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Key Points The CD34+ compartment of human cord blood contains a range of HSPC immunophenotypes, among which the Lin−CD34+CD38+CD127+ CLP is rare. There is no correlation between the frequencies of CD34+ cells and immunophenotypic HSC in umbilical cord blood products.

The thymic stroma is composed of epithelial and non-epithelial cells that collectively provide separate microenvironments controlling the homing of blood-born precursors to the tissue, and their subsequent differentiation to functionally mature and correctly selected T cells. While thymic epithelial cells are well characterized for their role in thymopoiesis, a comparably comprehensive analysis of the non-epithelial thymic stroma is lacking. Here we explore at single cell resolution the complex composition and dynamic changes that occur over time in the non-epithelial stromal compartment. We detail across different developmental stages in human and mouse thymus, and in an experimental model of Di George syndrome, the most common form of human thymic hypoplasia, the separate transcriptomes of mouse mesothelium, fibroblasts, neural crest cells, endothelial and vascular mural cells. The detected gene expression signatures identify novel stromal subtypes and relate their individual molecular profiles to separate differentiation trajectories and functions. Specifically, we demonstrate an abundance and unprecedented heterogeneity of diverse fibroblast subtypes that emerge at discrete developmental stages and vary in their expression of key regulatory signalling circuits and components of the extracellular matrix. Taken together, these findings highlight the dynamic complexity of the non-epithelial thymus stroma and link the cells’ specific gene expression profiles to separate instructive roles essential for normal thymus organogenesis and tissue maintenance.TeaserSingle cell profiling of thymic stroma identifies a dynamic contribution from neural crest cells to the thymic mesenchyme.

During their intrathymic development, nascent T cells are empowered to protect against pathogens and to be operative for a life-long acceptance of self. While autoreactive effector T (Teff) cell progenitors are eliminated by clonal deletion, the intrathymic mechanisms by which thymic regulatory T cell (tTreg) progenitors maintain specificity for self-antigens but escape deletion to exert their regulatory functions are less well understood. Both tTreg and Teff development and selection result from finely coordinated interactions between their clonotypic T cell receptors (TCR) and peptide/MHC complexes expressed by antigen-presenting cells, such as thymic epithelial cells and thymic dendritic cells. tTreg function is dependent on expression of the FOXP3 transcription factor, and induction of FOXP3 gene expression by tTreg occurs during their thymic development, particularly within the thymic medulla. While initial expression of FOXP3 is downstream of TCR activation, constitutive expression is fixed by interactions with various transcription factors that are regulated by other extracellular signals like TCR and cytokines, leading to epigenetic modification of the FOXP3 gene. Most of the understanding of the molecular events underlying tTreg generation is based on studies of murine models, whereas gaining similar insight in the human system has been very challenging. In this review, we will elucidate how inborn errors of immunity illuminate the critical non-redundant roles of certain molecules during tTreg development, shedding light on how their abnormal development and function cause well-defined diseases that manifest with autoimmunity alone or are associated with states of immune deficiency and autoinflammation.

There is a critical need for more effective therapy for acute myelogenous leukemia (AML). Although many patients achieve remission, most relapse with poor outcomes. Even after allogeneic Stem Cell Transplantation (SCT), 30-50% of patients relapse due to the persistence of residual disease. To address the poor immunogenicity of AML cells and the diminished immune responsiveness of patients, our candidate autologous AML vaccine is lentivirally engineered, in each patient's leukemic cells, to express CD80, IL-15, and IL-15 Receptor alpha (IL-15Rα). In prior studies in a syngeneic 32Dp210 murine AML model, CD80-mediated co-stimulation of T-cells combined with immune activation by the IL-15/IL-15Rα heterodimer showed unprecedented synergy in induction of anti-leukemic cytolytic activity (Shi, Y. et al, 2018). This was observed in both ex vivo co-culture and in vivo where vaccinated leukemic mice had >80% cure rates. No local skin, organ, or systemic toxicity was observed, nor was there evidence of systemic cytokine release of IL-6 or TNFα after SC or IV injection of up to 10 8 transduced irradiated AML cells. We confirmed the feasibility of producing patient-derived AML vaccines by transduction of 16 independent AML samples with a tri-cistronic lentiviral vector (TLV) that contains human CD80, IL-15 and IL-15Rα. Transduction levels were 11-71% of cells (median 38.6%). To define the minimum transduction level required for PBMC activation and to assess synergy of co-expressed human CD80, IL-15, and IL-15Rα, allogenic U937 leukemia cells were initially used as stimulators. Transduced U937 (U937-TLV) had high-level surface expression of CD80 and IL-15, secreted IL-15 (7 ng/ml/24 hours from 2 x 10 6 cells/ml) and activated CD3+ T-cells from an AML patient (Fig.1). Mixtures of irradiated U937-TLV with non-transduced U937 were created at fixed ratios (100%, 80%, 40%, 20%, 10%, 5%, 0%) for overnight co-culture with patient PBMC. At 24 hours, the T-cells were analyzed for activation by measurement of the frequency of CD69+ CD4 or CD8 T cells (Fig. 1), normalized to expression of unstimulated PBMC (0%) and the percentage of maximal CD69 expression with 100% U937-TLV (100%). Background levels of activation due to the presence of allogenic U937 were negligible. Co-culture with as little as 10% transduced U937-TLV reliably activated patient T-cells. To assess the synergy of CD80, IL-15 and IL-15Rα expression, parallel experiments were performed with PBMC co-cultured in IL-15 containing supernatants from U937-TLV cells (Fig. 1). The frequencies of activated T-cells were significantly higher after co-culture with U937/U937-TLV cells than after stimulation with IL-15-containing supernatants from similar ratios of U937/U937-TLV, confirming the synergy of CD80 and IL-15/IL-15Rα in the transduced cells. To better, model the clinical setting, we assessed induction of immune responses of patient T cells to autologous transduced AML. PBMC were stimulated with transduced or non-transduced autologous AML cells vs stimulation with allogeneic U937-TLV, or with anti-CD3/CD28 beads to define maximal stimulation. Negative controls included culture of PBMC alone. All patients had T-cell activation, as measured by induction of CD69, HLA-DR and CD95 (Fas) expression, although there was heterogeneity in the nature of responses, e.g., disparate induction of the markers in individual patients (Fig. 2A and B). Induction of cytotoxic effector pathways was confirmed by detection of CD178 (FasL) and perforin expression (Figure 2C and D). Overall, all patients' PBMC had the capacity to mount T-cell responses of similar magnitude to both allogeneic U937-TLV and autologous vaccine. These studies establish that autologous AML cells transduced with CD80, IL-15 and IL-15Rα can elicit specific anti-leukemic T-cell responses, even in the face of prior lymphodepleting chemotherapy. A strength of this autologous vaccine strategy is that it is agnostic to which AML proteins are immunogenic for each patient. Although uniformly detected, there was heterogeneity in the induction of activation markers and effector pathways, which may reflect host and/or disease-related differences. The mechanisms underlying differences in the nature of responses in patients will be important to understand and will provide the basis for future immune correlative studies for our Phase 1 vaccine trial in transplant ineligible AML patients. Figure 1 Figure 1. Disclosures Kohn: Lyrik Therapeutics: Membership on an entity's Board of Directors or advisory committees; MyoGene Bio: Membership on an entity's Board of Directors or advisory committees; ImmunoVec: Membership on an entity's Board of Directors or advisory committees; Pluto Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; UC Regents: Patents & Royalties; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity's Board of Directors or advisory committees.

Solid organ transplantation (SOT) treats over 1500 children/year in the US. While short-term outcomes have improved, long-term outcomes still remain poor. With the rare exception of monozygotic twins as donors, recipients require lifelong immunosuppression with its accompanying toxicities, the risk of nonadherence, and chronic rejection. Further, up to half of recipients require a second transplant. We have pioneered translational approaches that abrogate rejection and the need for post-transplant immunosuppression. Previous attempts in adult patients utilizing allogeneic hematopoietic stem cell transplantation (HSCT) to eliminate the need for post-SOT immunosuppression in non-histocompatible kidney transplant (KT) recipients have failed(Lowsky R, BMT 2019). The establishment of mixed lymphoid chimerism has permitted a reduction in the number of immunosuppressive drugs needed, but not their discontinuation, whereas complete donor engraftment after HSCT has resulted in fatal graft-versus-host disease in some patients (Leventhal JR, Eur J Pediatr 2000). The use of HSCT to eliminate the need for immunosuppression following pediatric KT has not been evaluated. We have pioneered sequential haploidentical HSCT followed by KT in three patients with Schimke immuno-osseous dysplasia (SIOD) to abrogate kidney rejection and the need for post-transplant immunosuppression. SIOD is a monogenic disorder with progressive nephropathy correctable by KT and a T-cell immunodeficiency curable by HSCT. All three patients received αβT-cell/CD19 B-cell depleted HSCT (αβhaplo-HSCT) from a parent prior to a KT from the same parental donor. The pre-HSCT reduced intensity preparative regimen consisted of fludarabine (a starting dose of 1 mg/kg x 4 days, which was adjusted based on AUC), total body irradiation (TBI) 200 cGy, cyclophosphamide 1200 mg/m 2, anti-thymocyte globulin (ATG) thymoglobulin® 7.5 mg/kg, and rituximab 200 mg/m 2. Immunosuppressive drugs were not prophylactically administered after HSCT. After confirmation of full donor lymphoid chimerism post-HSCT, the patients received a living donor KT from their parental HSCT donor. Post-KT immunosuppression included intraoperative methylprednisolone and post-operative low-dose oral prednisone (0.5 mg/kg/day with taper) and tacrolimus (target serum level of 3-5 ng/ml) to reduce potential reperfusion inflammation. All immunosuppressive drugs were tapered off by Day +30 post-KT. To demonstrate functional tolerance after KT, Mixed Lymphocyte Cultures (MLC) were performed between peripheral blood mononuclear cells (PBMC) and patient/parent-derived irradiated EBV transformed lymphoblastoid cells lines (EBV-LCL) as stimulators (Figure 1). PBMC were isolated from SIOD patients (n=3) by Ficoll Hypaque separation and were labelled with Invitrogen™ CellTrace Violet Proliferation Kit (CTV). EBV-LCL were established from SIOD patients, parents and healthy donors. The assay was performed in round bottom microtiter plates with 100,000 labelled PBMC as responder cells (R) and 50,000 irradiated (30gy) EBV-LCL in RPMI medium with 10% fetal calf serum. Cells were harvested on Day 6, and T-cell proliferation determined by CTV expression of CD3+ T cells using a Novocyte Penteon flow cytometer (Agilent). Donor-derived T cells isolated from the peripheral blood of all three recipients > 1-year post-HSCT/KT, did not respond to the donor cells, while they did proliferate to the non-donor parental and control cells (Fig. 1). These results demonstrate that post-HSCT/KT the circulating donor-derived T cells are functionally tolerant to the transplanted kidney and, therefore, are unable to mediate graft rejection even in the absence of immune suppression. We have achieved two goals: first, ablating the recipient immune system before HSCT we safely established a new donor-derived immune system - that could also, if required, cure a patient's underlying disease- and, second, functional tolerance to the transplanted kidney. At 12 to 24 months after KT, all patients have full donor lymphoid and myeloid chimerism, normal renal function without immunosuppression, MLC demonstrated tolerance towards donor cells, and normal proliferative responses to non-donor parental cells. These findings suggest that the combination of αβhaplo-HSCT and SOT could be extended to other solid transplantation like liver and small intestine. Figure 1 Figure 1. Disclosures Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees. Shah: OrchardTherapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Dr. Shah currently serves on the medical advisory board for Orchard Therapeutics . Parkman: Jasper Biotech: Consultancy.

Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death(1). Despite rapid advances over recent years, many of the molecular and cellular processes which underlie progressive loss of healthy physiology are poorly understood(2). To gain a better insight into these processes we have created a single cell transcriptomic atlas across the life span of Mus musculus which includes data from 23 tissues and organs. We discovered cell-specific changes occurring across multiple cell types and organs, as well as age related changes in the cellular composition of different organs. Using single-cell transcriptomic data we were able to assess cell type specific manifestations of different hallmarks of aging, such as senescence(3), genomic instability(4) and changes in the organism’s immune system(2). This Tabula Muris Senis provides a wealth of new molecular information about how the most significant hallmarks of aging are reflected in a broad range of tissues and cell types.

Aldehyde dehydrogenase 2 deficiency (ALDH2*2) causes facial flushing in response to alcohol consumption in approximately 560 million East Asians. Recent meta-analysis demonstrated the potential link between ALDH2*2 mutation and Alzheimer’s Disease (AD). Other studies have linked chronic alcohol consumption as a risk factor for AD. In the present study, we show that fibroblasts of an AD patient that also has an ALDH2*2 mutation or overexpression of ALDH2*2 in fibroblasts derived from AD patients harboring ApoE ε4 allele exhibited increased aldehydic load, oxidative stress, and increased mitochondrial dysfunction relative to healthy subjects and exposure to ethanol exacerbated these dysfunctions. In an in vivo model, daily exposure of WT mice to ethanol for 11 weeks resulted in mitochondrial dysfunction, oxidative stress and increased aldehyde levels in their brains and these pathologies were greater in ALDH2*2/*2 (homozygous) mice. Following chronic ethanol exposure, the levels of the AD-associated protein, amyloid-β, and neuroinflammation were higher in the brains of the ALDH2*2/*2 mice relative to WT. Cultured primary cortical neurons of ALDH2*2/*2 mice showed increased sensitivity to ethanol and there was a greater activation of their primary astrocytes relative to the responses of neurons or astrocytes from the WT mice. Importantly, an activator of ALDH2 and ALDH2*2, Alda-1, blunted the ethanol-induced increases in Aβ, and the neuroinflammation in vitro and in vivo. These data indicate that impairment in the metabolism of aldehydes, and specifically ethanol-derived acetaldehyde, is a contributor to AD associated pathology and highlights the likely risk of alcohol consumption in the general population and especially in East Asians that carry ALDH2*2 mutation.

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl., Gene correction in hematopoietic stem cells could be a powerful way to treat monogenic diseases of the blood and immune system. Here the authors develop a strategy using CRISPR-Cas9 and an aAdeno-Associated vVirus(AAV)-delivered IL2RG cDNA to correct X-linked sSevere Ccombined iImmunodeficiency (SCID-X1) with a high success rate.

Purpose To understand whether patients who have undergone Fontan palliation may have a higher susceptibility to post-HT infections given thymectomy in infancy and known depletions of T-cell subsets. Methods This was a single-center, retrospective cohort analysis of pediatric patients undergoing HT for dilated cardiomyopathy (DCM) or Fontan failure, who underwent induction with anti-thymoglobulin (ATG). The primary endpoint was infection in the first 180 days post-HT, defined as (1) positive blood/urine/respiratory culture; (2) positive viral PCR (excluding CMV and EBV); (3) skin or wound infection; and/or (4) culture-negative infection if a full antibiotic course (>5 days) was completed. Secondary endpoints included (1) absolute lymphocyte (ALC) and CD3 counts after 3 and 5 doses of anti-thymocyte gammaglobulin (ATG); (2) the incidence of post-transplant lymphoproliferative disorder (PTLD); and (3) rejection (≥ Grade 2R ACR or pAMR2) in the first 180 days post-HT. Results From 2014 to 2019, 59 pts (26 Fontan, 33 DCM) underwent HT at a median age of 14.7 (IQR 10.6, 19.5) and 11.7 (IQR 1.4, 13.6) years, respectively. The median total ATG received was 7.4 (IQR 4.9, 7.7) vs 7.5 (IQR 7.3, 7.6) mg/kg (p=NS). The median CD3 [8 (IQR 5, 14) vs 16 (IQR 10, 39); p=0.01] after 3 doses of ATG and ALC [172 (IQR 98, 400) vs 427 (IQR 205, 824); p=0.014] after 5 doses of ATG was lower in Fontan vs DCM patients (A,B). Twenty-three patients (39%) developed ≥1 infection within 180 days post-HT, with a higher rate of infection in Fontan patients (54% vs 27%, p=0.03; C, D). Adjusted for pre-transplant ALC, the risk for infection within 30 days post-HT for Fontan patients was OR 7.62, 95% CI 1.13-51.48, p=0.037. There was no difference in the incidence of PTLD (12% vs 0%; p=0.08) or rejection (12% vs 21%; p=0.49) between Fontan vs DCM patients. Conclusion Compared to DCM patients, Fontan patients have lower CD3 and ALC after induction, as well as a higher risk of infection. Modifications to induction therapy for Fontan patients may be considered.

Invasive fungal disease (IFD) remains a major cause of morbidity and mortality in pediatric patients after allogeneic hematopoietic stem cell transplant (HSCT). We analyzed the outcome of 152 consecutive pediatric patients who underwent allogeneic HSCT from 2005 to 2012: 126 of these without a history of IFD and 26 with IFD before HSCT. Antifungal prophylaxis agent was determined by the primary transplant attending. The rate of IFD after HSCT among patients with or without prior IFD was similar (7.7% with and 7.1% without a history of fungal disease before transplant). Mortality in these 2 populations did not differ (35% vs. 28%, P=0.48, χ). Patients deemed at higher risk for IFD were generally placed on voriconazole prophylaxis; however, this did not affect rates of posttransplant IFD. All-cause mortality in patients with posttransplant IFD was significantly higher than those without posttransplant IFD (67% vs. 21%, P

Maintenance of the hematopoietic stem cell (HSC) compartment depends on the ability to metabolize exogenously and endogenously generated toxins, and to repair cellular damage caused by such toxins. Reactive aldehydes have been demonstrated to cause specific genotoxic injury, namely DNA interstrand cross-links. Aldehyde dehydrogenase 2 (ALDH2) is a member of a 19 isoenzyme ALDH family with different substrate specificities, subcellular localization, and patterns of expression. ALDH2 is localized in mitochondria and is essential for the metabolism of acetaldehyde, thereby placing it directly downstream of ethanol metabolism. Deficiency in ALDH2 expression and function are caused by a single nucleotide substitution and resulting amino acid change, called ALDH2*2. This genetic polymorphism affects 35–45% of East Asians (about ~560 million people), and causes the well-known Asian flushing syndrome, which results in disulfiram-like reactions after ethanol consumption. Recently, the ALDH2*2 genotype has been found to be associated with marrow failure, with both an increased risk of sporadic aplastic anemia and more rapid progression of Fanconi Anemia. This review discusses the unexpected interrelationship between aldehydes, ALDH2 and hematopoietic stem cell biology, and in particular its relationship to Fanconi anemia.

Successful hematopoietic stem cell transplantation (HSCT) requires vacating recipient hematopoietic stem cell (HSC) niches in the bone marrow to permit donor HSC engraftment that can provide life-long hematopoietic and immune function. Currently, HSCT in SCID relies on DNA damaging chemotherapy to eliminate recipient HSC and achieve niche clearance. We have pursued a non-toxic approach to target and deplete HSC using a humanized monoclonal antibody, JSP191, that binds human CD117 (c-Kit). We previously showed the safety and successful HSC engraftment in a Phase 1 trial of the first 6 patients with severe combined immunodeficiency (SCID), who underwent a second transplant because of HSC engraftment failure and poor immunity after their first transplantation. In these re-transplant patients even a low level of stringently measured myeloid chimerism resulted in significant and sustained generation of naive T cells and clinical improvement. Based on these results, the study of JSP191 (NCT#02963064)has opened a cohort of newly diagnosed infants with SCID. Here we report data from the first patient in this cohort, a SCIDX1 patient who received a primary HSCT with haploidentical CD34+ cells after conditioning with JSP 191. The patient had a c.270-15A>G variant in the IL2RG gene, which is predicted to cause a null phenotype. Besides a T- B+ NK- phenotype typical of SCIDX1 including dysfunctional B cells, the patient had anemia and intermittent neutropenia and thrombocytopenia. Despite evidence of maternal T cell engraftment, the patient had no clinical graft-versus-host disease (GVHD). The patient was initially enrolled in a trial of lentiviral gene therapy, but harvested bone marrow cells died in vitro during transduction and culture. The patient also mobilized poorly with G-CSF/Plerixafor. Further investigation revealed heterozygosity for loss-of-function mutations in two genes involved in DNA repair, BRCA1 and RAD51; Diepoxybutane (DEB) breakage study showed greater than normal pathologic chromosomal breaks, but less than that seen in Fanconi anemia. Because of concern for possible hypersensitivity to alkylating agent-based conditioning, the patient was referred for transplant with JSP191 conditioning. The patient received a CD34+ peripheral blood HSCT from his father after conditioning with 0.3 mg/kg of JSP 191 antibody intravenously over an hour on Day -8 and rATG (Thymoglobulin) on Day -5, -4, -3 and -2 (3.5 mg/kg total) to prevent rejection by the maternal T cells. The cryopreserved donor CD34+ cells were administered after sufficient clearance of the JSP191 serum level. The antibody infusion was well tolerated without toxicity, and the post-transplant course was uneventful without acute toxicities or GVHD. As a surrogate marker for HSC engraftment, CD15+ myeloid cells from peripheral blood were stringently sorted by flow cytometry and donor levels were quantified by short-tandem repeat (STR) analysis. Progressive levels of myeloid engraftment were observed beginning at Week 4. The level of donor chimerism at 12 weeks was 8% in the sorted CD15+ blood cells, and a marrow aspirate showed 25% donor CD34+ cells. By 3 months pre-existing abnormal CD19-CD20+ host B lymphocytes were significantly reduced, and CD19+ donor-derived B lymphocytes were emerging. At 2 months, CD4+ recent thymic emigrant and naïve T lymphocytes were observed, and by 3 months, overall T and NK lymphocyte numbers were 390/uL and 117/uL, respectively. Normal blastogenic responses to the T cell mitogen PHA were observed at 3 months. These first-in-class results provide proof of concept of the safety and efficacy of the use of JSP191 antibody to clear host marrow niche space to enable sufficient donor HSC engraftment and immune reconstitution as primary therapy of SCID. Non-genotoxic conditioning with JSP191 may replace conventional conditioning for newly diagnosed infants with SCID, thereby avoiding toxicities of chemotherapy. Disclosures Kohn: Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. De Oliveira:Orchard Therapeutics: Research Funding; bluebird bio, Inc.: Research Funding. Czechowicz:Rocket Pharmaceuticals, Inc.: Research Funding. Brown:Merck: Membership on an entity's Board of Directors or advisory committees; Ansun: Membership on an entity's Board of Directors or advisory committees; Cidara: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; Cellerant Therapeutics: Membership on an entity's Board of Directors or advisory committees. Shizuru:Jasper Therapeutics, Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees.

Allogeneic hematopoietic stem cell transplantation (HSCT) from an HLA-matched donor, either related or unrelated, has been extensively used to treat patients with genetic disorders. However, only 25% of patients eligible to receive allogeneic HSCT have an HLA-identical sibling, and an HLA-matched unrelated donor can be identified for less than 60% of other patients. Mismatched HSCT has been associated with a high risk of graft loss, graft-versus-host disease (GvHD), and transplant-related mortality. Graft manipulation strategies have been used over the last decade to reduce these risks. HLA-haploidentical HSCT after ab T-cell/CD19 B-cell depletion (abhaplo-HSCT) has been shown to be effective in curing up to 90% of children with a variety of non-malignant disorders [Bertaina A, Blood 2014]. However, a scarcity of data are available for patients with rare and complex genetic diseases, including Tregopathies, an emerging new class of primary immunodeficiencies. Here we report preliminary data on the single center experience developed at Lucile Packard Children's Hospital, Stanford, CA, USA on the use of abhaplo-HSCT for rare and complex monogenic diseases (Table 1). All the cell products were manufactured at the Laboratory for Cell and Gene Medicine (LCGM) at Stanford (Table 2). Between 05/2018 and 08/2019, 5 patients with complex monogenic disorders were referred to our Center for HSCT including: a 23 year-old with Fanconi Anemia, two with Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) and two with Schimke Immuno-Osseous Dysplasia (SIOD). Remarkably, all these patients were previously considered not eligible for a HSCT, and one of the IPEX patients was in palliative care. At a median follow-up of 240 days (range 39-520), all patients are alive and disease-free. One patient (SIOD) developed Grade I/stage II skin only graft-versus-host disease (GvHD), while none of the evaluable patients had developed chronic GvHD. Five months after abhaplo-HSCT and with 100% of the circulating T cells being donor derived, the first SIOD patient received a living kidney transplant from the stem cell donor (mother) using minimal immunosuppression with tacrolimus and steroid. Four weeks after the kidney transplant all immunosuppression was stopped, and the patient remains off immunosuppression 12 weeks post-kidney transplantation. Our preliminary data on the use of abhaplo-HSCT in patients with rare and complex monogenic diseases provide a strong rationale for expanding this therapeutic approach to diseases not previously considered as an indication for allogeneic HSCT, to patients in very poor clinical condition and to patients requiring both HSCT and solid organ transplant.

Patients who fail to adequately reconstitute the donor-derived immune system after allogeneic hematopoietic stem cell transplantation (HSCT) are at increased risk for infections and leukemia relapse. In the past, pan T-cell depleted haploidentical grafts were associated with delayed immune reconstitution (IR). Recently, the majority of patients receiving αβ T-cell/B-cell depleted haploidentical HSCT (αβhaplo-HSCT) reach a threshold of 200 CD3+ T cells/mcl by 100 days after HSCT (Bertaina A et al. Blood 2014 Jul 31;124(5):822-6). However, a proportion of patients experience a slower IR with consequent higher morbidity and mortality. Early prediction of delayed IR may permit prompt clinical intervention such as infusion of donor lymphocytes or of virus-specific cytotoxic T cells. Flow cytometry, the most widely applied approach for IR analysis, suffers from intrinsic limitations, such as high lymphocyte number requirement, degradation of samples, and insufficient standardization due to technical and operator variability. To overcome these limitations, we used a DNA methylation-based quantitative PCR that detects the epigenetic signature of different peripheral blood immune cell subsets (epigenetic quantification). This technique provides relative and absolute immune cell counts applicable to fresh, frozen, or paper-spotted dried blood. Epigenetic measurements are based on a per cell DNA copy number and provide a clear positive or negative signal rather than arbitrarily defined thresholds for "positivity" as in flow cytometry. We hypothesize that epigenetic quantification at day 15 after αβhaplo-HSCT could predict flow-based IR at day 100. Patients were consented at Lucile Packard Children's Hospital (Stanford, CA). Blood was collected between days 10-17 for epigenetic quantification and days 82-124 for flow cytometry. Bisulfite treated DNA underwent qPCR quantification of cell type-specific DNA regions of de-methylation (Baron U et al. Sci Transl Med 2018 Aug 1;10(452):eaan3508). Flow cytometry was performed using directly conjugated antibodies. Absolute cell counts were determined, plotted, and then analyzed using a linear regression model. In the first 5 αβhaplo-HSCT patients evaluated, we found a direct correlation between the epigenetic quantification at day 15 and flow cytometry at day 100 for CD4+ T cells (P=0.01), while the early epigenetic quantification of CD3+ and CD8+ T cells was not informative (Fig. 1). Preliminary data suggest that the use of epigenetic quantification early after αβhaplo-HSCT can predict the IR of CD4+ T cells at day 100 in αβhaplo-HSCT recipients. Ongoing analysis on a larger cohort of both αβhaplo-HSCT and unmanipulated HSCT recipients, will confirm if epigenetic quantification results obtained early post-HSCT can be used as a clinical biomarker of delayed IR and guide physicians in the use of post-HSCT adoptive immunotherapy.

Age-related thymic involution is characterized by a decrease in thymic epithelial cell (TEC) number and function parallel to a disruption in their spatial organization, resulting in defective thymocyte development and proliferation as well as peripheral T cell dysfunction. Deficiency of Klotho, an antiaging gene and modifier of fibroblast growth factor signaling, causes premature aging. To investigate the role of Klotho in accelerated age-dependent thymic involution, we conducted a comprehensive analysis of thymopoiesis and peripheral T cell homeostasis using Klotho-deficient (Kl/Kl) mice. At 8 wk of age, Kl/Kl mice displayed a severe reduction in the number of thymocytes (10–100-fold reduction), especially CD4 and CD8 double-positive cells, and a reduction of both cortical and medullary TECs. To address a cell-autonomous role for Klotho in TEC biology, we implanted neonatal thymi from Klotho-deficient and -sufficient mice into athymic hosts. Kl/Kl thymus grafts supported thymopoiesis equivalently to Klotho-sufficient thymus transplants, indicating that Klotho is not intrinsically essential for TEC support of thymopoiesis. Moreover, lethally irradiated hosts given Kl/Kl or wild-type bone marrow had normal thymocyte development and comparably reconstituted T cells, indicating that Klotho is not inherently essential for peripheral T cell reconstitution. Because Kl/Kl mice have higher levels of serum phosphorus, calcium, and vitamin D, we evaluated thymus function in Kl/Kl mice fed with a vitamin D–deprived diet. We observed that a vitamin D–deprived diet abrogated thymic involution and T cell lymphopenia in 8-wk-old Kl/Kl mice. Taken together, our data suggest that Klotho deficiency causes thymic involution via systemic effects that include high active vitamin D levels.

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing, allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. The most widely used homologs of the Cas9 protein are derived from the bacteriaStaphylococcus aureus(S. aureus) andStreptococcus pyogenes(S. pyogenes). Based on the fact that these two bacterial species cause infections in the human population at high frequencies, we looked for the presence of pre-existing adaptive immune responses to their respective Cas9 homologs, SaCas9 (S. aureushomolog of Cas9) and SpCas9 (S. pyogeneshomolog of Cas9). To determine the presence of anti-Cas9 antibodies, we probed for the two homologs using human serum and were able to detect antibodies against both, with 79% of donors staining against SaCas9 and 65% of donors staining against SpCas9. Upon investigating the presence of antigen-specific T-cells against the two homologs in human peripheral blood, we found anti-SaCas9 T-cells in 46% of donors. Upon isolating, expanding, and conducting antigen re-stimulation experiments on several of these donors’ anti-SaCas9 T-cells, we observed an SaCas9-specific response confirming that these T-cells were antigen-specific. We were unable to detect antigen-specific T-cells against SpCas9, although the sensitivity of the assay precludes us from concluding that such T-cells do not exist. Together, this data demonstrates that there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

Epstein Barr virus (EBV) is the etiological agent of acute infectious mononucleosis (IM). Since acute IM is a self-resolving disease with most patients regaining health in one to three weeks there have been few studies examining molecular signatures in early acute stages of the disease. microRNAs have been shown, however, to influence immune cell function and consequently the generation of antibody responses in IM. In this study, we performed a comprehensive analysis of differentially expressed microRNAs in early stage uncomplicated acute IM. microRNAs were profiled from patient peripheral blood obtained at the time of IM diagnosis and at subsequent time points, and pathway analysis performed to identify important immune and cell signaling pathways. We identified 215 differentially regulated microRNAs at the most acute stage of infection when the patients initially sought medical help. The number of differentially expressed microRNAs decreased to 148 and 68 at one and two months post-primary infection, with no significantly changed microRNAs identified at seven months post-infection. Interferon signaling, T and B cell signaling and antigen presentation were the top pathways influenced by the microRNAs associated with IM. Thus, a dynamic and regulated expression profile of miRNA accompanies the early acute immune response, and resolution of infection, in IM.

Allogeneic hematopoietic stem cell transplantation for patients with a hemoglobinopathy can be curative but is limited by donor availability. Although positive results are frequently observed in those with an HLA-matched sibling donor, use of unrelated donors has been complicated by poor engraftment, excessive regimen-related toxicity, and graft-versus-host disease (GVHD). As a potential strategy to address these obstacles, a pilot study was designed that incorporated both a reduced-intensity conditioning and mesenchymal stromal cells (MSCs). Six patients were enrolled, including 4 with high-risk sickle cell disease (SCD) and 2 with transfusion-dependent thalassemia major. Conditioning consisted of fludarabine (150 mg/m2), melphalan (140 mg/m2), and alemtuzumab (60 mg for patients weighing > 30 kg and .9 mg/kg for patients weighing

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34

HSC loss in FA is due to failure to resolve DNA inter-strand crosslinks (ICL), which can be induced by reactive aldehydes, radiation, or other clastogenic agents. Aldehyde exposure may occur through environmental sources, e.g. ingestion, absorption, and inhalation, or endogenously as a byproduct of cellular metabolism. The ALDH2*2 genotype, a dominant-negative point mutation in the aldehyde dehydrogenase 2 (ALDH2) gene, causes the "Asian flushing syndrome" when ethanol (EtOH) is ingested, due to decreased metabolism of small aldehydes, particularly acetaldehyde. ALDH2*2 is a disease modifier in FA, causing more rapid bone marrow failure and earlier leukemia onset in doubly affected children. Similarly, mice experimentally doubly knocked out for FANCD2 and ALDH2 demonstrate increased HSC loss, which is accelerated by EtOH exposure. To reduce aldehyde exposure, we developed a small molecule ALDH activator, Alda-1, which increases the enzymatic activity of both wild type (WT) and mutant ALDH2. We hypothesized that DNA damage and HSC loss in FA would be prevented by reduction of the aldehyde load. To test the effects of Alda-1 mediated ALDH2 activation, we generated a novel murine FA model with FANCD2 KO and knock-in of the ALDH2*2 mutation into the murine locus. The FANCD2-/- ALDH2*1/*2 genetic model and parental controls were then tested after exogenous aldehydic challenge and/or therapeutic intervention with Alda-1. Increased aldehydic load was experimentally induced by EtOH administration 10 mg/kg/day IP, while Alda-1 10 ug/kg/day was continuously administered via osmotic pump. For each of these conditions, marrow was analyzed for HSC and progenitor cell (HSPC) number, HSC gene expression, and function. The importance of the altered aldehyde metabolism due to ALDH2*2 genotype was demonstrated by progressive loss of HSPC in ALDH2*2/*2 and FANCD2-/- ALDH2*1/*2 mice, e.g., 5-fold and 2-fold decline in long-term HSC (LT-HSC), respectively, by 36 weeks. Experimental EtOH challenge to increase the aldehyde load precipitously decreased HSC numbers of all genotypes. After 5 weeks of EtOH challenge, LT-HSC decreased 35-fold in FANCD2-/- ALDH2*1/*2, 12.5-fold in FANCD2-/-ALDH2*1/*1, and 10.5-fold in WT mice. Long-term Alda-1 treatment to decrease aldehydic load rescued FA mice from HSC loss. After 7 months of Alda-1 treatment, LT-HSC numbers in FANCD2-/-ALDH2*1/*2 and FANCD2-/-ALDH2*1/*1 were approximately 10-fold higher than untreated controls. There were no clinically observed adverse effects. Aldehyde exposure and Alda-1 treatment altered gene expression of HSC. Principal component analysis and clustering of HSC gene expression showed that the first principal component representing 40% of the variation in gene expression could be attributed to increased aldehydic load, either genetically (ALDH2*2 genotype) or environmentally (EtOH administration) induced, while Alda-1 treatment obviated these effects. HSC from Alda-1 treated mice clustered with those from control WT mice. To test whether Alda-1 improved HSC function as well as phenotypic number, engraftment potential was assessed with competitive repopulation assays of sorted HSC from congenic untreated donors vs short-term (3 weeks) Alda-1 treated donors. HSC from Alda-1 treated mice had 2-4 fold greater granulocyte repopulating capacity than those from the untreated donors. Our results demonstrate that Alda-1 treatment rescues HSC from aldehyde induced loss, whether from genetic variation (FANCD2- and/or ALDH2*2) or experimental challenge (EtOH administration). Furthermore, Alda-1 treatment prevents the abnormal HSC gene expression induced by increased aldehydic load. HSC function is improved by Alda-1 with greater repopulating capacity observed even after short-term treatment. These preclinical experiments provide compelling proof-of-concept that sustained activation of ALDH2 can prevent HSC loss in FA. Potential applications include long-term administration to prevent the development of marrow failure or leukemia, and HSC expansion to increase the number of cells available for gene therapy with autologous HSC. Our results suggest that a clinical trial of ALDH2 activation in FA patients to prevent marrow failure is warranted. Disclosures Van Wassenhove: U.S. Patent Office: Patents & Royalties: patent pending - submitted for ALDH2 activators to expand hematopoietic stem cells. Chen:Foresee Pharmaceuticals: Patents & Royalties: patents licensed to Foresee related to compounds that activate aldehyde dehydrogenase 2 (ALDH2) and correct the dysfunction in ALDH2*2; U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Mochly-Rosen:Foresee Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: patents licensed to Foresee related to compounds that activate aldehyde dehydrogenase 2 (ALDH2) and correct the dysfunction in ALDH2*2; U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Weinberg:U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells.

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit transplanted HSC to engraft. Currently, DNA damaging radiation or chemotherapy are used to eliminate recipient HSC and achieve niche clearance. We have pursued a non-genotoxic approach to target and deplete HSC using a humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit), a receptor tyrosine kinase expressed on the surface of HSC and progenitor cells (HSPC). We have shown that AMG 191 suppresses human hematopoiesis in vitro, depletes human HSC in mice xenografted with human cells, and safely depletes HSC of non-human primates. We have initiated a Phase I dose escalation trial to test AMG 191 as the sole conditioning agent to achieve donor CD34-enriched HSPC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID) (ClinicalTrials.gov: NCT02963064). SCID is a severe genetic immune disorder curable only by HCT. Because of toxicity concerns, infants with SCID often receive donor hematopoietic grafts without conditioning, resulting in a lack of donor HSC engraftment. Instead, mature T lymphocytes and possibly lymphoid progenitors engraft but support only donor T cell development. This approach is associated with incomplete and poorly sustained immune reconstitution, and many patients have either no donor B cells and/or poor B cell function requiring life-long immunoglobulin replacement therapy. Second unconditioned donor HSC "boosts" can be performed, but they do not result in HSC engraftment and immune defects may persist. The primary endpoint of our study is to assess the safety and tolerability of AMG 191 as a conditioning agent in SCID patients. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and the determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor blood granulocyte chimerism in peripheral blood at 24 weeks. Seven patients have been treated to date who are >12 weeks post-HCT (Table 1): three in each of the first two dose cohorts (0.1 and 0.3 mg/kg AMG 191), and one patient in the third cohort (1.0 mg/kg). An eighth patient has been treated at the 1.0 mg/kg dose and is three weeks post-HCT. Patients have a mixture of SCID genotypes. All patients treated to date had prior HCT with lack of donor HSC engraftment as evidenced by 0% donor sorted granulocyte chimerism at study entry. AMG 191 administration and infusion of original donor CD34+-selected cells were uniformly well tolerated. Pre- and post-infusion marrow analyses in five evaluable patients demonstrated dose-dependent decline in CD117+HSPC following AMG 191 treatment. Table 1 shows that four of six patients, who are >24 weeks post-HCT, reached the predefined endpoint of >5% granulocyte chimerism at 24 weeks, demonstrating donor HSC engraftment. The two patients who did not show donor engraftment at 24 weeks had detectable, low level (36 weeks show the production of recent thymic emigrants and/or de novo production of naïve T and/or B cells. In addition to improved lymphocyte values, patients have demonstrated clinical improvement including resolution of chronic diarrhea, significant weight gain, and reduced IVIG requirements. Conclusion: This study is the first demonstration of HSC engraftment following monoclonal antibody-based conditioning of patients without chemo(radio)therapy. Specifically, this first-in-human HCT trial shows that an anti-CD117 antibody safely clears HSC niches and facilitates donor HSPC engraftment in patients with SCID. Clinical benefit has been observed with minimal to no toxicity. Four of six evaluable patients have sustained evidence of donor myeloid engraftment along with T and B lymphopoiesis, indicative of engraftment of multipotent HSC. These results suggest that antibody conditioning for HCT may be preferable to traditional chemo(radio)therapy conditioning, especially in patients with non-malignant diseases and/or increased risk of toxicities due to such agents, such as certain forms of SCID, Fanconi anemia and sickle cell disease. Anti-CD117 antibody conditioning may also be applicable to gene therapy with genetically corrected autologous HSC. The AMG 191 study is actively enrolling previously transplanted SCID patients and newly diagnosed SCID patients. Disclosures Dvorak: Alexion Inc: Consultancy; Jazz Pharmaceuticals: Consultancy. Prohaska:Forty Seven Inc: Equity Ownership, Patents & Royalties. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Cowan:Rocket Pharma: Consultancy; Homology Medicine: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; California Institute Of Regenerative Medicine: Research Funding; UpToDate: Honoraria; Leadiant: Consultancy; NIH NIAD: Research Funding. Logan:Kadmon: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Research Funding; TeneoBio: Consultancy; Novartis: Consultancy; Astellas: Research Funding; Abbvie: Consultancy; Incyte: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Research Funding; Kiadis: Consultancy; Jazz: Research Funding; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. Weinberg:U.S. Patent Office: Patents & Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Shizuru:Forty Seven Inc: Equity Ownership, Patents & Royalties.

Successful hematopoietic cell transplantation (HCT) requires vacating recipient hematopoietic stem cell (HSC) niches to permit donor HSC engraftment to provide life-long hematopoietic and immune function. Currently HCT relies on DNA damaging radiation or chemotherapy to achieve HSC niche clearance. We have pursued a non-toxic approach to target and deplete HSC using humanized monoclonal antibody, AMG 191, that binds human CD117 (c-Kit). We opened a Phase 1 dose escalation trial using AMG 191 as the sole conditioning agent to achieve donor HSC engraftment in patients undergoing HCT for severe combined immunodeficiency (SCID). The primary endpoint is to assess the safety of administering AMG 191. Secondary endpoints include AMG 191 pharmacokinetics (PK), host HSC depletion, and determination of the dose of AMG 191 that achieves adequate donor HSC engraftment, defined as >5% donor granulocyte chimerism at 24 weeks. We have completed the first dose cohort of patients receiving 0.1 mg/kg AMG 191 and treated the first two patients in the second cohort (0.3 mg/kg). All five patients tolerated the AMG 191 infusion and the subsequent infusion of their CD34-selectd donor cells without clinical problems. Here we report efficacy in the first two patients, who have reached the 24-week timepoint. Both patients had T-B-NK+ SCID with mutations in the DCLRE1C (Artemis) gene. Both previously received unconditioned HCT as infants, failed to develop donor B cells and remained dependent on exogenous immunoglobulin. CD34-selected mobilized peripheral blood cells from the original donors were infused when the AMG 191 serum level was Conclusion: These data are proof of concept that a humanized monoclonal antibody targeting CD117 can safely clear human HSC niches and facilitate donor HSC engraftment. This study is ongoing and open for enrollment.

RB family genes control T cell production and promote thymic involution through reducing Foxn1 expression in thymic epithelial cells., Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow–independent mechanism, identifying a novel pathway to target to increase thymic function in patients.

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Autologous hematopoietic stem cell transplantation (HSCT) has been used for the treatment of both adult and pediatric autoimmune diseases. However, HSCT has significant side effects (neutropenia, thrombocytopenia, infertility, cardiotoxicity) and costs (HSC collection/harvesting, blood product support). In an attempt to avoid the toxicities and costs associated with HSCT, we investigated whether immune ablation similar to that achieved following myeloablative HSCT could be achieved by the intensive administration of an anti-CD52 antibody (Campath-1H antibody). The first patient treated with the treatment regime, who had refractory juvenile polymyositis, achieved immune ablation (the elimination of pre-therapy antigen-specific T lymphocyte immunity) and has had stable clinical improvement for more than 6 years.

Following hematopoietic stem cell transplantation (HSCT), thymic reconstitution of peripheral T lymphocytes is essential to avoid a chronically immunodeficient state and disease recurrence. The purpose of this study was to determine if children and adolescents with treatment refractory SSc, awaiting HSCT, have sufficient thymic function to reconstitute T lymphocyte function after transplantation. Thirteen children with systemic scleroderma were enrolled and assessed by physical exam, chest MRI, measurement of autoantibodies, B and T cell immuno-phenotyping, and quantization of T cell receptor rearrangement excision circles (TREC) as a marker of thymopoiesis. MRI detected thymic tissue in 9/13 children. TREC levels were detectable in all but one child but were significantly reduced (p

Hemophagocytic lymphohistiocytosis (HLH) is a rare autosomal recessive disorder of infancy and childhood that is invariably fatal if not treated. We report on the first patient to receive post-natal HSCT for HLH after receiving in utero chemotherapy for disease stabilization. Pediatr Blood Cancer 2009;52:139–142. © 2008 Wiley-Liss, Inc.

Neurocognitive function of pediatric patients is of great concern after hematopoietic stem cell transplantation (HSCT). We evaluated the neurocognitive function of pediatric patients pre-HSCT, 1, 3, and 5 years post-HSCT. All patients had a hematologic malignancy and received therapy to their central nervous system. Healthy siblings were tested as a comparison group. Pediatric patients with a hematologic malignancy did not have a significant decrease in their cognitive function before HSCT compared with their siblings except in areas of academic achievement. Our study population had significant declines in visual motor skills and memory test scores within the first year post-HSCT. By 3 years post-HSCT, there was an improvement in the visual motor development scores and memory scores, but there were new deficits in verbal skills. By 5 years post-HSCT, there were progressive declines in verbal skills (P=0.005), performance skills (0.04), and new deficits seen in long-term verbal memory scores (0.04). On the basis of the raw scores, most of these tests showed that patients had an inability to acquire new skills at a rate comparable to their age-matched healthy peers. However, long-term memory scores showed definite declines. The greatest decline in neurocognitive function occurred in those patients who received cranial irradiation either as part of their initial therapy or as part of their HSCT conditioning. Pediatric patients who received HSCT for hematologic malignancies have neurocognitive deficiencies that are both acute and chronic. Although some patients have acute deficits that appear and improve over time, other patients have progressive declines in neurocognitive function that are chronic.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a standard treatment for a variety of hematologic conditions. However, very young children may experience different complications of HSCT compared to older patients. The authors retrospectively analyzed the results of 51 transplants performed on children less than 3 years of age between June 1987 and October 2005. Donors were matched-related (n = 21), partially mismatched related (n = 3), and unrelated (n = 27). The majority of patients had one or more grade III organ toxicities, but all nonrelapse deaths were attributable to infection. Perineal dermatitis was found in a large number (73%) of recipients of cyclophosphamide-based conditioning regimens. The 1-year transplant-related mortality (TRM) was 14%, but significantly declined in the more modern period. Grades II-IV acute graft-versus-host-disease (GvHD) was seen in 22% of patients, while chronic extensive GvHD developed in only 7% of patients. Relapse was seen in 40% of transplants performed for a malignant condition, most commonly in those patients not in remission at time of HSCT. The 5-year event-free survival (EFS) and overall survival (OS) were 53 and 64%, respectively. Recipients of fractionated total body irradiation (fTBI) were more likely to have at least one long-term sequelae than patients who received chemotherapy-based regimens (p = .014). These data demonstrate that HSCT can be performed safely in very young children, especially as supportive-care techniques improve. Cyclophosphamide-related perineal dermatitis is a unique complication in very young children. Finally, the incidence of acute and chronic GvHD in this population is low.

A patient with adenosine deaminase–deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells.

Inosine, a naturally occurring purine with anti-inflammatory properties, was assessed as a possible modulator of hyperoxic damage to the pulmonary alveolar epithelium. Rats were treated with inosine, 200 mg/kg ip, twice daily during 48-h exposure to >90% oxygen. The alveolar epithelial type 2 cells (AEC2) were then isolated and cultured. AEC2 isolated from inosine-treated hyperoxic rats had less DNA damage and had increased antioxidant status compared with AEC2 from hyperoxic rats. Inosine treatment during hyperoxia also reduced the proportion of AEC2 in S and G2/M phases of the cell cycle and increased levels of the DNA repair enzyme 8-oxoguanine DNA glycosylase. Bronchoalveolar lavage (BAL) recovered from hyperoxic, inosine-treated rats contained threefold higher levels of active transforming growth factor-β than BAL from rats exposed to hyperoxia alone, and Smad2 was activated in AEC2 isolated from these animals. ERK1/2 was activated both in freshly isolated and 24-h-cultured AEC2 by in vivo inosine treatment, whereas blockade of the MAPK pathway in vitro reduced the protective effect of in the vivo inosine treatment. Together, the data suggest that inosine treatment during hyperoxic exposure results in protective signaling mediated through pathways downstream of MEK. Thus inosine may deserve further evaluation for its potential to reduce hyperoxic damage to the pulmonary alveolar epithelium.

Allogeneic stem cell transplantation is an accepted treatment modality for selected malignant and non-malignant diseases. However, the ability to identify suitably matched related or unrelated donors can be difficult in some patients. Alternative sources of stem cells such as cord blood provide a readily available graft for such patients. Data accumulated over the past several years have demonstrated that the use of cord blood is an accepted source of stem cells for pediatric patients. Since the cell numbers of hematopoietic progenitors in cord blood is limited and the collection can occur only in a single occasion, its use in adult patients can be more problematic. Here, new developments in the use of cord blood for adults and studies aimed at expansion of cord blood cells and immune reconstitution are described.In Section I, Dr. Nelson Chao describes the early data in cord blood transplantation in adult patients. The patient outcomes are reviewed and analyzed for various factors such as cell dose, HLA typing, and patient selection that could have contributed to the final outcome of these adult patients. Myeloablative as well as nonmyeloablative approaches are presented. Discussion of the various benefits and risks are presented. More recent data from multiple single institutions as well as larger registry data comparisons are also provided. Analyses of these studies suggest methods to improve on the outcome. These newer data should lead to a logical progression in the use of cord blood cells in adult patients.In Section II, Dr. Stephen Emerson describes the historical efforts associated with expansion of hematopoietic stem cells, specifically with cord blood cells. These efforts to expand cord blood cells continue with novel methods. Moreover, a better understanding of stem cell biology and signaling is critical if we are to be able to effectively expand these cells for clinical use. An alternative, more direct, approach to expanding stem cells could be achieved by specific genetic pathways known or believed to support primitive HSC proliferation such as Notch-1 receptor activation, Wnt/LEF-1 pathway induction, telomerase or the Homeobox (Hox) gene products. The clinical experience with the use of expanded cord blood cells is also discussed.In Section III, Dr. Kenneth Weinberg describes immune reconstitution or lack thereof following cord blood transplantation. One of the hallmarks of successful hematopoietic stem cell transplantation is the ability to fully reconstitute the immune system of the recipient. Thus, the relationship between stem cell source and the development of T lymphocyte functions required for protection of the recipient from infection will be described, and cord blood recipients will be compared with those receiving other sources of stem cells. T cell development is described in detail, tracking from prethymic to postthymic lymphocytes with specific attention to umbilical cord blood as the source of stem cells. Moreover, a discussion of the placenta as a special microenvironment for umbilical cord blood is presented. Strategies to overcome the immunological defects are presented to improve the outcome of these recipients.

Autoimmune hemolytic anemia due to warm reactive IgM autoantibodies is unusual, severe, and often fails to respond to standard immunosuppressive therapies in both adults and children. A 6-year-old girl with common variable immunodeficiency had longstanding steroid dependent, splenectomy-unresponsive, warm IgM autoantibody-mediated autoimmune hemolytic anemia. Rituximab, a monoclonal antibody directed against CD20 antigen, was used to deplete B lymphocytes and reduce autoantibody production. She received a total of six doses of rituximab (375 mg/m2). Therapy was well tolerated, and B-lymphocytes were effectively depleted from the peripheral blood. The patient was completely tapered off glucocorticoids. The patient has remained off immunosuppressive agents for 16 months despite the return of B lymphocytes to the peripheral circulation. She continues to require IVIG. Early treatment with rituximab might be an option for patients with warm reactive IgM autoantibody-mediated autoimmune hemolytic anemia not responding to other treatments or experiencing untoward side effects from those treatments.

At diagnosis, clonal chromosomal abnormalities are found in the bone marrow blasts in more than two thirds of children with acute lymphoblastic leukemia (ALL). Practically, however, failure to detect these abnormalities is frequent and usually attributed to poor marrow sampling, inadequate metaphases, and/or a preponderant mitotic activity among nonleukemic cells. The authors applied fluorescence in situ hybridization (FISH) techniques to re-examine 30 cases of karyotypically "normal" childhood ALL to explore the role of preponderant mitotic activities of nonleukemic cells in failures to detect clonal abnormalities. The FISH test were performed using TEL/AML1 fusion gene probe and the centromere probes for chromosome 8 and 10 to detect the t(12;21) translocation and/or hyperdiploidy. Half of the karyotypically "normal" ALL cases examined have been found to have abnormal clones with t(12;21) rearrangement and/or hyperdiploidy by this specially designed FISH assay. Contrary to expectation, the authors found a higher incidence (52%) of clonal abnormalities in cases where over 20 metaphases had been examined than in cases (44%) where fewer than 20 metaphases had been analyzed. These findings suggest that a preponderant mitotic activity of nonleukemic cells plays an important role in failures to detect an abnormal clone by conventional cytogenetic studies. Therefore, karyotypically "normal" childhood ALL patients should undergo FISH studies to rule out the presence of t(12;21) and/or hyperdiploid clone.

Cell cycle G(1) exit is a critical stage where cells commonly commit to proliferate or to differentiate, but the biochemical events that regulate the proliferation/differentiation (P/D) transition at G(1) exit are presently unclear. We previously showed that MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), modulates CAK activities to regulate G(1) exit. Here we find that the retinoid-induced G(1) arrest and differentiation activation of cultured human leukemic cells are associated with a switch to CAK hypophosphorylation of retinoic acid receptor alpha (RARalpha) from CAK hyperphosphorylation of RARalpha. The switch to CAK hypophosphorylation of RARalpha is accompanied by decreased MAT1 expression and MAT1 fragmentation that occurs in the differentiating cells through the all-trans-retinoic acid (ATRA)-mediated proteasome degradation pathway. Because HL60R cells that harbor a truncated ligand-dependent AF-2 domain of RARalpha do not demonstrate any changes in MAT1 levels or CAK phosphorylation of RARalpha following ATRA stimuli, these biochemical changes appear to be mediated directly through RARalpha. These studies indicate that significant changes in MAT1 levels and CAK activities on RARalpha phosphorylation accompany the ATRA-induced G(1) arrest and differentiation activation, which provide new insights to explore the inversely coordinated P/D transition at G(1) exit.

Despite potent intensive conditioning regimens, hematopoietic stem cell transplantation (HSCT) may fail because of either relapse of the malignancy or the rejection of the graft. We report on 27 pediatric patients who received a second HSCT from an allogeneic donor for relapsed malignancy or graft failure. One-year, 5-year, and 10-year probabilities of survival for all patients were 53%, 36%, and 24%, respectively. Twenty patients received second HSCTs for relapsed malignancy, of whom 6 were alive and disease free at the time of this report. Seven patients received a second HSCT for graft failure, of whom 3 were alive and well as of this report. Twenty-five patients were tested for immune reconstitution following their second HSCT. Sixteen patients developed antigen-specific T-lymphocyte responses; the median time to development of antigen-specific responses was 13 months. There was no significant neurocognitive decline in patients tested 1 to 3 years following their second HSCT. Endocrine evaluations revealed deficiencies in growth hormone (7 patients), gonadal function (3 patients), and thyroid function (2 patients). Three patients developed significant abnormalities of tooth development, including absence of secondary teeth. These results show that a second HSCT offers curative therapy for selected pediatric patients whose first HSCT failed. Although toxicity is considerable following a second transplantation, the major causes of mortality continue to be relapse and infection. Biol Blood Marrow Transplant 2002;8(4):221-8.

Blood and marrow transplantation (BMT) is a standard curative therapy for patients with nonmalignant genetic diseases. Myeloablative conditioning has been associated with significant regimen-related toxicity (RRT), whereas reduced-intensity conditioning regimens have been associated with graft failure. In this prospective pilot trial conducted at 2 centers between 2006 and 2013, we report the outcome of 22 patients with nonmalignant genetic diseases who were conditioned with a novel reduced-toxicity regimen: i.v. busulfan (16 mg/kg), alemtuzumab (52 mg/m2), fludarabine (140 mg/m2), and cyclophosphamide (105 mg/kg). The median age of the study population was 3.5 years (range, 5 months to 26 years). No cases of sinusoidal obstruction syndrome, severe or chronic graft-versus-host disease (GVHD), or primary graft failure were reported. Median time to neutrophil engraftment (>500 cells/μL) and platelet engraftment (>20K cells/μL) were 19 (range, 12 to 50) and 23.5 (range, 14 to 134) days, respectively. The median length of follow-up was 3 years (range, .2 to 6.3). The overall survival rates were 95% at 100 days (95% confidence interval, .72 to .99) and 90% at 6 years (95% confidence interval, .68 to .98). RRT and chronic GVHD are significant barriers to BMT for patients with nonmalignant genetic diseases. This alemtuzumab-based reduced-toxicity regimen appears to be promising with durable engraftment, effective cure of clinical disease, low rates of RRT, and no observed chronic GVHD.

Interleukin-7 (IL-7) is the major thymopoietic cytokine. Injections of IL-7 after murine bone marrow transplantation (BMT) correct defects in thymic differentiation, including thymic hypocellularity, abnormal differentiation of CD3− CD4−CD8− (triple-negative [TN]) thymocytes into CD4+ CD8+ (double-positive [DP]) cells, and antigen-specific mature T-lymphocyte proliferation. To determine whether IL-7 production is decreased in BMT recipients, BMT was performed with congenic murine donor-recipient strains and escalating doses of pre-BMT conditioning. Increasing doses of radiation resulted in decreased thymic cellularity and maturation from the TN to the DP stage. Quantitative reverse transcription–polymerase chain reaction analyses demonstrated that intrathymic production of IL-7 was significantly decreased in irradiated mice than in nonirradiated controls. Decline in IL-7 transcript levels was correlated with the dose of radiation administered. Analyses of the numbers of CD45− major histocompatibility complex class II+ thymic stromal cells suggested that the mechanism for the decreased IL-7 production was loss of IL-7–producing thymic stromal cells. Experiments indicated that pre-BMT conditioning with radiation led to decreased stromal production of IL-7 and consequent blocks in the maturation of thymocytes. They provided a mechanism for both the abnormal thymopoiesis observed after BMT and the previously observed beneficial effects of IL-7 administration in murine models. Impaired production of IL-7 by thymic stroma may be a general model for the clinically observed adverse effects of cytotoxic therapy on thymopoiesis.

Hematopoietic stem cell transplantation (HSCT) is followed by profound immunodeficiency. Thymic function is necessary for de novo generation of T cells after HSCT. Circulating CD45RA+ naive T-cell levels are predictive of antigen-specific T-cell responses in the absence of graft-versus-host disease (GVHD). These T cells may not represent recent thymic emigrants, since naive T cells may maintain this phenotype if not antigen-activated. To accurately measure thymic output after HSCT and determine the factors that influence thymic function, T-cell receptor excision circles (TRECs) were examined in CD4+ and CD8+ cells from a cross-section of patients following HSCT. TREC levels rose weeks after HSCT and could be detected in patients 6 years after HSCT. TREC levels correlated with the frequency of phenotypically naive T cells, indicating that such cells were not expanded progeny of naive T cells present in the donor graft. Chronic GVHD was the most important factor that predicted low TREC levels even years after HSCT. Patients with a history of resolved GVHD had decreased numbers of TREC, compared with those with no GVHD. Because few adults had no history of GVHD, it was not possible to determine whether age alone inversely correlated with TREC levels. Recipients of cord blood grafts had no evidence of decreased TREC induced by immunosuppressive prophylaxis drugs. Compared with unrelated donor grafts, recipients of matched sibling grafts had higher TREC levels. Collectively, these data suggest that thymopoiesis is inhibited by GVHD. Larger studies will be needed to determine the independent contributions of age and preparative regimen to post-transplant thymopoietic capacity.

Beta-thalassemia major is characterized by ineffective erythropoiesis, although it is difficult to define the dynamics of this process from the static information revealed by analysis of bone marrow (BM) aspirates. We aimed to study the kinetics of sequential erythroid differentiation in beta-thalassemia major. We isolated the progenitor cells (CD34(+) and CD34(+)CD38(-) cells) from BM of thalassemia major patients and studied in vitro erythropoiesis. This is the first report of an in vitro study in human beta-thalassemia major from purified BM CD34(+) progenitor cells, using erythroid culture conditions, which allow unilineage differentiation to mature enucleated red blood cells. In contrast to normal donors, a high proportion of BM CD34(+) and CD34(+)CD38(-) progenitors from beta-thalassemia major coexpressed the late erythroid lineage-specific protein glycophorin A and generated a higher proportion of erythroid colonies. However, despite the marked increase in erythroid clonogenicity of the progenitor population, erythroid cultures initiated from beta-thalassemia major BM CD34(+) cells expanded 10- to 20-fold less than from normal BM. There were less viable cells during differentiation, specifically after the polychromatophilic normoblast stage. There was a progressive increase in the apoptotic erythroid progeny with differentiation, and apoptosis occurred predominantly at the polychromatophilic normoblast stage. In thalassemia major, BM progenitor cells show increased erythroid clonogenicity, increased expression of late erythroid lineage-specific proteins, and accelerated erythroid differentiation. However, despite the apparent increased erythroid commitment, ineffective erythropoiesis occurs due to apoptosis at the polychromatophil stage. Identification of the differentiation stage at which apoptosis occurs will permit further studies of the underlying mechanisms and target therapeutic strategies to improve red cell production.

The Wiskott-Aldrich syndrome (WAS) is an uncommon X-linked recessive disease characterized by thrombocytopenia, eczema and immunodeficiency. The biochemical defect of this disorder primarily affects cells derived from bone marrow. To understand better the molecular mechanisms underlying this disease and to evaluate the possibility of correcting the genetic defects in hematopoietic cells, a Moloney murine leukemia virus (MoMLV)- based retroviral vector carrying a functional Wiskott-Aldrich syndrome protein (WASp) cDNA driven by an SV40 promoter (LNS-WASp) was constructed. A packaging cell line containing this vector produced a stable level of WAS protein and maintained a high titer of viral output. Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCL) from WAS patients, which lack expression of the WAS protein, were transduced by the LNS-WASp retroviral vector and showed expression of WASp by Western blot. Analysis of the O-glycan pattern on cell surface glycoproteins from WAS patients' B-LCL showed an altered glycosylation pattern, due to increased activity of beta-1, 6-N-acetylglucosaminyltransferase (C2GnT). Transduction by the retroviral vector carrying the functional WASp cDNA partially restored the abnormal glycosylation pattern, and was accompanied by a decreasing C2GnT activity. These findings imply a functional linkage between the WAS protein and the expression of the glycosyltransferase involved in the O-glycosylation, and also suggest a potential gene therapy via transferring a functional WASp cDNA into hematopoietic cells for Wiskott-Aldrich syndrome. Gene Therapy (2000) 7, 314-320.

As the field of human gene therapy has developed over the past 15 years, T-cell immunodeficiencies have been a frequent target of investigation (Table 1). The first of these was adenosine deaminase (ADA) deficiency (severe combined immunodeficiency [SCID]) because of the availability of the responsible gene. As the genes responsible for several of the other T-cell immune deficiencies have been identified and cloned, these deficiencies also have been investigated in preclinical studies, and are now the subject of clinical trials. This article reviews the work that has been performed to date and future directions.

The lack of human B lymphocyte development in beige/nude/XID (bnx) mice is in sharp contrast to the robust development observed in another immune deficient strain, the NOD/SCID mouse. The ability to generate human B lymphocytes in the NOD/SCID, but not bnx mouse has been hypothesized to be caused by differences in the microenvironments or systemic cytokine concentrations. In the current studies we report that the differences in development can be primarily attributed to the source of the progenitors transplanted into the mice. The prior studies in bnx mice used cultured pediatric or adult bone marrow (BM) as the source of the CD34+ cells, whereas the NOD/SCID studies have predominantly used fresh or cultured umbilical cord blood (UCB). We have analyzed BM and UCB for the number of human CD34+/CD38- cells capable of in vitro B lymphocyte development, and have found a lower frequency of B lymphocyte generation in BM. The individual B lymphocyte clones that developed from bone marrow produced 100-fold fewer cells than the UCB-derived clones. In agreement with the in vitro studies, human B lymphocytes developed in bnx mice from both CD34+ and CD34+/CD38- cells isolated from human umbilical cord blood, but not from equivalent numbers of CD34+ and CD34+/CD38- progenitors from bone marrow. Therefore, the lower generative capacity, and frequency of B lymphocyte precursors in human marrow may be responsible for the previous results that showed a lack of B lymphocyte development in bnx mice.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, and a progressive deterioration of immune function. WAS is caused by mutations in an intracellular protein, WASP, that is involved in signal transduction and regulation of actin cytoskeleton rearrangement. Because immune dysfunction in WAS may be due to an accelerated destruction of lymphocytes, we examined the susceptibility to apoptosis of resting primary lymphocytes isolated from WAS patients in the absence of exogenous apoptogenic stimulation. We found that unstimulated WAS lymphocytes underwent spontaneous apoptosis at a greater frequency than unstimulated normal lymphocytes. Coincident with increased apoptotic susceptibility, WAS lymphocytes had markedly attenuated Bcl-2 expression, whereas Bax expression did not differ. A negative correlation between the frequency of spontaneous apoptosis and the level of Bcl-2 expression was demonstrated. These data indicate that accelerated lymphocyte destruction by spontaneous induction of apoptosis may be one pathogenic mechanism by which the progressive immunodeficiency in WAS patients develops.

The survival of type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic injury is regulated by signals from the cellular environment. Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. 274 ( Lung Cell. Mol. Physiol. 18): L714–L720, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC2 on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with growth on plastic. In contrast, the synthetic substrate poly-d-lysine conferred no protection. Hyperoxic AEC2 cultured on laminin showed an increased ratio of expression of Bcl-2 to interleukin-1β-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-d-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the cells started to attach to laminin and was sustained after 24 h of culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 μM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.