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Late-onset inflammatory toxicities resembling hemophagocytic lymphohistiocytosis (HLH) or macrophage activation syndrome (MAS) occur after chimeric antigen receptor T cell (CAR T cell) infusion and represent a therapeutic challenge. Given the established link between perforin deficiency and primary HLH, we investigated the role of perforin in anti-CD19 CAR T cell efficacy and HLH-like toxicities in a syngeneic murine model. Perforin contributed to both [CD8.sup.+] and [CD4.sup.+] CAR T cell cytotoxicity but was not required for in vitro or in vivo leukemia clearance. Upon CAR-mediated in vitro activation, perforin-deficient CAR T cells produced higher amounts of proinflammatory cytokines compared with WT CAR T cells. Following in vivo clearance of leukemia, perforin-deficient CAR T cells reexpanded, resulting in splenomegaly with disruption of normal splenic architecture and the presence of hemophagocytes, which are findings reminiscent of HLH. Notably, a substantial fraction of patients who received anti-CD22 CAR T cells also experienced biphasic inflammation, with the second phase occurring after the resolution of cytokine release syndrome, resembling clinical manifestations of HLH. Elevated inflammatory cytokines such as IL-1[beta] and IL-18 and concurrent late CAR T cell expansion characterized the HLH-like syndromes occurring in the murine model and in humans. Thus, a murine model of perforin-deficient CAR T cells recapitulated late-onset inflammatory toxicities occurring in human CAR T cell recipients, providing therapeutically relevant mechanistic insights., Introduction Chimeric antigen receptor T cells (CAR T cells) are highly effective against B cell malignancies (1-8) but are frequently associated with cytokine-mediated toxicities or hyperinflammatory conditions (9, 10). The [...]

Posttransplantation cyclophosphamide (PTCy) recently has had a marked impact on human allogeneic hematopoietic cell transplantation (HCT). Yet our understanding of how PTCy prevents graft-versus- host disease (GVHD) largely has been extrapolated from MHC-matched murine skin-allografting models that were highly contextual in their efficacy. Herein, we developed a T cell-replete, MHC-haploidentical, murine HCT model (B6C3F'WB6D2F1) to test the putative underlying mechanisms: alloreactive T cell elimination, alloreactive T cell intrathymic clonal deletion, and suppressor T cell induction. In this model and as confirmed in four others, PTCy did not eliminate alloreactive T cells identified using either specific V[beta]s or the 2C or 4C T cell receptors. Furthermore, the thymus was not necessary for PTCy's efficacy. Rather, PTCy induced alloreactive T cell functional impairment, which was supported by highly active suppressive mechanisms established within one day after PTCy that were sufficient to prevent new donor T cells from causing GVHD. These suppressive mechanisms included the rapid, preferential recovery of [CD4.sup.+][CD25.sup.+][Foxp3.sup.+] regulatory T cells, including those that were alloantigen specific, which served an increasingly critical function over time. Our results prompt a paradigm shift in our mechanistic understanding of PTCy. These results have direct clinical implications for understanding tolerance induction and for rationally developing novel strategies to improve patient outcomes., Introduction Although potentially curative for many otherwise incurable hematologic malignancies, allogeneic hematopoietic cell transplantation (HCT) historically was inaccessible to many patients for lack of HLA-matched donors (1, 2). Nearly all [...]

Iron regulatory proteins (Irps) 1 and 2 posttranscriptionally control the expression of transcripts that contain iron-responsive element (IRE) sequences, including ferritin, ferroportin, transferrin receptor, and hypoxia-inducible factor 2α (HIF2α). We report here that mice with targeted deletion of Irp1 developed pulmonary hypertension and polycythemia that was exacerbated by a low-iron diet. Hematocrits increased to 65% in iron-starved mice, and many polycythemic mice died of abdominal hemorrhages. Irp1 deletion enhanced HIF2α protein expression in kidneys of Irp1(-/-) mice, which led to increased erythropoietin (EPO) expression, polycythemia, and concomitant tissue iron deficiency. Increased HIF2α expression in pulmonary endothelial cells induced high expression of endothelin-1, likely contributing to the pulmonary hypertension of Irp1(-/-) mice. Our results reveal why anemia is an early physiological consequence of iron deficiency, highlight the physiological significance of Irp1 in regulating erythropoiesis and iron distribution, and provide important insights into the molecular pathogenesis of pulmonary hypertension.

Chuvash polycythemia is an inherited disease caused by a homozygous germline [VHL.sup.R200W] mutation, which leads to impaired degradation of HIF2[alpha], elevated levels of serum erythropoietin, and erythrocytosis/polycythemia. This phenotype is recapitulated by a mouse model bearing a homozygous [Vhl.sup.R200W] mutation. We previously showed that iron- regulatory protein 1-knockout (lrp1-knockout) mice developed erythrocytosis/polycythemia through translational derepression of Hif2[alpha], suggesting that IRP1 could be a therapeutic target to treat Chuvash polycythemia. Here, we fed [Vhl.sup.R200W] mice supplemented with Tempol, a small, stable nitroxide molecule and observed that Tempol decreased erythropoietin production, corrected splenomegaly, normalized hematocrit levels, and increased the lifespans of these mice. We attribute the reversal of erythrocytosis/polycythemia to translational repression of Hif2[alpha] expression by Tempol-mediated increases in the IRE-binding activity of Irp1, as reversal of polycythemia was abrogated in [Vhl.sup.R200W] mice in which /rp1 was genetically ablated. Thus, a new approach to the treatment of patients with Chuvash polycythemia may include dietary supplementation of Tempol, which decreased Hif2[alpha] expression and markedly reduced life-threatening erythrocytosis/polycythemia in the [Vhl.sup.R200W] mice., Introduction Chuvash polycythemia is an autosomal recessive disorder that is endemic to the Chuvash population of the Russian Federation (1) and the Italian island of Ischia, but has also been [...]

Post-transplantation cyclophosphamide (PTCy) reduces the incidence and severity of graft-versus-host disease (GVHD), thereby improving the safety and accessibility of allogeneic hematopoietic cell transplantation (HCT). We have shown that PTCy works by inducing functional impairment and suppression of alloreactive T cells. We also have identified that reduced proliferation of alloreactive CD4+ T cells at day +7 and preferential recovery of CD4+CD25+Foxp3+ regulatory T cells (Tregs) at day +21 are potential biomarkers associated with optimal PTCy dosing and timing in our B6C3F1→B6D2F1 MHC-haploidentical murine HCT model. To understand whether the effects of PTCy are unique and also to understand better the biology of GVHD prevention by PTCy, here we tested the relative impact of cyclophosphamide compared with five other optimally dosed chemotherapeutics (methotrexate, bendamustine, paclitaxel, vincristine, and cytarabine) that vary in mechanisms of action and drug resistance. Only cyclophosphamide, methotrexate, and cytarabine were effective in preventing fatal GVHD, but cyclophosphamide was superior in ameliorating both clinical and histopathological GVHD. Flow cytometric analyses of blood and spleens revealed that these three chemotherapeutics were distinct in constraining conventional T-cell numerical recovery and facilitating preferential Treg recovery at day +21. However, cyclophosphamide was unique in consistently reducing proliferation and expression of the activation marker CD25 by alloreactive CD4+Foxp3- conventional T cells at day +7. Furthermore, cyclophosphamide restrained the differentiation of alloreactive CD4+Foxp3- conventional T cells at both days +7 and +21, whereas methotrexate and cytarabine only restrained differentiation at day +7. No chemotherapeutic selectively eliminated alloreactive T cells. These data suggest that constrained alloreactive CD4+Foxp3- conventional T-cell numerical recovery and associated preferential CD4+CD25+Foxp3+ Treg reconstitution at day +21 may be potential biomarkers of effective GVHD prevention. Additionally, these results reveal that PTCy uniquely restrains alloreactive CD4+Foxp3- conventional T-cell proliferation and differentiation, which may explain the superior effects of PTCy in preventing GVHD. Further study is needed to determine whether these findings also hold true in clinical HCT. [ABSTRACT FROM AUTHOR]

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein [Alpha]-subunit ([G.sub.s][Alpha]) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous [G.sub.s] deficiency is embryonically lethal. Heterozygotes with maternal (m-/+) and paternal (+/p-) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m-/+, but not +/p-, mice. [G.sub.s][Alpha] expression in the renal cortex (the site of PTH action) is markedly reduced in m-/+ but not in +/p- mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m-/+ and +/p- mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein [Epsilon] (C/EBP[Epsilon]) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBP[Epsilon] develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3-5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBP[Epsilon] is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.

The CBFB-MYH11 gene was introduced into mouse genome by inserting the 3' part of the human MYH11 cDNA into the end of exon 5 of the mouse Cbfb gene through homologous recombination. This led to the creation of a half genomic (Cbfb), half cDNA (MYH11) fusion gene, which, in turn, resulted in the generation of a Cbf(beta)-SMMHC fusion protein under the control of the Cbfb promoter. Cbf(beta)-SMMHC is shown to dominantly suppress the normal function of Cbf gene products and inhibits the differentiation of hematopoietic stem cells.

The Atm locus was disrupted via gene targeting to create a murine model of the human autosomal recessive disorder ataxia telangiectasia. Growth retardation, neurologic dysfunction, male and female infertility, the absence of mature gametes, defects in T lymphocyte maturation and extreme sensitivity to gamma-irradiation were observed in mice that are homozygous for the disrupted Atm allele. The Atm-disrupted mice were found to recapitulate the ataxia telangiectasia phenotype in humans. This provides a mammalian model for the pathophysiologic study of ataxia telangiectasia.