Embryonic lethality and impairment of haematopoiesis in mice heterozygous for an AML1-ETO fusion gene.

Acute myeloid leukaemia (AML) is a major haematopoietic malignancy characterized by the proliferation of a malignant clone of myeloid progenitor cells. A reciprocal translocation, t(8;21)(q22;q22), observed in the leukaemic cells of approximately 40% of patients with the M2 subtype of AML disrupts both the AML1 (CBFA2) gene on chromosome 21 and the ETO (MTG8) gene on chromosome 8 (refs 3-5). A chimaeric protein is synthesized from one of the derivative chromosomes that contains the N terminus of the AML1 transcription factor, including its DNA-binding domain, fused to most of ETO, a protein of unknown function. We generated mice that mimic human t(8;21) with a "knock-in' strategy. Mice heterozygous for an AML1-ETO allele (AML1-ETO/+) die in midgestation from haemorrhaging in the central nervous system and exhibit a severe block in fetal liver haematopoiesis. This phenotype is very similar to that resulting from homozygous disruption of the AML1 (Cbfa2) or Cbfb genes, indicating that AML1-ETO blocks normal AML1 function. However, yolk sac cells from AML1-ETO/+ mice differentiated into macrophages in haematopoietic colony forming unit (CFU) assays, unlike Cbfa2-/- or Cbfb-/-cells, which form no colonies in vitro. This indicates that AML1-ETO may have other functions besides blocking wild-type AML1, a property that may be important in leukaemogenesis.

Role of the PD-1 pathway in the immune response.

Understanding immunoregulatory mechanisms is essential for the development of novel interventions to improve long-term allograft survival. Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, have emerged as critical inhibitory signaling pathways that regulate T cell response and maintain peripheral tolerance. PD-1 signaling inhibits alloreactive T cell activation, and can promote induced regulatory T cell development. Furthermore, the upregulation of PD-L1 on nonhematopoietic cells of the allograft may actively participate in the inhibition of immune responses and provide tissue-specific protection. In murine transplant models, this pathway has been shown to be critical for the induction and maintenance of graft tolerance. In this review, we discuss the current knowledge of the immunoregulatory functions of PD-1 and its ligands and their therapeutic potential in transplantation.

Essential role of PDL1 expression on nonhematopoietic donor cells in acquired tolerance to vascularized cardiac allografts.

The PD1:PDL1 pathway is an essential negative costimulatory pathway that plays a key role in regulating the alloimune response. PDL1 is expressed not only on antigen-presenting cells (APCs) but also cardiac endothelium. In this study, we investigated the importance of PDL1 expression on donor cardiac allograft in acquired transplantation tolerance in a fully MHC-mismatched model. We generated PDL1 chimeric mice on B6 background that expressed PDL1 on either hematopoietic cells or nonhematopoietic cells of the heart. Sham animals were used as controls. These hearts were then transplanted into BALB/c recipients and treated with CTLA4-Ig to induce tolerance. Cardiac endothelium showed significant expression of PDL1, which was upregulated upon transplantation. While the absence of PDL1 on hematopoietic cells of the heart resulted in delayed rejection and prevented long-term tolerance in most but not all recipients, we observed an accelerated and early graft rejection of all donor allografts that lacked PDL1 on the endothelium. Moreover, PDL1-deficient endothelium hearts had significant higher frequency of IFN-γ-producing alloreactive cells as well as higher frequency of CD8(+) effector T cells. These findings demonstrate that PDL1 expression mainly on donor endothelium is functionally important in a fully allogeneic mismatched model for the induction of cardiac allograft tolerance.

B7-1 or B7-2 is required to produce the lymphoproliferative phenotype in mice lacking cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).

The costimulatory molecules B7-1 and B7-2 regulate T lymphocyte activation by delivering activating signals through CD28 and inhibitory signals through cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The importance of CTLA-4-mediated inhibition was demonstrated by the uncontrolled T cell activation and lymphoproliferative disease that develops in CTLA-4-deficient (-/-) mice. To examine the role of B7 signaling in the activation of CTLA-4-deficient T cells, we bred CTLA-4(-/-) mice with mice lacking B7-1, B7-2, or both B7 molecules. The CTLA-4/B7-1(-/-) and the CTLA-4/B7-2(-/-) mice develop lymphoproliferation and enhanced T cell activation. Mice lacking CTLA-4, B7-1, and B7-2 have a normal life-span, and do not have lymphocytic infiltrates in any organs, or increased T cell activation. Therefore, the two B7 molecules have overlapping functions, since either B7-1 or B7-2 alone can cause the CTLA-4(-/-) phenotype. Elimination of both B7-1 and B7-2 from the CTLA-4- deficient mouse abrogates the lymphocyte activation and disease, and does not reveal evidence for additional stimulatory CD28 ligands. The CTLA-4(-/-) phenotype can be reproduced with anti-CD28 antibody in mice lacking CTLA-4, B7-1, and B7-2, but wild-type mice are unaffected by the same treatment. This suggests that the inhibitory function of CTLA-4 can overcome strong CD28-mediated signaling in vivo.

Studies in B7-deficient mice reveal a critical role for B7 costimulation in both induction and effector phases of experimental autoimmune encephalomyelitis.

The importance of B7 costimulation in regulating T cell expansion and peripheral tolerance suggests that it may also play a significant regulatory role in the development of autoimmune disease. It is unclear whether B7 costimulation is involved only in the expansion of autoreactive T cells in the periphery, or if it is also required for effector activation of autoreactive T cells in the target organ for mediating tissue injury and propagating autoimmune disease. In this study, the role of B7-CD28 costimulation and the relative importance of B7 costimulators for the induction and effector phases of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide were examined. Wild-type, B7-1/B7-2-deficient mice, or CD28-deficient C57BL/6 mice were immunized with MOG 35-55 peptide. Mice lacking both B7-1 and B7-2 or CD28 showed no or minimal clinical signs of EAE and markedly reduced inflammatory infiltrates in the brain and spinal cord. However, mice lacking either B7-1 or B7-2 alone developed clinical and pathologic EAE that was comparable to EAE in wild-type mice, indicating overlapping functions for B7-1 and B7-2. Resistance to EAE was not due to a lack of induction of T helper type 1 (Th1) cytokines, since T cells from B7-1/B7-2(-/-) mice show reduced proliferative responses, but greater interferon gamma production compared with T cells from wild-type mice. To study the role of B7 molecules in the effector phase of the disease, MOG 35-55-specific T lines were adoptively transferred into the B7-1/B7-2(-/-) and wild-type mice. Clinical and histologic EAE were markedly reduced in B7-1/B7-2(-/-) compared with wild-type recipient mice. These results demonstrate that B7 costimulation has critical roles not only in the initial activation and expansion of MOG-reactive T cells, but also in the effector phase of encephalitogenic T cell activation within the central nervous system.

Syngeneic monoclonal antiidiotype can induce cellular immunity to reovirus.

A syngeneic monoclonal antiidiotypic antibody was generated in BALB/c mice after repeated immunization with a BALB/c monoclonal anti-reovirus hemagglutinin (HA) antibody. The resultant syngeneic monoclonal antiidiotypic antibody, in the absence of adjuvant, was found to be capable of priming both BALB/c (H-2d, Igh-1a) and C3H/Hej (H-2k, Igh-1j) mice for Lyt-1+- and Lyt-2+-dependent responses against the mammalian reovirus. By the use of intertypic reassortants and variant virus analysis, the specificity of the response was finely mapped to the neutralization domain of the viral hemagglutinin (HA). Using purified monoclonal antiidiotype, we were able to compare the potency of antiidiotype to virus in terms of induction of immunity. 8 X 10(8) protein molecules were able to prime for cellular responses to reovirus. These studies indicate that in the reovirus system, T cells and B cells share idiotypic configurations, and that antiidiotypic antibodies of the type described herein may be useful in the development of vaccines against certain viral infections.

Retroviruses and mouse embryos: a rapid model for neurovirulence and transplacental antiviral therapy.

A murine model in which neurotropic retroviral infection can be studied over short periods of time was developed. Microinjection of Cas-Br-E virus into midgestation mouse embryos caused paralysis and death within 25 days after birth, in contrast to virus-infected neonates which develop disease only after 4 months. To evaluate whether antiviral drugs could cross the placental barrier and influence the course of the disease, the drug 3'-azido-3'-deoxythymidine (AZT) was administered to infected embryos through the drinking water of pregnant females. AZT treatment markedly retarded the onset and course of virus-induced central nervous system disease, permitting animals to survive beyond 4 months of age. These results are evidence for effective antiviral treatment during gestation and in the perinatal period and are of potential significance for the management of maternal transmission of the acquired immune deficiency syndrome (AIDS) virus.

Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3.

Biochemical studies of signaling mediated by many cytokine and growth factor receptors have implicated members of the Jak family of tyrosine kinases in these pathways. Specifically, Jak3 has been shown to be associated with the interleukin-2 (IL-2) receptor gamma chain, a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Mice lacking Jak3 showed a severe block in B cell development at the pre-B stage in the bone marrow. In contrast, although the thymuses of these mice were small, T cell maturation progressed relatively normally. In response to mitogenic signals, peripheral T cells in Jak3-deficient mice did not proliferate and secreted small amounts of IL-2. These data demonstrate that Jak3 is critical for the progression of B cell development in the bone marrow and for the functional competence of mature T cells.

Intestinal M cells: a pathway for entry of reovirus into the host.

Thirty minutes after inoculation of reovirus type 1 into the intestinal lumen of the mouse, viruses were found adhering to the surface of intestinal M cells but not other epithelial cells. Within 1 hour, viruses were seen in the M cell cytoplasm and were associated with mononuclear cells in the intercellular space adjacent to the M cell. These findings suggest that M cells are the site where reovirus penetrates the intestinal epithelium.

B7-dependent T-cell costimulation in mice lacking CD28 and CTLA4.

To examine whether B7 costimulation can be mediated by a molecule on T cells that is neither CD28 nor CTLA4, we generated mice lacking both of these receptors. CD28/CTLA4(-/-) mice resemble CD28(-/-) mice in having decreased expression of T-cell activation markers in vivo and decreased T-cell proliferation in vitro, as compared with wild-type mice. Using multiple approaches, we find B7-dependent costimulation in CD28/CTLA4(-/-) mice. The proliferation of CD28/CTLA4(-/-) T cells is inhibited by CTLA4-Ig and by the use of antigen-presenting cells lacking both B7-1 and B7-2. CD28/CTLA4(-/-) T-cell proliferation is increased by exposure to Chinese hamster ovary cells transfected with B7-1 or B7-2. Finally, administration of CTLA4-Ig to CD28/CTLA4(-/-) cardiac allograft recipients significantly prolongs graft survival. These data support the existence of an additional receptor for B7 molecules that is neither CD28 nor CTLA4.

Analysis of lymphocyte costimulation in vivo using transgenic and 'knockout' mice.

The use of transgenic technologies in the functional evaluation of the contributions of costimulatory pathways to T-cell activation in vivo has recently undergone a rapid expansion. During the past two years, mice deficient in costimulatory molecules and their receptors have been generated. These mice have revealed novel and critical in vivo functions of costimulatory pathways and have provided valuable models in which to test therapeutic strategies involving costimulatory pathway blockade. Transgenic mice constitutively expressing costimulatory molecules have provided insights into their role in peripheral tolerance.

Costimulation and autoimmunity.

The past year has seen significant advances in our understanding of the role of the B7-CD28/CTLA-4 pathway in T cell activation and self-tolerance. Recent studies have demonstrated that CTLA-4 is a critical negative regulator of T cell activation and autoreactivity, revealing a previously unsuspected means by which costimulation is involved in the maintenance and breakdown of self-tolerance. Manipulation of this costimulatory pathway in animal models of autoimmunity has shown an important role for this pathway in both the initiation and progression of autoimmune diseases.

The B7-CD28/CTLA-4 costimulatory pathways in autoimmune disease of the central nervous system.

The past year has seen significant advances in our understanding of the role of the B7-CD28/CTLA-4 pathway in regulating the responses of self-reactive T cells, giving impetus to manipulation of this pathway for treating human autoimmune diseases. Recent studies have demonstrated that B7-CD28 costimulation has critical roles in stimulating both the initiation and effector phases of autoimmunity and that CD28 regulates the threshold for activation of self-reactive T cells. Recent work has also revealed critical roles for CTLA-4 in limiting the extent of Th1/Th2 cell differentiation and in downregulating the responses of self-reactive T cells during both the initiation and progression of autoimmune disease.

CTLA-4 and T cell activation.

The past year has seen significant advances in our understanding of the role of cytotoxic T lymphocyte antigen 4 (CTLA-4) in regulating T cell activation and tolerance. Recent studies indicate that CTLA-4 not only counterbalances CD28 signals but also can inhibit T cell responses independently of CD28. Recent work has also revealed a role for CTLA-4 in regulating Th1/Th2 differentiation. Manipulation of CTLA-4 in animal models of autoimmunity has shown that CTLA-4 regulates both the initiation and the progression of autoimmune diseases.

Retroviral transduction of the human c-Ha-ras-1 oncogene into midgestation mouse embryos promotes rapid epithelial hyperplasia.

Infection of mouse embryos at 8 days of gestation with a replication-defective retrovirus carrying the human c-Ha-ras-1 oncogene led to efficient and rapid induction of hyperplastic lesions. Twenty-four percent of viable off-spring developed abnormal growths after infection with purified virus. The lesions contained a single integrated provirus and produced viral RNA and the Ha-ras oncogene product (p21). The latency period between the time of infection and appearance of the lesions suggested that secondary alterations in addition to activated ras were necessary for neoplasms to develop. The earliest and most abundant growths were cutaneous and appeared from 4 to 36 weeks of age, with a median of 4 weeks of age. A number of subcutaneous lesions also developed over the same time span but at a median of 18 weeks of age. The rapid development of cutaneous lesions in response to transduction of the ras oncogene contrasts with other studies in which adult skin required secondary treatment with promoters prior to ras induction of epithelial hyperplasia. These results demonstrate that infection of midgestation mouse embryos allows rapid analysis of oncogene potency in skin.

Requirement of CDC45 for postimplantation mouse development.

CDC45 is required for the initiation of DNA replication in Saccharomyces cerevisiae and functions as a DNA polymerase alpha loading factor in Xenopus, but its role in mammalian DNA replication is unknown. To investigate the genetic and physiological functions of CDC45, we used a gene targeting strategy to generate mice lacking a functional CDC45 gene. Homozygous mutant mice lacking a functional CDC45 gene underwent uterine implantation and induced uterine decidualization but did not develop substantially thereafter. Detailed analysis of CDC45 null embryos cultured in vitro revealed impaired proliferation of the inner cell mass. These findings make CDC45 the only putative replication factor experimentally proven to be essential for mammalian development. The CDC45 gene localizes to human chromosome 22q11.2 in the DiGeorge syndrome critical region (DGCR). Almost 90% of individuals with congenital cardiac and craniofacial defects have a monoallelic deletion in the DGCR that includes CDC45. We report here that heterozygous mutant mice develop into adulthood without any apparent abnormalities, so that it is unlikely that hemizygosity of CDC45 alone is responsible for the cardiac and craniofacial defects in the congenital syndromes.

Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice.

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.

Transplacental antiretroviral therapy with 9-(2-phosphonylmethoxyethyl)adenine is embryotoxic in transgenic mice.

Transgenic Mov-14 mice, which carry the provirus of Moloney murine leukemia virus (Mo-MuLV) in the germ line and begin to produce infectious virus on embryonic day 14, were used to evaluate the ability of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) to cross the placenta and protect embryos from viremia. We have used the Mov-14 model previously to demonstrate the antiviral efficacy and lack of teratogenicity of transplacental therapy with 3'-azido-3'-deoxythymidine (zidovudine, ZDV). PMEA was administered to pregnant females by daily intraperitoneal injection or by osmotic pump. In contrast to ZDV, PMEA was either noneffective in preventing viremia in the offspring or embryotoxic, depending on the dose. The specific toxic effects seen were resorption of pregnancy, low birth weight, and neonatal death. Histopathological analysis of neonatal mice exposed to PMEA showed severe lymphoid depletion of the thymus. We conclude that PMEA therapy is contraindicated for use during pregnancy.

Absence of host B7 expression is sufficient for long-term murine vascularized heart allograft survival.

CD28 antagonists have been shown to promote long-term graft survival and induce donor-specific tolerance. In this study, the role of CD28/B7 costimulation and the relative importance of host versus donor B7 expression in allograft rejection was assessed in a murine abdominal vascularized heterotopic heart transplant model. Wild-type, CD28-deficient, or B7-1/B7-2-deficient C57BL/6 (B6) mice were grafted with allogeneic wild type or B7-1/B7-2-deficient hearts. The results demonstrate allogeneic heart grafts survive long-term in mCTLA4Ig-treated B6 and untreated B7-1/B7-2-deficient B6 recipients but not CD28KO B6 mice. B7-1/B7-2KO B6 recipients treated with anti-CD28 (PV-1) or recombinant human IL-2 rejected the heart transplants indicating that these mice are immunologically competent to reject grafts if costimulatory signals are supplied or bypassed. Finally, there was no difference in rejection between normal animals transplanted with wild-type versus B7-1/B7-2-deficient hearts. These results support a critical role for B7-expressing host antigen presenting cells in the rejection of heart allografts in mice and differences among B7KO and CD28KO animals.