Zinc Treatment Stimulates Thymic Regeneration after Bone Marrow Transplant

Prolonged T cell reconstitution after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Therefore, strategies to enhance thymic reconstitution in allo-HSCT recipients are clinically desirable, although currently limited. Zinc, the second most abundant trace metal in the body, plays an important role in T cell homeostasis and thymic function. In a mouse model of allo-HSCT (Fig. a), we demonstrated that zinc supplementation can significantly improve thymic regeneration (Fig. b). Importantly, these findings in thymus were translated to the periphery as mice that received zinc supplementation showed increased numbers of naïve T cells as well as increased recent thymic emigrants (demonstrated using RAG2-GFP BM donors) model (Fig. c-d) 5-8 weeks after allo-HSCT. We have previously demonstrated that endothelial cells (EC), which are extremely resistant to damage, can promote endogenous thymic regeneration after acute injury via their production of BMP4, a growth factor that targets thymic epithelial cells (TECs), a key population crucial for T cell development. Interestingly, when stimulated in vitro for 24 hours with zinc sulphate, ECs could be directly induced to produce BMP4, but not when exposed to increased zinc import with the cell-permeable zinc pyrithione (Fig. e). This latter finding suggests a role for extracellular zinc in stimulating the endogenous response to damage. To explore this, we first measured the content of zinc in whole mouse thymus by mass spectrometry. Interestingly, when we examined a lysate of the entire thymus, total zinc concentration sharply declined early after total body radiation (TBI), followed by a steady increase that mirrored the reconstitution of the thymic cellularity (Fig. f). However, if we looked at zinc in the extracellular fraction of thymic dissociation (referred to as supernatants, SN), we saw that zinc increased significantly after TBI, revealing an inverse correlation with thymic cellularity (Fig. g), and providing a rationale for how zinc might contribute to the endogenous regenerative response. Given these findings, we hypothesized that zinc is normally used and stored in the T-cell precursors, a population of highly-replicating cells that account for approximately 98% of thymic cellularity in young mice and require the import of intracellular zinc for their proliferation. T-cell precursors are radio-sensitive and might release zinc in the extracellular space after cell death due to TBI, thereby triggering the production of regenerating factors from radio resistant cells, such as EC. Zinc supplementation could help this loop by increasing endogenous zinc levels. This hypothesis was confirmed when we co-cultured EC in presence of thymic SN there was no difference in BMP4 expression in cocultures with SN from control and zinc-treated mice at day 0, whereas BMP4 increased in presence of SN harvested from mice that had previously received TBI and even more when mice also received zinc supplement (Fig h). In conclusion, our findings demonstrate that zinc supplementation can improve T-cell regeneration in mice receiving allo-HSCT by reinforcing endogenous mechanisms of thymic regeneration. These results could be readily clinically translated into better outcomes for recipients of allo-HCT. Figure Disclosures No relevant conflicts of interest to declare.

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Zinc Supplementation Improves T Cell Reconstitution after Allogeneic HSCT By Stimulating Endogenous Pathways of Thymic Regeneration

Blood ◽

10.1182/blood-2018-99-112707 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3321-3321 ◽

Cited By ~ 1

Author(s):

Lorenzo Iovino ◽

Kirsten Cooper ◽

Sinéad Kinsella ◽

Paul DeRoos ◽

Reema Jain ◽

...

Keyword(s):

T Cell ◽

Zinc Supplementation ◽

Conflicts Of Interest ◽

The Body ◽

Thymic Epithelial Cells ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Thymic Function ◽

Allogeneic Hsct ◽

T Cell Reconstitution

Abstract Delayed T cell reconstitution after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Optimal T cell recovery requires a functional thymus, and strategies to enhance T cell reconstitution have the potential to improve overall outcome in allo-HSCT recipients, however, at the present time such strategies are limited. Hence one of the most significant clinical challenges is the need for rapid regeneration of thymopoiesis following induced immunodepletion and transplantation. Zinc is the second most abundant trace metal in the body, binding to more than 300 proteins involved in DNA synthesis and repair, gene transcription, cell proliferation as well as differentiation and apoptosis. Zinc deficiency (ZD) is a clinical condition causing immunosuppression and thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). Furthermore, mild ZD is one of the causes of the reduction in thymic function in the elderly and the role of zinc in tissue regeneration after damage has been clearly demonstrated in liver, skin, and intestinal diseases. In a pilot clinical trial, we demonstrated that patients receiving oral zinc supplementation after autologous HSCT showed increased thymic-dependent T cell reconstitution in the absence of adverse clinical events (Iovino 2018, Leuk Res). Although a clear clinical benefit was observed, the mechanisms underlying this process are poorly understood. Thus, we used a murine model to evaluate the effect of zinc supplementation in thymic reconstitution after acute damage. Using a model of thymic damage caused by sub-lethal total body irradiation (SL-TBI, 550 cGy), we found that mice that received zinc supplementation demonstrated increased thymic cellularity when compared to untreated age-matched mice (Fig. 1a). Importantly, this finding was also confirmed in a clinically-applicable model of MHC-matched allogeneic HSCT (Fig. 1b). We have previously demonstrated endothelial cells (EC), which are extremely resistant to damaged, are able to trigger thymic endogenous reconstitution after damage by producing regenerative factors such as BMP4, which targets thymic epithelial cells (TECs), a key population crucial for T cell development (Wertheimer 2018, Science Immunol). Interestingly, in our model of zinc administration, we found an increase in the number of regeneration-initiating ECs (Fig. 1c), and increased proliferation of TECs (Fig. 1d), which can occur in response to BMP4. Consistent with the hypothesis that zinc supplementation is activating the BMP4 pathway, when stimulated in vitro for 24 hours with supraphysiological doses of zinc sulfate, ex vivo propagated ECs (exECs) were directly induced to produce BMP4 (Fig. 1e), suggesting a likely mechanism by which zinc supplementation promotes thymic reconstitution. In conclusion, we demonstrate a mechanism by which zinc supplementation can improve thymic function and offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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NOD2 Acts As a Master Regulator of Endogenous Thymic Regeneration

Blood ◽

10.1182/blood-2018-99-115386 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 478-478

Author(s):

Sinéad Kinsella ◽

Kirsten Cooper ◽

Paul DeRoos ◽

Reema Jain ◽

Lorenzo Iovino ◽

...

Keyword(s):

T Cell ◽

Rho Gtpase ◽

Acute Injury ◽

Thymic Epithelial Cells ◽

Common Mechanism ◽

High Morbidity ◽

Hematopoietic Stem ◽

Common Cancer ◽

Wide Range ◽

Thymic Regeneration

Abstract Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. Our recent studies have revealed that innate lymphoid cells (ILCs) and endothelial cells (ECs), through their production of the regeneration-associated factors (RAFs) IL-22 and BMP4, respectively, have profound reparative effects in the thymus after acute injury; and can be utilized individually as therapeutic strategies of immune regeneration (Dudakov 2012 Science 336:91; Dudakov 2017 Blood 130:933; Wertheimer 2018 Sci Immunol 3:19). These two pathways act by stimulating thymic epithelial cells (TECs), a heterogeneous population of stromal cell in the thymus critical for thymopoiesis. However, the regulation of these endogenous regenerative responses is still poorly understood. Here we reveal an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in governing multiple pathways of thymic regeneration. Analysis of thymic recovery following acute injury in mice deficient for NOD2 revealed increased intrathymic BMP4 and IL-23 (Fig. 1a), a key regulator of IL-22 production, and commensurate improved ability to regenerate (Fig. 1b). Although NOD2 is expressed ubiquitously across all populations within the thymus, in regeneration-initiating DCs and ECs, but not thymocytes (damage-targets), we identified a specific reduction in the expression of miR29c after damage, which previous reports suggest could mediate NOD2-induced suppression of IL-23 (Brain 2013 Immunity 39:521). Consistent with these findings, miR29c expression was decreased in the thymus of NOD2-deficient mice (Fig. 1d), and overexpression of miR29c in either ECs or DCs reduced their expression of Bmp4 or Il23, respectively (Fig. 1e). Canonical ligands for NOD2 are peptidoglycans found in the cell wall of bacteria; however, these are unlikely to serve as a NOD2 activator in the thymus since it is typically thought of as a sterile organ. One recently described alternate function of NOD2 is as a cytosolic sensor of activated Rho GTPases. The Rho GTPase family is responsible for a wide range of physiological processes, including the intrathymic regulation of b-selection and positive selection, and inhibition of Rho GTPase signaling is of considerable clinical interest. Consistent with a role in suppressing regeneration, unbiased transcriptome analysis revealed significant downregulation of many members of the RhoGTPase family after damage, corresponding to the increase in production of RAFs. Importantly, suggestive of a potential clinical application, pharmacological suppression of RhoGTPase in vitro significantly induced the expression of Bmp4 in ECs, and Il23 in DCs (Fig. 1f), as well as suppressing the expression of miR29c (Fig. 1g). Although several pathways have been described as contributing to endogenous thymic regeneration, the specific mechanisms regulating their induction has been poorly understood. Here we reveal a common mechanism triggering production of multiple distinct regeneration pathways such as those centered on production of BMP4 and IL-22. Therefore, the mechanistic and pre-clinical studies described not only define an important regulatory mechanism governing endogenous tissue regeneration, but could also offer an innovative therapeutic strategy to boost thymic function and T cell reconstitution in recipients of allo-HSCT, as well as for individuals with T cell deficiencies due to aging, autoimmune diseases, genetic causes, infectious disease, shock, radiation injury (nuclear accident, terrorism) and common cancer treatments such as chemo- and radiation-therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Mechanisms Governing Endogenous Thymic Regeneration

Blood ◽

10.1182/blood.v130.suppl_1.66.66 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 66-66

Author(s):

Tobias Wertheimer ◽

Enrico Velardi ◽

Jennifer Tsai ◽

Kirsten Cooper ◽

Katja J Ottmüller ◽

...

Keyword(s):

T Cell ◽

Lymphoid Cells ◽

Thymic Epithelial Cells ◽

High Morbidity ◽

Paracrine Factors ◽

Hematopoietic Stem ◽

Thymic Function ◽

Common Cancer ◽

Thymic Regeneration

Abstract Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. We have recently described a central role for group 3 innate lymphoid cells (ILC) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in their recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also contribute to thymic repair. Unbiased transcriptome analysis on the damage-resistant non-hematopoietic compartemtn of the thymus revealed significant upregulation of Bmp4 and its downstream signalling targets (Fig. 1a). Further interrogation revealed that while thymic expression of BMP4 was restricted to fibroblasts and endothelial cells (ECs), only ECs increase their expression of Bmp4 after damage; and specific and inducible deletion of BMP4 in ECs led to significantly worse regeneration (Fig. 1b). Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration has thus far remained largely unclear. Careful interrogation of ECs after damage revealed that, much like ILCs, ECs are extremely resistant to multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and TBI. However, whole organ imaging analysis using light sheet field microscopy suggested that even though the number of ECs remain unchanged after damage, there is considerable structural changes to the vasculature including shortening of the vessels and reduced branching. Although BMP4 receptors are widely expressed in the thymus, there was enriched expression for BMP4 receptor subunits on TECs, which is consistent with the role of BMP4 in thymus ontogeny by promoting TEC development, at least partially due to its ability to induce expression of Foxn1 (Fig. 1c), a key transcription factor for the development and maintenance of TECs. Consistent with these findings, after thymic damage we observed a significant increase in the expression of Foxn1 after damage as well as GSEA enrichment for downstream FOXN1 target genes (Fig. 1d); including Dll4, the Notch ligand critical for T cell development and whose concentration we have previously shown can directly regulate thymic size (Velardi 2014 J Exp Med 211:2341). Finally, using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function; but only when the exEC were derived from the thymus but not from heart or kidney (Fig. 1e). Consistent with endogenous regeneration, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 and Dll4 . Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic renewal. These studies thus not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy. Figure 1 Figure 1. Disclosures van den Brink: PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy.

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Damage-Induced Pyroptotic Cell Death Facilitates Regeneration of the Thymus

Blood ◽

10.1182/blood-2020-143051 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-28

Author(s):

Sinéad Kinsella ◽

Cindy Evandy ◽

Kayla S Hopwo ◽

Kirsten Cooper ◽

Lorenzo Iovino ◽

...

Keyword(s):

T Cells ◽

Cell Death ◽

T Cell ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Acute Injury ◽

T Cell Reconstitution ◽

Increased Risk ◽

Metabolic Remodeling ◽

Thymic Regeneration

T cell reconstitution after transplant is critically dependent on the thymus; an inverse relationship between a transplant recipient's age and their capacity to generate T lymphocytes (in particular CD4+T cells) has been found in several studies, and thymic function pre-transplant can have a significant impact on clinical outcomes. Although the thymus has a remarkable ability to repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Despite this regenerative capacity, delayed T cell reconstitution is associated with an increased risk of infections, relapse of malignancy and the development of secondary malignancies. Therefore, there is a clinical demand for therapeutics that restore immune function after damage. Our recent studies have identified two key pathways driving thymic regeneration; centered on the secretion of BMP4 by endothelial cells (ECs) and IL-22 by innate lymphoid cells (Dudakov 2012 Science 336:91; Dudakov 2017 Blood130:933; Wertheimer 2018 Sci Immunol3:19). However, the specific regulatory mechanisms that trigger these regeneration-associated factors after damage remain unclear. Our previous work identified that the presence of homeostatic apoptotic CD4+CD8+ (DP) thymocytes, as apoptotic thymocytes form the bulk of developing T cells, suppress the production of IL-23 in dendritic cells (DCs), a key downstream mediator for IL-22, and BMP4 in ECs (Fig. 1A), and that the depletion of apoptotic thymocytes after damage precedes the production of these regenerative factors. Therefore, together with our findings that the metabolic needs of key thymus populations alter drastically following injury due to damage-induced metabolic remodeling, we hypothesized that further to the loss of DP-specific suppression, metabolic dysfunction in DPs after damage triggers mitochondrial-induced pyroptotic cell death, which can directly promote regeneration of the thymus. Consistent with this hypothesis, our preliminary data shows increased levels of cl-caspase 1 (pyroptotic caspase) and a decrease in cl-caspase 3 (apoptotic caspase) in DPs after SL-TBI (550 cGy), demonstrating a preferential induction of pyroptotic cell death in DPs after damage (Fig. 1B). Furthermore, we demonstrated an increase in extracellular lactate dehydrogenase (LDH) levels, HMGB-1 and TNF⍺[canonical damage-associated molecular patterns (DAMPs) released during ICD] acutely after damage caused by SL-TBI (Fig. 1C).Given our previous findings that stromal cells are more radio-resistant than DP thymocytes (Wertheimer 2018 Sci Immunol3:19), and evidence for mitochondrial-induced pyroptosis, we identified hyperpolarization of the mitochondrial membrane potential accompanied by increased levels of ROS in DPs, an effect not observed in TECs, suggesting metabolic stability confers protection against acute damage (Fig. 1D). Furthermore, co-culture of pyroptotic thymocytes results in increased IL12p40+ DCs and increased Foxn1 expression in TECs (Fig. 1E), strengthening our hypothesis that cell-cell communication drives thymic regeneration after damage by inducing regenerative factors as well as directly promoting TEC function via secreted factors from pyroptotic DPs. One way in which DAMPs, such as ATP, can initiate cell signaling is by the activation of cell surface purinergic receptors, including P2Y2 which is widely expressed on TECs, and here we demonstrate that in vitro treatment with ATP or P2Y2 agonist increases Foxn1 in cTECs, and P2Y2 antagonism reverses this effect (Fig 1F). As P2Y2 activation promotes Ca2+efflux from the ER, we have further demonstrated that stimulating the intracellular release of Ca2+, using tunicamycin, induced Foxn1 expression in cTECs, which was reversed upon inhibition of Ca2+release (Fig. 1G). Importantly, we demonstrate here that this pathway can be therapeutically targeted by activating P2Y2 signaling in vivo with MRS2568 or ATP enhances thymus cellularity and expands cTECs in models of acute injury (Fig. 1H&I). These findings not only reveal a novel metabolic-mediated molecular mechanism governing tissue regeneration; but also by targeting FOXN1 directly offers a potentially superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by HCT, infection or cytoreductive therapy. Disclosures No relevant conflicts of interest to declare.

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A Comparison Study Between Allogeneic and Autologous Hematopoietic Stem Cell Transplant for High-Risk Peripheral T-Cell Lymphomas (PTCL)

Blood ◽

10.1182/blood.v128.22.5845.5845 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5845-5845 ◽

Cited By ~ 2

Author(s):

Haiwen Huang ◽

Qiangli Wang ◽

Zhengming Jin ◽

XiaoWen Tang ◽

Huiying Qiu ◽

...

Keyword(s):

T Cell ◽

High Risk ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Cell Transplant ◽

Term Survival ◽

Hematopoietic Stem ◽

Significant Difference

Abstract Objective: To evaluate the efficacy of auto-HSCT and allo-HSCT in the treatment of high risk peripheral T cell lymphoma (PTCL). Methods: From July 2007 to July 2014, 60 cases of high risk PTCL were analyzed retrospectively. Results: All 60 patients were at high risk group (carried with IPI≥3), with a median age of 31 (13-55) years old. Of the 60 cases, 22 were PTCL-not otherwise specified (PTCL-NOS), 22 with ALK negative anaplastic large cell lymphoma (ALK-negative ALCL) and 16 with angioimmunoblastic T-cell lymphoma (AITL). Twenty-one (21/60) received allo-HSCT and thirty-nine patients (39/60) received auto-HSCT. Before receiving transplantation, 40/60 patients were in complete remission (CR), 2/60 patients were partial remission (PR) and 18/60 patients were not remission (NR). In the 40 CR patients before transplant, 10 patients received allo-HSCT and 30 patients received auto-HSCT. In the 20 PR/NR patients before transplant, 11 patients received allo-HSCT and 9 patients received auto-HSCT. After a median follow-up of 39 (range 1-96) months, the K-M analysis showed that the 5-year PFS for auto-HSCT and allo-HSCT were 61% and 60% (P = 0.724). The 5-year OS for auto-HSCT and allo-HSCT were 62% and 61% (P = 0.724). There were no statistically significant differences between the auto-HSCT and allo-HSCT. And the cumulative TRM of allo-HSCT and auto-HSCT were 16.5% and 0 (P=0.250) within 5-years after transplantation. At the end of the last follow-up, 7 patients relapsed in auto-HSCT group and 2 patients relapsed in allo-HSCT group, the 5-year cumulative recurrence rates of auto-HSCT and allo-HSCT transplantation were 37.2% and 10.1% (P=0.298), respectively. Conclusion: There was no significant difference in the long-term survival between auto-HSCT and allo-HSCT for high risk PTCL patients. The effect of allo-HSCT may be better for NR patients. Disclosures No relevant conflicts of interest to declare.

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Relapsed subcutaneous panniculitis-like T cell lymphoma: role of haploidentical hematopoietic stem cell transplant

Annals of Hematology ◽

10.1007/s00277-017-3119-2 ◽

2017 ◽

Vol 96 (12) ◽

pp. 2125-2126 ◽

Cited By ~ 3

Author(s):

Bhagirathbhai Dholaria ◽

Raj J. Patel ◽

Jason C. Sluzevich ◽

Sikander Ailawadhi ◽

Vivek Roy

Keyword(s):

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell ◽

Hematopoietic Stem Cell Transplant ◽

Stem Cell Transplant ◽

Cell Lymphoma ◽

T Cell Lymphoma ◽

Cell Transplant ◽

Hematopoietic Stem

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The Platelet Millionaire: A Rare Cause of Thrombocytosis in an Adolescent

Blood ◽

10.1182/blood-2019-129278 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5391-5391

Author(s):

Ritika Walia ◽

Theresa Sepulveda ◽

Sharon Wretzel ◽

Philip H Brandt

Keyword(s):

Bone Marrow ◽

Bone Marrow Biopsy ◽

Primary Care Physicians ◽

Conflicts Of Interest ◽

Abdominal Ultrasound ◽

Cell Transplant ◽

Peripheral Smear ◽

Hematopoietic Stem ◽

Marrow Fibrosis

Objectives: Primary myelofibrosis is rare in pediatrics, often manifesting as persistent idiopathic thrombocytosis.Transitions from pediatric to adult medical care can be complicated by workup requiring invasive procedures. J.M., an 18-year-old healthy male, presented for excessive gingival bleeding after wisdom tooth extraction. Workup revealed persistent thrombocytosis to 1,165K, prompting a referral to hematology-oncology. A peripheral smear was notable for many platelets but normal RBC morphology. He had splenomegaly on abdominal ultrasound and a decreased von-Willebrand's activity to antigen ratio, suggesting acquired vWD. A bone marrow biopsy was advised; however, J.M. became lost to follow up for over 9 months owing to self-reported anxiety about the procedure. He remained asymptomatic in this interim until he re-presented to clinic for easy bruising, with no other evidence of bleeding at the time. The biopsy was pursued, revealing hypercellular marrow for age with left shifted granulocytic and erythroid maturation, abnormal megakaryocytes, and 3% blasts. This was consistent with primary early myelofibrosis (PMF), positive for MF-1, CALR, and TP53 mutations and negative for JAK2 and BCR-ABL. He was transitioned to adult hematology, maintained on baby aspirin, and referred for potential allogeneic hematopoietic stem cell transplant (HSCT). PMF is characterized by marrow fibrosis due to secretion of fibroblast growth factor by clonally proliferative megakaryocytes. It is a disease of adulthood, with 67 years being the median age at diagnosis. Only 100 cases have been reported in children, most of which are secondary to AML, ALL or other malignancies.1 Most patients present with complications of extramedullary hematopoiesis or bleeding.2 Diagnosis is suggested by a leukoerythroblastic picture on peripheral smear and confirmed with a bone marrow biopsy "dry tap" revealing marrow fibrosis.3 Prognosis in pediatric PMF is difficult to predict but outcomes tend to be worse;4 TP53 mutation is rare and based on limited adult studies may portend a poorer prognosis.5 Our young patient with this rare mutation was therefore referred for HSCT evaluation. Further complicating this case was J.M.'s anxiety, which delayed definitive diagnosis by biopsy. He only agreed to it when, at the med-peds clinic, the concept of local pain management was discussed. Anticipation of upcoming procedures by primary care physicians and close follow-up is especially important for patients transitioning from pediatric to adult providers. Disclosures No relevant conflicts of interest to declare.

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1098. Norovirus Infection in Cancer Patients Undergoing Chimeric Antigen Receptor T-cell Immunotherapy (CAR-T)

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1284 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S578-S579

Author(s):

Divya S Kondapi ◽

Sasirekha Ramani ◽

Adilene Olvera ◽

Robert L Atmar ◽

Mary K Estes ◽

...

Keyword(s):

T Cell ◽

Lymphoblastic Leukemia ◽

Lymphocytic Leukemia ◽

Nucleic Acid Amplification ◽

High Dose ◽

Cell Transplant ◽

Research Support ◽

Hematopoietic Stem ◽

Car T Cell ◽

Car T

Abstract Background CAR-T is used to treat certain refractory hematological malignancies. B-cell aplasia and immunosuppression used to treat CAR-T side effects increase infection risk. Little data are available describing Norovirus (NoV) infections in CAR-T recipients. Methods We reviewed the medical records of 134 patients with NoV diarrhea (identified by nucleic acid amplification test) between 2016-2019. Of these patients, nine received CAR-T prior to developing NoV. Here we describe their demographics, clinical characteristics, treatments, and complications. Results The median age was 49 years (Table 1). Patients’ underlying malignancies included Non-Hodgkin’s Lymphoma (4), Acute Lymphoblastic Leukemia (3), Chronic Lymphocytic Leukemia (1) and metastatic Sarcoma (1). Prior to development of NoV, six patients had undergone hematopoietic stem cell transplant, and 1 had received checkpoint inhibitor therapy. Five patients experienced cytokine release syndrome after CAR-T, and 1 experienced CAR-T-related encephalopathy syndrome (Table 2). Two patients received interleukin-6 antagonist therapy, and one received high dose steroids. Time to diarrhea onset post-CAR-T cell infusion was variable(median 256days, IQR 26-523 days).Six had an absolute lymphocyte count< 1000/mm3 at diarrhea onset. Three had diarrhea for >14 days; median diarrhea duration in the other 6 patients was 4 days. Other GI complaints included abdominal pain (3), nausea (4), and vomiting (3). For NoV treatment, three received oral immunoglobulin, and 8 received Nitazoxanide. Complications included development of concomitant GI-GVHD(5), ileus (2), need for TPN (3), renal failure requiring dialysis (2), ICU stay (3), and death (2). Two patients were co-infected with other enteropathogens such as rotavirus, enteropathogenic and enteroaggregative E.Coli and Clostridioides difficile. Three patients with diarrhea lasting >14 days had serial samples collected over time; NoV shedding lasted 81-546 days. NoV was genotyped in 6 patients(Table 3) and included GII.2(2), GII.4(2), GII.6(1) and GII.12(1). Table 1: Patient characteristics (N=9) Table 2: CAR-T related factors Table 3: NoV Genotypes Conclusion NoV belonging to various genotypes is an important cause of acute and chronic diarrhea in patients receiving CAR-T cell therapy. Disclosures Adilene Olvera, MPH MLS (ASCP), MERK (Grant/Research Support, Scientific Research Study Investigator) Robert L. Atmar, MD, Takeda Vaccines, Inc. (Grant/Research Support) Mary K. Estes, PhD, Takeda Vaccines (Consultant, Grant/Research Support)

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CongenitalSTAT3mutation Mediates Primary Persistent Polymorphic Inflammatory Activation and T Cell Leukemogenesis

Blood ◽

10.1182/blood-2019-132138 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1054-1054 ◽

Cited By ~ 1

Author(s):

Hongxing Liu

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cell ◽

Lymph Nodes ◽

Conflicts Of Interest ◽

Sh2 Domain ◽

Janus Kinase ◽

Gingival Hyperplasia ◽

Hematopoietic Stem ◽

T Cell Leukemogenesis

Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways play a pivotal role in inflammation and immunity, among which, JAK/STAT3 pathway is the most potent and leads the crosstalk of immunity and oncogenesis. Somatic STAT3 activatingmutations have been found in about 40% of T cell large granular lymphocytic leukemia (T-LGLL) patients, most of which are located in exon 21 which encodes Src homology 2 (SH2) domain leading to the increased activity of aberrant STAT3 protein and the upregulation of its transcriptional targets. While germline STAT3activatingmutations represent a newly defined entity of immune dysregulations named infantile-onset multisystem autoimmune disease-1 (ADMIO1, #MIM 615952). Both the two diseases are rare and poorly understood. Here, we report a pedigree including a proband, a six-year-old girl, primarily manifesting as thrombocytopenia and lymphadenopathy and her father diagnosed as T-LGLL with pure red cell aplastic anemia without autoimmune disorders preceding or during his disease course. Morphology of the bone marrow smears of the proband indicated normal hyperplasia without evident dyspepsia or increased blast cells. However, the vacuoles in monocytes and the density and size of granules in neutrophils increased, and megaloblast transformation was observed in some neutrophils. (Fig. 1A, 1B) Biopsy of an enlarged lymph node showed the reactive follicular hyperplasia. (Fig. 1C) Whole exon sequencing and pedigree analysis of the family revealed the germline STAT3 c.833G>A/p.R278Hmutation harbored by the proband which originated de novo from her father who additionally carried a germline TAL1G62Rmutation and somatically accumulated an FLT3-ITD mutation. (Fig. 2) Through single-cell RNA sequencing, we also found the increase of circulating CD8+ T cells and the decrease of NK cells of the proband. (Fig. 3) The STAT3 target genes were generally overactivated, and the expression of cytokines decreased in transcription level. In the genes participating in JAK/STATs pathways, the expression of JAK3, STAT1, and STAT3was up-regulated significantly. (data not shown) Immunophenotype of the proband by flow cytometry confirmed change in immunocyte compartments, (Fig. 4) but the serum cytokine concentrations measured by flow cytometry yielded controversial results, that most of cytokines were moderately elevated, and IL-1β, IL-5, TNF-α, and IFN-γ were of the most evident. (data not shown) During the treatment and follow-up, Cyclosporin A (CsA) was efficient in maintaining her circulating platelets in the range of 166×109/L to 302×109/L, but the enlarged lymph nodes and hepatosplenomegaly had no response. Eleven months later, CsA was replaced by tacrolimusfor the severe gingival hyperplasia, which has efficiently stabilized her platelets count and normalized the enlarged lymph nodes, liver, and spleen. On the contrary, in the three and a half years' span of illness, the father was refractory to CsA and methotrexate (MTX), moreover, lethal bone marrow suppression was induced by one course of fludarabine. For the high level of HLA-I and HLA-II antibodies in the circulation, plantlets transfusions were only efficient after plasmapheresis. The father eventually died from pulmonary and gastrointestinal infection due to the failure of maternal HLA-haploidentical hematopoietic stem cell transplantation (HSCT). We comprehensively elaborated the immunophenotype of the proband and thoroughly elucidated the genetic alternations of the father which led to the T cell leukemogenesis, which brought new insight on these two rare diseases and highlighted a more scrupulous therapeutic strategy in T-LGLL with congenital mutations. Figure 1 Disclosures No relevant conflicts of interest to declare.

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