further this possibility, hearts from H-2(b)--into--H-2(k) irradiation bone marrow (BM) chimera were transplanted in transgenic recipients. A direct correlation was observed between the presence of I-A(b)-positive EC within myocardial microvessels and the induction of acute rejection of chimeric H-2(k) male cardiac allografts transplanted in transgenic recipients. CONCLUSIONS: We conclude that graft endothelium replacement by recipient-type cells is required for the rejection of cardiac allograft mediated by indirect pathway alloreactive CD4(+) T cells.

Effect of X- and Y-box deletions on the development of diabetes in H-2Ealpha-chain transgenic nonobese diabetic mice.

The development of type 1 diabetes in nonobese diabetic (NOD) mice is influenced by major histocompatibility complex (MHC) class II genes. The NOD-E transgenic mouse, which expresses H2-E as a result of the introduction of an Ead gene, is protected from development of type 1 diabetes. While the mechanism of protection remains unclear, the effect has been regarded as a model system for MHC protection from autoimmunity. We have investigated the effect of deletions of the Ea promoter region, which, in turn, affect H2-E expression patterns in transgenic NOD mice. We have constructed transgenic NOD mice where the X (DeltaX) and Y (DeltaY) boxes of the Ead gene have, respectively, been functionally deleted. Previous reports, using X- or Y-box-deleted H2-E transgenic mice, made by crossing the appropriate transgenes onto the NOD background from C57BL/6 transgenic mice, indicated that promoter mutation abrogated the H2-E-mediated protection seen in NOD-E. The NOD DeltaX and NOD DeltaY transgenic mice generated in the present study differ in susceptibility to diabetes from wild-type NOD mice. NOD DeltaY1 animals are protected from diabetes development, while DeltaX mice remain susceptible, albeit to a lesser extent than the parental NOD strain.

The regulatory functions of Ly-49A, Ly-49D and Ly-49G2 on NK cells in the recognition and rejection of the alloantigen in vivo.

Ly-49A is an inhibitory receptor that binds H-2Dd and H-2Dk. The downregulation of Ly-49A is thought to mediate NK self tolerance in vivo. In this study, we analyzed the regulation of Ly-49A, D and G2 on NK cells in an in vivo rejection model. After injection with 1 x 10(8) B10.D2 spleen cells (SC) into B 10 mice, we found Ly-49A downregulated within 3 h on NK cells of B10 mice, whereas expressions of Ly-49D and G2 were augmented. To investigate effects of different expression patterns of Ly-49 receptors on NK cells, Ly-49A, D or G2-depleted B10 mice were inoculated with B10.D2 SC. NK cells from SC of Ly-49A-depleted and B10.D2 SC-injected B10 mice showed enhanced cytotoxicity to Dd-positive targets in vitro. Furthermore, reduced numbers of B10.D2 SC were observed in Ly-49A or G2-depleted B10 mice, whereas increased numbers of B10.D2 SC were observed in Ly-49D-depleted B10 mice after inoculation with B10.D2 SC in vivo. These findings indicated that the downregulation of Ly-49A and the augmentation of Ly-49D expression may mediate NK cells to recognize and kill Dd antigen efficiently. In conclusion, each Ly-49 isoform may play independent roles in the regulation of activation or inhibition on NK cells.

Intratumoral injection of IL-secreting syngeneic/allogeneic fibroblasts transfected with DNA from breast cancer cells prolongs the survival of mice with intracerebral breast cancer.

Prior studies have revealed the immunotherapeutic properties of a vaccine prepared by transfer of genomic DNA from breast cancer cells into a highly immunogenic cell line. The rationale for this type of vaccine is that genes specifying an array of weakly immunogenic, unique tumor antigens associated with the malignant cells will be expressed in a highly immunogenic form by the transfected cells. Here, the immunotherapeutic properties of a vaccine prepared by transfection of mouse fibroblasts with DNA from a breast carcinoma (SB-5b) that arose spontaneously in a C3H/He mouse (H-2Kb) were tested in mice with intracerebral breast cancer. To augment their nonspecific immunogenic properties, before DNA transfer, the fibroblasts (of C3H/He mouse origin) were modified to express allogeneic MHC class I H-2Kb-determinants and to secrete IL-2, IL-18 or GM-CSF. The results indicate that C3H/He mice injected intracerebrally (i.c.) with the breast cancer cells and syngeneic/allogeneic-transfected fibroblasts modified to secrete IL-2 survived significantly longer (P < .005) than mice in various control groups, including mice injected i.c. with the breast cancer cells alone. The immunotherapeutic properties of transfected fibroblasts modified to secrete IL-18 or GM-CSF were less efficacious. The results of two independent in vitro cytotoxicity assays indicate that systemic cellular antitumor immunity was generated in mice injected i.c. with the transfected cells, and the immunity was mediated predominantly by CD8+ T cells.

Macrophage colony-stimulating factor (M-CSF), as well as granulocyte colony-stimulating factor (G-CSF), accelerates neovascularization.

It has been reported that bone marrow cells (BMCs) differentiate into endothelial cells of blood vessels, and that granulocyte colony-stimulating factor (G-CSF) mobilizes progenitors in the BMCs to the peripheral blood, while macrophage colony-stimulating factor (M-CSF) augments the production of monocytes. We examined whether M-CSF augments the differentiation of BMCs into endothelial cells of blood vessels using a hindlimb-ischemic model. Either G-CSF or M-CSF, or both, was administered to the hindlimb-ischemic mice for 3 days. Both M-CSF and G-CSF augmented the differentiation of BMCs into endothelial cells of blood vessels through vascular endothelial cell growth factor (VEGF), resulting in early recovery of blood flow in the ischemic limbs.

Prolonged survival of mouse skin allografts after transplantation of fetal liver cells transduced with hIL-10 gene.

INTRODUCTION: Interleukin-10 (IL-10) is a cytokine with a moleculary weight of 18 kDa, that was first identified as being produced by Th2 cells. It appears to have anti-inflammatory action by diminishing the production of pro-inflammatory cytokines produced by Th1 cells. IL-10 also regulates the differentiation and proliferation of several immune cells such as T cells, B cells, natural killer cells, antigen-presenting cells, mast cells and granulocytes. Recent data suggest, however, that IL-10 also has immunostimulatory properties with important consequences on the prognosis of disease. In this study, we demonstrate the importance of injection of hematopoietic fetal liver cells transduced with the human IL-10 (hIL-10) gene into an allogenic recipient subsequently transplanted with allogenic skin grafts. The immaturity of stem cells and precursor cells from fetal liver and their transient survival in the host, due to the production of hIL-10, may afford 'prope' tolerance. It also explains the lack of graft-vs.-host reaction (GvHR) and the delay in rejection of the specific donor skin grafts after virtual disappearance of donor hematopoietic cells. OBJECTIVES: Transduction of CBA hematopoietic fetal cells with the human IL-10 gene was used with the aim of inducing tolerance to donor antigen in recipient BALB/c mice. The observed effects were prolonged IL-10 production, donor cell chimerism in the host and delayed rejection of skin grafts from the specific donor strain. MATERIALS AND METHODS: To prevent or delay rejection of highly incompatible skin allografts, we used IL-10 gene transfer to establish chimerism with donor hematopoietic cells. Fetal liver cells from CBA mice were transduced with the human IL-10 gene and injected into BALB/c mice. RESULTS: Human IL-10, which is active in mice but does not cross-react with murine IL-10 in ELISA, was produced in vivo for 3 weeks. Donor cells were identified in the recipients during the same time period, on the basis of presence of the H-2 k gene and human IL-10 intracellular protein. Skin allografts from CBA or C57BL/6 mice survived for a mean of 9.5 days in recipient mice injected with non-transduced cells. In contrast, survival of CBA allograft was extended to 18.9+/-1.8 days in recipients injected with hIL-10-transduced fetal liver cells from CBA mice. Human IL-10 alone, without donor hematopoietic cell engraftment, did not prolong graft survival (9.6+/-1.2 days). CONCLUSIONS: IL-10 transduction of donor hematopoietic stem cells resulted in production of IL-10, cell engraftment and chimerism. Although full tolerance was not obtained at this level of donor cell development in the host, a specific and highly significant (P<0.001) prolongation of the survival of donor skin allografts was observed.

Consequences of Notch-mediated induction of Jagged1.

Notch signaling is initiated upon contact of cells expressing Notch receptors with those expressing ligands. While examining the dynamic response of NIH 3T3 cells to cells expressing the Notch ligand Jagged1, we found that Notch signaling resulted in increased levels of the ligand Jagged1. Induction of Jagged1 was delayed relative to the generation of active Notch and dependent on the transcription factor p63. The induced Jagged1 had no apparent autocrine effects on Notch signaling but could promote signaling in naïve cells. These results describe a mechanism through which Notch signaling can be relayed from cell to cell.

[Allogeneic chimerism induced by B7 antisense peptide pre-treated splenocytes prolongs the survival of allograft in mice].

OBJECTIVE: To investigate the roles of B7 antisense peptide (B7AP) in blocking the CD28-B7 pathway and inducing the allogeneic chimerism. METHODS: B7 antisense peptide was synthesized by solid phase synthetic methods and purified with HPLC. The C57BL/6 splenocytes of mice were pre-treated by B7AP, and subsequently injected in travenously to BALB/c mice. Three days later the mice were injected with fresh-made bone marrow cells derived from C57BL/6 mice. The B7 expression and allogeneic chimerism were analyzed with FACS. The lymphocyte proliferation reaction and the mice pinna cardiac transplantation model were exerted to study the relation between chimerism and prolongation of allograft in vitro and in vivo. RESULTS: Lymphoproliferation of the splenocytes derived from BALB/c mice immunized with the B7AP pretreated C57BL/6 splenocytes versus splenocytes from C57BL/6 mice was inhibited dramatically with a inhibition rates up to 43%. Under this condition, the allogeneic chimerism was successfully induced after BMT. Both the chimerism and the survival of allogeneic cardiac grafts were prolonged over 100 days (n = 6). CONCLUSION: Synthetic B7 antisense peptide can induce allogeneic chimerism in mice and consequently prolong the survival of allogeneic cardiac grafts.

Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells.

The classical paradigm for dendritic cell function derives from the study of Langerhans cells, which predominate within skin epidermis. After an encounter with foreign agents, Langerhans cells are thought to migrate to draining lymph nodes, where they initiate T cell priming. Contrary to this, we show here that infection of murine epidermis by herpes simplex virus did not result in the priming of virus-specific cytotoxic T lymphocytes by Langerhans cells. Rather, the priming response required a distinct CD8alpha+ dendritic cell subset. Thus, the traditional view of Langerhans cells in epidermal immunity needs to be revisited to accommodate a requirement for other dendritic cells in this response.

Cell surface expression of heat shock protein gp96 enhances cross-presentation of cellular antigens and the generation of tumor-specific T cell memory.

Gp96 is an endoplasmic reticular heat shock protein (HSP). We have shown previously that surface expression of gp96 (96tm) on tumor cells led to the activation of dendritic cells and increased anti-tumor immunity. In this report, we have found that protective immunity elicited by 96tm+ tumor cells was tumor-specific and long-lasting. Both CD4+ and CD8+ T cell memory were elicited. By immunizing with tumor cells loaded with the chicken ovalbumin (ova) model antigen, we demonstrated that the priming of adoptively transferred ova-specific CD8+ T cells could occur across MHC haplotypes. The efficiency of this cross priming can be significantly increased when mice were immunized with whole cells that express both ova and cell surface gp96 (ova+96tm+). Mere mixture of soluble ova with 96tm-expressing tumor cells (ova-96tm+) was insufficient, arguing for further processing of ova and perhaps the participation of 96tm-ova complexes in this process. We further compared the relative efficiency of two whole cell vaccines based on the manipulation of gp96 expression in one system: 96tm+ whole cells and cells that secrete the gp96-Ig fusion protein. We found that both vaccines are effective in a prophylactic model against tumors. Our study has reinforced the notion that the manipulation of the site of expression of HSPs may be an effective approach for cancer immunotherapy.

H2-M3-restricted memory T cells: persistence and activation without expansion.

H2-M3-restricted T cells respond more rapidly to primary Listeria monocytogenes infection than conventional MHC class Ia-restricted T cells. Reinfection with L. monocytogenes, while inducing explosive proliferation of H2-K(d)-restricted T cells, does not stimulate significant expansion of H2-M3-restricted CTL. These disparate responses to reinfection are apparent within 5 days of primary L. monocytogenes infection. However, H2-M3-restricted memory T cells are generated, and are indistinguishable from classically restricted T cells in terms of cell surface memory markers and longevity. Early responses of H2-M3- and H2-K(d)-restricted memory T cells to reinfection are similar, with increases in size and expression of activation markers. Interestingly, priming of H2-M3-restricted T cells with an L. monocytogenes-derived N-formyl peptide plus anti-CD40 generates memory T cells that expand upon re-exposure to Ag during L. monocytogenes infection. Our data indicate that disparate H2-M3- and MHC class Ia-restricted memory T cell responses reflect intrinsic differences between these T cell populations. Although distinct proliferative programs appear to be hardwired in these populations during primary L. monocytogenes infection, under different inflammatory circumstances M3-restricted T cell populations can maintain the ability to expand upon re-exposure to Ag.

Intrinsic susceptibility of mouse trophoblasts to natural killer cell-mediated attack in vivo.

Protecting the fetus and placenta from the maternal immune system has long been considered a function of placental trophoblasts. Here, we present two related lines of evidence that contradict this assumption. First, we show that transformed mouse trophoblast cell lines akin to human choriocarcinomas form tumors in syngeneic and immunodeficient mice, yet are rejected in immunocompetent allogeneic mice. Second, we show that wild-type trophoblasts are rapidly killed after i.v. injection into allogeneic mice. In both cases, the pattern of trophoblast killing in different strains of immunodeficient mice indicated that rejection involved host natural killer cells, and this was corroborated by in vitro killing assays. The apparent intrinsic susceptibility of mouse trophoblasts to immune attack strongly suggests that it is instead some property of the pregnant uterus that is of primary importance in preventing rejection of the fetus.

Cytokine-dependent bystander hepatitis due to intrahepatic murine CD8 T-cell activation by bone marrow-derived cells.

BACKGROUND & AIMS: Intrahepatic accumulation of CD8+ T cells following antigen-specific activation has been demonstrated in a number of transgenic models and also in extrahepatic viral infections. In some transgenic models, intrahepatic accumulation of cytotoxic T lymphocytes is associated with hepatitis. This observation suggests that hepatocellular damage may occur in some forms of immune-mediated hepatitis on the basis of a "bystander injury," whereby cytotoxic T lymphocytes accumulating in the liver mediate injury to hepatocytes in a nonspecific manner. Mouse transgenic models were therefore developed to investigate whether bystander damage to non-antigen-bearing hepatocytes occurs in vivo. METHODS: T cell receptor transgenic T cells were adoptively transferred into transgenic mice ubiquitously expressing the specific antigen, or into bone marrow radiation chimeras in which hepatocytes did not express the antigen. RESULTS: Selective accumulation of transgenic CD8+ T cells in the liver of intact recipients could be detected within 2 hours of transfer, despite ubiquitous antigenic expression. T cells retained in the liver were activated and induced hepatitis. Similar results were obtained using bone marrow chimeras, suggesting that antigen expression by hepatocytes was not required either for intrahepatic accumulation or for subsequent hepatitis. This "bystander hepatitis" was dependent on tumor necrosis factor alpha and interferon gamma. CONCLUSIONS: Intrahepatic accumulation of activated CD8+ T cells and subsequent hepatitis can result from primary activation of CD8+ T cells by liver resident bone marrow-derived cells, inducing bystander damage to non-antigen-bearing hepatocytes. This mechanism may play a role in some forms of biologically significant hepatitis, including autoimmune hepatitis and hepatitis associated with extrahepatic diseases.

Inhibitory effects of cytomegalovirus proteins US2 and US11 point to contributions from direct priming and cross-priming in induction of vaccinia virus-specific CD8(+) T cells.

The extent to which naive CD8(+) CTLs (T(CD8)(+)) are primed by APCs presenting endogenous Ags (direct priming) or Ags acquired from other infected cells (cross-priming) is a critical topic in basic and applied immunology. To examine the contribution of direct priming in the induction of VV-specific T(CD8)(+), we generated recombinant vaccinia viruses that express human CMV proteins (US2 and US11) that induce the destruction of newly synthesized MHC class I molecules. Expression of US2 or US11 was associated with a 24-63% decrease in numbers of primary or secondary VV-specific T(CD8)(+) responding to i.p. infection. Using HPLC-isolated peptides from VV-infected cells, we show that US2 and US11 selectively inhibit T(CD8)(+) responses to a subset of immunogenic VV determinants. Moreover, VV-US2 and lysates from VV-infected histoincompatible cells elicit T(CD8)(+) specific for a similar subset of VV determinants. These findings indicate that US2 and US11 can function in vivo to interfere with the activation of virus-specific T(CD8)(+). Furthermore, they suggest that 1) both cross-priming and direct priming contribute significantly to the generation of VV-specific T(CD8)(+), 2) the sets of immunogenic vaccinia virus determinants generated by cross-priming and direct priming are not completely overlapping, and 3) cross-priming overrides the effects of cis-acting viral interference with the class I Ag presentation pathway.

Mutant MHC class I molecules define interactions between components of the peptide-loading complex.

Class I histocompatibility molecules, consisting of a heavy chain, beta2-microglobulin and peptide, are assembled in the endoplasmic reticulum (ER) with the assistance of several molecular chaperones and accessory proteins. Peptide binding occurs when assembling class I molecules associate with a loading complex consisting of the transporter associated with antigen processing (TAP) peptide transporter, tapasin, ERp57 and calreticulin (CRT)/calnexin. To assess the physical organization of this complex, we generated a series of mutants in the murine H-2Dd heavy chain and assessed their association with components of the complex. Seven mutations, clustered between amino acids 122 and 136 in the heavy chain alpha2 domain plus one mutation at position 222 in the alpha3 domain, resulted in loss of interaction with tapasin. Association with TAP was always lost simultaneously, supporting the view that tapasin acts as an obligatory bridge between class I molecules and TAP. Compared with previous studies on the HLA-A2 molecule, some differences in points of tapasin interaction were observed. Failure of the H-2Dd mutants to bind tapasin resulted in low cell-surface expression and altered intracellular transport. Most mutants retained a substantial degree of peptide loading, consistent with the view that although tapasin may promote peptide binding to class I, it is not required. A surprising observation was that all mutants lacking tapasin interaction retained normal association with CRT. This contrasts with previous observations on other class I molecules and, combined with differences in tapasin interaction, suggests that the organization of the ER peptide-loading complex can vary depending on the specific class I molecule examined.

Characterization of the Fas ligand/Fas-dependent apoptosis of antiretroviral, class I MHC tetramer-defined, CD8+ CTL by in vivo retrovirus-infected cells.

C57BL/6 (B6; H-2(b)) mice mount strong AKR/Gross murine leukemia virus (MuLV)-specific CD8(+) CTL responses to the immunodominant K(b)-restricted epitope, KSPWFTTL, of endogenous AKR/Gross MuLV. In sharp contrast, spontaneous virus-expressing AKR.H-2(b) congenic mice are low/nonresponders for the generation of AKR/Gross MuLV-specific CTL. Furthermore, when viable AKR.H-2(b) spleen cells are cocultured with primed responder B6 antiviral precursor CTL, the AKR.H-2(b) cells function as "veto" cells that actively mediate the inhibition of antiviral CTL generation. AKR.H-2(b) veto cell inhibition is virus specific, MHC restricted, contact dependent, and mediated through veto cell Fas ligand/responder T cell Fas interactions. In this study, following specific priming and secondary in vitro restimulation, antiretroviral CD8(+) CTL were identified by a labeled K(b)/KSPWFTTL tetramer and flow cytometry, enabling direct visualization of AKR.H-2(b) veto cell-mediated depletion of these CTL. A 65-93% reduction in the number of B6 K(b)/KSPWFTTL tetramer(+) CTL correlated with a similar reduction in antiviral CTL cytotoxicity. Addition on sequential days to the antiviral CTL restimulation cultures of either 1) AKR.H-2(b) veto cells or 2) a blocking Fas-Ig fusion protein (to cultures also containing AKR.H-2(b) veto cells) to block inhibition demonstrated that AKR.H-2(b) veto cells begin to inhibit B6 precursor CTL/CTL expansion during days 2 and 3 of the 6-day culture. Shortly thereafter, a high percentage of B6 tetramer(+) CTL cocultured with AKR.H-2(b) veto cells was annexin V positive and Fas(high), indicating apoptosis as the mechanism of veto cell inhibition. Experiments using the irreversible inhibitor emetine demonstrated that AKR.H-2(b) cells had to be metabolically active and capable of protein synthesis to function as veto cells. Of the tetramer-positive CTL that survived veto cell-mediated apoptosis, there was no marked skewing from the preferential usage of Vbeta4, 8.1/8.2, and 11 TCR normally observed. These findings provide further insight into the complexity of host/virus interactions and suggest a fail-safe escape mechanism by virus-infected cells for epitopes residing in critical areas of viral proteins that cannot accommodate variations of amino acid sequence.

[Biological characterization of mouse erythroblastic leukemia cells in haploiden tical mice].

Using transplantable erythroblastic leukemia cells of EL9611(H-2d), the cells were inoculated to CB6F(1)(H-2d/b) generation of BALB/c x C57BL/6 mouse, the biological characterization of erythroblastic leukemia in haploidentical mouse was studied, that provides an experimental model for the study of graft-versus leukemia (GVL) with bone marrow or stem cell transplantation. When 10(3) - 10(8) of the spleen cells of EL9611(H-2d) had been intravenously inoculated to CB6F(1) mouse, the erythroblastic leukemia cells were transplanted successively and the F(1) generation of erythroblastic leukemia model in mice was established with 100% incidence of erythroblastic leukemia. There was a linear relationship between the survival time and the number of leukemic cell. The survival time of the mice was (9.6 +/- 0.8) days when 10(6) cells were inoculated. If the CB6F(1) mouse was transplanted successively for four generations, the incidence was 100%. The main targets for the leukemic EL9611(H-2d) cells were liver, spleen and marrow. The reaction of the erythroblastic leukemia cells for hemoglobin staining was positive, while the peroxidase reaction was negative. These cells were sensitive to some chemotherapeutic drugs, such as cytosine arabinoside and cyclophosphamide. This study presents the convenience for the studies on the GVL with haplo-allogeneic transplantation, in the F(1) generation of erythroblastic leukemia model of the commonly-used CD57BL/6 x BALB/c mouse.