pre-T Cell Research Articles

Synchronized development of thymic eosinophils and thymocytes.

The thymus is an organ required for T cell development and is also an eosinophil-rich organ; however, the nature and function of thymic eosinophils remain unclear. Here, we characterized the gene expression and differentiation mechanism of thymic eosinophils in mice. Thymic eosinophils showed a distinct gene expression profile compared with other organ-resident eosinophils. The number of thymic eosinophils was controlled by medullary thymic epithelial cells. In Rag-deficient mice, the unique gene expression signature of thymic eosinophils was lost but restored by pre-T cell receptor signaling, which induces CD4+ CD8+ thymocyte differentiation, indicating that T cell differentiation beyond the CD4- CD8- stage is necessary and sufficient for the induction of thymic eosinophils. These results demonstrate that thymic eosinophils are quantitatively and qualitatively regulated by medullary thymic epithelial cells and developing thymocytes, respectively, suggesting that thymic eosinophils are a distinct, thymus-specific cell subset, induced by interactions with thymic cells.

JAK2 V617F Impairs Lymphoid Differentiation in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) arise from the clonal acquisition of a driver mutation in a hematopoietic stem cell (HSC), often decades before the emergence of clinical disease. A defining characteristic of an HSC is its potential to give rise to both myeloid and lymphoid hematopoietic lineages. Yet the most common MPN driver mutation, JAK2 V617F, has only rarely been observed in lymphocytes. We conducted this study to determine why the JAK2 V617F clone does not contribute to mature lymphocytes. We directly measured myeloid and lymphoid lineage contribution of normal and MPN HSCs in 133 patients with JAK2 V617F MPN. Peripheral blood subpopulations were fractionated via fluorescence-activated cell sorting (FACS) and JAK2 V617F mutation allele frequency (MAF) was measured in each subpopulation by droplet digital PCR. JAK2 V617F was enriched in neutrophils and maintained in erythroid progenitors (EPs) relative to HSCs. In contrast, JAK2 V617F alleles were largely absent in T and B cells relative to HSCs. Lymphopoiesis declines with age, potentially explaining the absence of JAK2 V617F in lymphocytes from older patients with MPN even if JAK2 V617F has no effect on lymphopoiesis. This “JAK2 V617F neutral” hypothesis predicts that patients who acquired the mutation at a younger age, and those who harbor the mutation for a longer duration, have a higher likelihood of accumulating JAK2mutated lymphocytes. However, we found that JAK2 V617F MAF in T and B cells across our cohort does not correlate with either patient age at diagnosis or duration of clinical disease. Thus, predictions of the “JAK2 V617F neutral” hypothesis are not supported by patient data. The alternative explanation is that JAK2 V617F impairs lymphocyte differentiation from mutated progenitors. We tested this “JAK2 V617F adverse” hypothesis by tracking JAK2 V617F during various stages of lymphopoiesis. To assess the relative bias of JAK2 V617F HSCs during the early stages of lymphoid and myeloid commitment, we measured JAK2 V617F MAF in common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs) and HSCs isolated by FACS from the peripheral blood of 79 patients with JAK2 V617F MPN. We found that while myeloid commitment from HSC to CMP was associated with an increase in mean JAK2 V617F MAF, there was no statistically significant change in mean MAF between CLPs and the parent HSCs across the cohort. Therefore, JAK2 V617F HSCs are not completely excluded from the earliest stages of lymphoid differentiation. To further define the extent to which JAK2 V617F affects lymphopoiesis, we used FACS to isolate CLPs and CMPs from 17 patients with JAK2 V617F polycythemia vera (PV) and differentiated each subpopulation in vitro into T cell progenitors (pro- and pre-T cells) and erythroblasts (EBs), respectively. We found that all CLP samples could be differentiated into T cell progenitors. However, we observed a marked depletion of JAK2 V617F alleles in progressively mature T cell progenitors (Figure 1). In contrast, JAK2 V617F alleles were further enriched in progressively mature erythroblasts. The in vitro differentiation potential of MPN progenitors thus supports the “JAK2V617F adverse” hypothesis. To determine the effect of JAK2 V617F on lymphopoiesis in vivo, we performed competitive transplantations of lethally irradiated CD45.1 mice with congenic CD45.2 donor whole bone marrow (WBM) cells harboring JAK2 V617F or wild type JAK2 (JAK2 WT). Measuring CD45.2 chimerism by FACS in lymphocytes and neutrophils from engrafted mice revealed that only CD45.2 cells harboring JAK2 V617F were preferentially depleted in lymphocytes (Figure 2). Therefore, JAK2 V617F HSCs displayed a competitive disadvantage relative to their JAK2 WT counterparts during lymphopoiesis in vivo. Myeloid proliferation in MPN driven by JAK2 V617F is well recognized but does not fully explain certain MPN complications such as increased risk of infections and second malignancies. Our findings demonstrate impaired lymphoid differentiation from JAK2 V617F stem and progenitor cells that may also contribute to clinical MPN phenotypes. Further study is ongoing to define actionable mechanisms through which JAK2 V617F interferes with T cell differentiation.

Pre-T cell receptor localization and trafficking are independent of its signaling.

Expression of the pre-T cell receptor (preTCR) is an important checkpoint during the development of T cells, an essential cell type of our adaptive immune system. The preTCR complex is only transiently expressed and rapidly internalized in developing T cells and is thought to signal in a ligand-independent manner. However, identifying a mechanistic basis for these unique features of the preTCR compared with the final TCR complex has been confounded by the concomitant signaling that is normally present. Thus, we have reconstituted preTCR expression in non-immune cells to uncouple receptor trafficking dynamics from its associated signaling. We find that all the defining features of the preTCR are intrinsic properties of the receptor itself, driven by exposure of an extracellular hydrophobic region, and are not the consequence of receptor activation. Finally, we show that transitory preTCR cell surface expression can sustain tonic signaling in the absence of ligand binding, suggesting how the preTCR can nonetheless drive αβTCR lineage commitment.

Clinical features and prognosis of pediatric acute lymphocytic leukemia with JAK-STAT pathway genetic abnormalities: a case series.

The objective of this study is to explore the clinical features and outcomes of pediatric patients with acute lymphoblastic leukemia (ALL) harboring JAK-STAT signaling pathway genetic abnormalities. This retrospective case series examined the clinical data of pediatric patients diagnosed with ALL harboring JAK-STAT pathway genetic abnormality at the Children's Hospital of the Capital Institute of Pediatrics between January 2016 and January 2022. Bone marrow next-generation sequencing was used to reveal the JAK pathway abnormalities. Descriptive statistics were used. From 432 children with ALL during the study period, eight had JAK-STAT pathway genetic abnormalities. Regarding immunotyping, there were four patients with common-B cell types and one with pre-B cell type. The three patients with T-ALL had early T-cell precursor(ETP) type, pre-T cell type, and T cell type. Gene mutations were more common than fusion genes. There was no central nervous system involvement in eight patients. All patients were considered at least at intermediate risk before treatments. Four patients underwent hematopoietic stem cell transplantation (HSCT). One child had a comprehensive relapse and died. The child had a severe infection and could not tolerate high-intensity chemotherapy. Another child relapsed 2years after HSCT and died. Disease-free survival was achieved in six children. JAK-STAT pathway genetic abnormalities in pediatric Ph-like ALL are rare. Special attention should be paid to treatment-related complications, such as infection and combination therapy (chemotherapy, small molecule targeted drugs, immunotherapy, etc.) to reduce treatment-related death and improve long-term quality of life.

HnRNPL is essential for T cell function in the Periphery

Abstract While RNA binding proteins (RBPs) have recently emerged as regulators of gene expression in immune cells, their effect on T cell fate needs further investigation. Heterogeneous Nuclear Ribonucleoprotein (hnRNPs) are RBPs that play a crucial role in many aspects of RNA biogenesis, alternative splicing, expression, and function. During T cell development hnRNPL is known to regulate the proliferation, and migration of thymic pre-T cells, as loss of hnRNPL in early T cell development results in a failure of T cells to reach the periphery. Little is known about how hnRNPL affects T cell activation, and function. Our lab has used CD4 Cre × hnRNPL Fl/Fl(KO) mice to understand the role of hnRNPL in peripheral T cells. Our initial study of the steady state profile of the KO mice shows normal migration of T cells form the thymus, but there is a reduction in peripheral T cell numbers. We also show that hnRNPL plays an important role in T cell activation and survival, since T cells lacking hnRNPL do not survive long enough to differentiate into T helper cell subsets. In vivo, using immunization studies, we show that KO CD4 T cells fail to fully differentiate into Tfh and are unable to support the formation of germinal center B cells. To ensure the effect that hnRNPL has on T cells in the periphery is not caused by the loss of hnRNPL during thymic T cell development we are using the CD4-Cre/ERT2 × hnRNPL (I-KO) to investigate how the loss of hnRNPL in peripheral T cell affects the ability of T cells to differentiate and survive post activation. Further studies will also focus on the mechanisms hnRNPL use to regulate T cells activation and survival. These studies suggest that hnRNPL plays an important role in the activation and survival of peripheral T cells. AAI Fellowship

Abstract 426: Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines

Abstract The prognosis of recurrent precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) is poor, mainly due to chemotherapy resistance. The specific mechanism(s) leading to chemoresistance at relapse is still poorly understood. We previously characterized a mouse strain that contains a CreERT2 cassette “knocked into” the 3′ UTR of Mcm2. Mice with 2 copies of this knock-in allele, designated Mcm2cre/cre show decreases Mcm2 protein expression. They die of pre-T LBL, due to acquired DNA copy number abnormalities (CNA), involving important tumor suppressor or oncogenes. We hypothesize this unique mutator phenotype may reveal specific genetic drivers leading to chemoresistance. We selected six drugs with different mechanisms that are known to be active against pre-T LBL, including cytarabine, etoposide, melphalan, methotrexate, prednisone, and vincristine. We used 4 mcm2 Pre-T LBL cell lines (#2883, 2696, 2641 and 2869). After 1 year of treatment, we obtained resistant cell lines that can tolerate drug concentrations which are up to 10,000X that of parental cell lines. These samples will be characterized by Sparse WGS (for CNA), RNA-Seq and WES. Before sequencing, we wanted to verify that cell lines had not become cross-contaminated during drug selection. During thymocyte differentiation, noncontiguous genomic V, D, and J gene segments become juxtaposed, and form an exon which encodes the variable region of a T cell receptor protein. The TCRβ VDJ rearrangement provides a unique identifier for pre-T LBL cell lines. For the 2869 cell line, we found that the VDJ sequences of cytarabine, vincristine, melphalan, and prednisone resistant 2869 were identical to the parental 2869 cells. However, analysis of 2883 cell lines were more complex. Although there were multiple VDJ rearrangements in parental 2883, they had an identical junction of D2J2S4, suggesting that the variable VDJ rearrangements were derived from a single clone with a unique DJ rearrangement, and subsequent different V-DJ rearrangements. Moreover, following drug selection, the VDJ rearrangement of chemo-resistant 2883 and parental 2883 were not identical. However, the chemo-resistant 2883 cells retain the fingerprint of parental 2883, namely the unique junction of D2J2S4. The VDJ rearrangement of etoposide resistant and vincristine resistant 2883 (2883ER and 2883VR, respectively) clones were identical, and could be identified as a subclone of parental 2883. Interestingly, the2883ER and 2883VR clones had nearly identical CNA of a number of genes, including Abcb1a (also known as Multi-Drug Resistance gene 1a), suggesting selection of a pre-existing resistant clone. Although the VDJ rearrangement of cytarabine 2883 was not found in the parental 2883 subclone, however, the unique D2J2S4 was present. These findings indicate that pre-T LBL cell lines do not invariably have stable TCRβ VDJ rearrangements, and can undergo V-DJ rearrangement event after several years of continuous cell culture. Citation Format: Dengchao Cao, Mianmian Yin, Toshihiro Matsukawa, Taimour Baslan, Peter Aplan. Ongoing TCRβ VDJ rearrangement in pre-T LBL cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 426.

Mechanisms of Thymic Repair of In Vitro-Induced Precursor T Cells as a Haploidentical Hematopoietic Stem Cell Transplantation Regimen

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is currently an effective treatment for malignant hematological disease, but the immune deficiency and severe infection triggered by slow immune reconstitution are the main causes of high mortality and transplant failure. One of these outstanding problems is thymus damage, which is associated with graft-versus-host disease (GVHD), and preconditioning including irradiation and chemotherapy. Therefore, rapid repair of damaged thymus and rapid proliferation of thymus-derived donor T cells after transplantation are key to solving the problem. This study is designed to accelerate the recovery of thymus-derived T cells after transplantation. Wild-type mice with normal immunity were used as recipients in a haplo-HSCT mouse model to mimic clinical haplo-HSCT. A modified cell culture system using Notch ligand Delta4 and IL-7 was established that is capable of inducing and amplifying the differentiation and proliferation of hematopoietic stem cells into precursor T (preT) cells in vitro. Haplo-HSCT protocol included the preT and G-CSF mobilized donor splenic mononuclear cells (MNC) co-infusion or MNC alone. Thymic GVHD, thymic repair, and thymus-derived T cell development were compared in two groups by polychromatic immunofluorescence tracking, flow cytometry and detection of T cell receptor Vβ. The thymus homing and T-cell regeneration of allogenic preT cells were observed. The functions of preT cells in accelerating immune reconstitution, restoring thymic architecture, weakening GVH effects, and enhancing immuno-tolerance after transplantation were demonstrated. Further studies revealed that allogeneic preT cells induced by a culture system containing IL7 and Delta4 highly express ccr9 and RANKL. Interestingly, the RANK expression was promoted after preT cells' thymus homing. These results suggested that the RANK/RANKL pathway may play an important role in thymus homing. Our results provide a potential therapeutic option to optimize haplo-HSCT. It further opened up a new field of T cell therapy for artificial induction of allogeneic precursor T cells in vitro to repair the damaged thymus from irradiation and chemotherapy, and to compensate for the recovery of immune function in patients with immune deficiency caused by multiple reasons.

Acute complications observed during intensive chemotherapy in pediatric patients with acute lymphoblastic leukemia: Single-center experience.

In childhood acute lymphoblastic leukemia (ALL), very promising results were obtained thanks to the developments in treatment strategies in recent years. However, acute complications during treatment continue to be the important causes of mortality and morbidity. In this study, acute complications that develop during the treatment of ALL in childhood were evaluated. Medical records of 47 patients treated according to (ALL Intercontinental Berlin-Frankfurt-Münster) 2009 protocol between 2016 and 2021 were evaluated retrospectively. Of 47 patients, 28 (59.6%) were male and 19 (40.4%) were female. The mean age at diagnosis was 5.9±4.2 years. Forty-four patients (93.6%) were pre-B cell ALL, 3 patients (6.4%) were pre-T cell ALL. Of 47 patients, 9 (19.1%) were high risk, 32 (68.1%) were intermediate risk, and 6 (12.8%) were standard risk. Acute complications developed in 38 patients (80.8%). Among these complications, infectious complications are the most common and these were followed by gastrointestinal complications, drug-related reactions, thrombotic, neurological, and endocrine/metabolic complications, respectively. In terms of complications that may develop, the threshold of suspicion should be kept low, and patients should be treated with the same medical team in fully equipped centers with a multidisciplinary approach. Inpatient treatment strategies should be applied especially in the early stages of treatment. The importance of inpatient treatment strategy, especially in the early stages of treatment, is emphasized.

Pre-T cell receptor self-MHC sampling restricts thymocyte dedifferentiation.

Programming T cells to distinguish self from non-self is a vital, multi-step process that occurs in the thymus1-4. Signalling through the pre-T cell receptor (preTCR), a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, is a critical early checkpoint in thymocyte development within the αβ T cell lineage5,6. PreTCRs arrayed on CD4-CD8- double-negative thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma, similar to αβ T cell receptors that appear on CD4+CD8+ double-positive thymocytes, but via a different molecular docking strategy7-10. Here we show the consequences of these distinct interactions for thymocyte progression using synchronized fetal thymic progenitor cultures that differ in the presence or absence of pMHC on support stroma, and single-cell transcriptomes at key thymocyte developmental transitions. Although major histocompatibility complex (MHC)-negative stroma fosters αβ T cell differentiation, the absence of preTCR-pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation. Highly proliferative double-negative and double-positive thymocyte subsets emerge, with antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1 MHC-knockout mice partially safeguards in vivo thymocyte progression, although disseminated double-positive thymic tumours may develop with ageing. Thus, as well as promoting β chain repertoire broadening for subsequent αβ T cell receptor utilization, preTCR-pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation and proliferation that, if absent, introduce developmental vulnerabilities.

Evolution of surrogate light chain in tetrapods and the relationship between lengths of CDR H3 and VpreB tails.

In the mammalian immune system, the surrogate light chain (SLC) shapes the antibody repertoire during B cell development by serving as a checkpoint for production of functional heavy chains (HC). Structural studies indicate that tail regions of VpreB contact and cover the third complementarity-determining region of the HC (CDR H3). However, some species, particularly bovines, have CDR H3 regions that may not be compatible with this HC-SLC interaction model. With immense structural and genetic diversity in antibody repertoires across species, we evaluated the genetic origins and sequence features of surrogate light chain components. We examined tetrapod genomes for evidence of conserved gene synteny to determine the evolutionary origin of VpreB1, VpreB2, and IGLL1, as well as VpreB3 and pre-T cell receptor alpha (PTCRA) genes. We found the genes for the SLC components (VpreB1, VpreB2, and IGLL1) only in eutherian mammals. However, genes for PTCRA occurred in all amniote groups and genes for VpreB3 occurred in all tetrapod groups, and these genes were highly conserved. Additionally, we found evidence of a new VpreB gene in non-mammalian tetrapods that is similar to the VpreB2 gene of eutherian mammals, suggesting VpreB2 may have appeared earlier in tetrapod evolution and may be a precursor to traditional VpreB2 genes in higher vertebrates. Among eutherian mammals, sequence conservation between VpreB1 and VpreB2 was low for all groups except rabbits and rodents, where VpreB2 was nearly identical to VpreB1 and did not share conserved synteny with VpreB2 of other species. VpreB2 of rabbits and rodents likely represents a duplicated variant of VpreB1 and is distinct from the VpreB2 of other mammals. Thus, rabbits and rodents have two variants of VpreB1 (VpreB1-1 and VpreB1-2) but no VpreB2. Sequence analysis of VpreB tail regions indicated differences in sequence content, charge, and length; where repertoire data was available, we observed a significant relationship between VpreB2 tail length and maximum DH length. We posit that SLC components co-evolved with immunoglobulin HC to accommodate the repertoire – particularly CDR H3 length and structure, and perhaps highly unusual HC (like ultralong HC of cattle) may bypass this developmental checkpoint altogether.

Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection pre-T cells

Abstract Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2 and regulating phosphorylation of Baf which functions to promote nuclear envelope integrity and suppress cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor controlling the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway driving apoptosis of developing T cells. Supported by grants from NIH R01-GM059638, P01-AI102853, RO1-AI099100, RO1-NS120417, and RO1-AI160737

Monoallelic Heb/Tcf12 Deletion Reduces the Requirement for NOTCH1 Hyperactivation in T-Cell Acute Lymphoblastic Leukemia.

Early T-cell development is precisely controlled by E proteins, that indistinguishably include HEB/TCF12 and E2A/TCF3 transcription factors, together with NOTCH1 and pre-T cell receptor (TCR) signalling. Importantly, perturbations of early T-cell regulatory networks are implicated in leukemogenesis. NOTCH1 gain of function mutations invariably lead to T-cell acute lymphoblastic leukemia (T-ALL), whereas inhibition of E proteins accelerates leukemogenesis. Thus, NOTCH1, pre-TCR, E2A and HEB functions are intertwined, but how these pathways contribute individually or synergistically to leukemogenesis remain to be documented. To directly address these questions, we leveraged Cd3e-deficient mice in which pre-TCR signaling and progression through β-selection is abrogated to dissect and decouple the roles of pre-TCR, NOTCH1, E2A and HEB in SCL/TAL1-induced T-ALL, via the use of Notch1 gain of function transgenic (Notch1ICtg) and Tcf12+/- or Tcf3+/- heterozygote mice. As a result, we now provide evidence that both HEB and E2A restrain cell proliferation at the β-selection checkpoint while the clonal expansion of SCL-LMO1-induced pre-leukemic stem cells in T-ALL is uniquely dependent on Tcf12 gene dosage. At the molecular level, HEB protein levels are decreased via proteasomal degradation at the leukemic stage, pointing to a reversible loss of function mechanism. Moreover, in SCL-LMO1-induced T-ALL, loss of one Tcf12 allele is sufficient to bypass pre-TCR signaling which is required for Notch1 gain of function mutations and for progression to T-ALL. In contrast, Tcf12 monoallelic deletion does not accelerate Notch1IC-induced T-ALL, indicating that Tcf12 and Notch1 operate in the same pathway. Finally, we identify a tumor suppressor gene set downstream of HEB, exhibiting significantly lower expression levels in pediatric T-ALL compared to B-ALL and brain cancer samples, the three most frequent pediatric cancers. In summary, our results indicate a tumor suppressor function of HEB/TCF12 in T-ALL to mitigate cell proliferation controlled by NOTCH1 in pre-leukemic stem cells and prevent NOTCH1-driven progression to T-ALL.

Anti-prion activity of cellulose ether is impaired in mice lacking pre T-cell antigen receptor α, T-cell receptor δ, or lytic granule function

The anti-prion activity of cellulose ether (CE) has been reported in rodents, but the mechanism of action is not well understood. As defects in early T-cell development have been reported in Tga20 mice which show only a slight effect of CE administration, we investigated the involvement of immune functions in the CE action. We confirmed an insertion of the prion protein transgene into the pre T-cell antigen receptor α gene of Tga20 mice, and its impaired expression in the thymus and other tissues. The influence of immune suppression on the CE effect was then examined in high CE-responder mice treated with immunosuppressive agents or neonatal thymectomy. As neonatal thymectomy significantly reduced the CE effect, we compared the influence of various T-cell defects in mice with similar genetic backgrounds. The CE effect was increased or unchanged in mice with defects in the αβ T-cell lineage, whereas it was abolished in T-cell receptor δ deficient mice. Further, when other immune defects were examined, the CE effect was reduced in mice with lysosomal trafficking dysfunction, but was unchanged in mice deficient in B-cell differentiation or toll-like receptor 4 signaling. These findings collectively suggest that the mechanism of CE action may involve γδ T cells and lytic granule function, as well as immune factors like natural killer T cells which are lacking in pre T-cell antigen receptor α deficient mice and neonatally thymectomized mice.

Effectiveness of interleukin-4 administration or zinc supplementation in improving zinc deficiency-associated thymic atrophy and fatty degeneration and in normalizing T cell maturation process.

Nutritional zinc deficiency induces thymic atrophy, but the underlying mechanisms remain unknown. In this study, we investigated the mechanism of thymic atrophy and fatty degeneration associated with zinc deficiency and its effect on T cell maturation. Building on previous research demonstrating the beneficial effect of IL-4 administration or zinc supplementation on the spleen in zinc-deficient rats, we further examined whether these supplements also improve thymic atrophy. Five-week-old male Sprague-Dawley rats were fed a standard diet, zinc-deficient diet (n=16 each) with either saline or IL-4, or a zinc-deficient diet for 6weeks followed by a standard diet for 4weeks. Relative thymus weights, serum thymulin concentrations, and the number of cytokeratin-8-positive cells, AIRE-positive cells, IL-7-positive cells, CD8+ T cells, CD4+ T cells, pre-T cells, and CD25+ CD44+ (DN3) cells in the thymus of zinc-deficient rats significantly decreased compared with those in all other groups. Conversely, PPAR-γ-positive cells, oil red O-positive areas, pro T cells, CD25- CD44+ cells, TUNEL-positive cells, Viobility 405/452 Fixable Dye-positive cells, CD68-, CD163- or CD169- macrophages, and IL-1β concentrations were significantly increased in the thymus of zinc-deficient rats as compared to those in the other groups. After IL-4 administration or zinc supplementation for zinc deficiency, all the measurement indices were recovered to levels in standard rats. It was demonstrated that zinc deficiency caused thymic atrophy, accompanied by fatty degeneration in the cortical regions and affected T cell maturation. IL-4 administration or zinc supplementation for zinc deficiency ameliorated thymic fatty degeneration.

1163. HIV-1 NAAT Pitfalls in B-cell Lymphoblastic Leukemia Patients Following CAR-T Cell Therapy

BackgroundCAR-T Cell Therapy is approved for the treatment of pediatric patients with relapsed/refractory B-ALL. Lentiviral vector technology, highly modified from HIV-1, is used to induce stable, long-term transgene expression by integration into the host genome. This integration may interfere with HIV-1 NAAT producing false-positive results. MethodsA retrospective chart review of Pre-B ALL patients who underwent to CAR T cell therapy with KYMRIAH(tisagenlecleucel) at a single institution between January 2019 and May 2021 to assess for patients whose CAR-T infusion interfered with post-infusion HIV-1 testing. All patients had HIV NAAT pre and post CAR T-cell therapy (using platform, Roche COBAS AmpliPrep/Quantitative TaqMan HIV-1 T). Reactive HIV NAAT by Roche test were subsequently tested using different HIV-1 assay platforms to rule out or confirm HIV infection.ResultsWe report three cases in which interpretation of HIV-1 NAAT testing was complicated by CAR T cell therapy. Case 1: 1-year-old male with refractory infantile leukemia was found to have a reactive HIV NAAT post CAR-T in routine infectious screening prior to SCT. Case 2: 3-year-old refractory ALL planned for SCT who had a reactive HIV NAAT 9 months post CART. Fourth-generation HIV-1 testing (targeting the p24 antigen and anti-HIV-1 antibodies) was negative pre and post CART. Viral loads were also undetectable indicating false-positive post-CAR-T HIV-1 NAT test results. Case 3: 21-year-old sexually active female with relapsed B cell ALL. Post CAR-T HIV NAAT was reactive, and a quantitative viral load was positive at 176 copies/mm3 one day prior to start of stem cell transplant conditioning regimen. Aptima HIV testing is typically used as confirmatory test for CAR-T associated false positive HIV NAT cases, but surprisingly, the Aptima test was also reactive. Additional testing with Abbott m2000 Realtime HIV-1 assay and 4th generation p24 antigen-based testing were both negative. Table 1 Summary of reported patients with false-positive HIV-1 NAAT test results following CAR T cell therapy and Table 2, HIV platforms used for screening and diagnosis with interpretations following CAR T cell therapy.ConclusionClinicians need to be aware of potential false-positive HIV testing after CAR-T therapy. HIV testing platforms with targets not used in lentiviral vectors (4th generation test/P24, or Abbott test/integrase region) are highly recommended to avoid delays in subsequent therapy and unwanted stress for the patients, families, and cliniciansDisclosures All Authors: No reported disclosures

Reference Mapping Pediatric Leukemia Using Single Cell Multiomic Atlas of Pediatric Hematopoiesis

Acute lymphoblastic leukemia is the most common pediatric cancer and leading cause of cancer related mortality in pediatric populations. A key challenge to bridge better therapies to patients that fail conventional therapy are to understand their tumor landscape and aberrations in cell signaling, particularly in relation to normal hematopoietic development.To address this gap, we produced a unified reference map of pediatric T, B, and myeloid cell development from the HSPC using single cell RNA-seq and single-cell ATAC-seq on healthy pediatric bone marrow and thymus. We employed 6 different FACS sorting strategies in order to capture rare, but informative, progenitor cell states, including those of the CCR9+ CD34+ CD1A- CD4- CD8- early-T-cell precursor, CD34+ CD1A- CD4- CD8- pro-T cell, and CD34+ CD1A+ CD4- CD8- pre-T cell and Lin-CD34+CD38- multipotent, lymphoid, and myeloid progenitors from the bone marrow.We mapped leukemic cells from patients from 4 different subtypes of pediatric leukemia (T-ALL, ETP-ALL, MPAL, AML) to our healthy reference and found that our reference map can distinguish between subtle differences in transcriptome and epigenome that were undetectable using surface marker or canonical gene expression. Notably, using trajectories inferred from our healthy reference map, we discovered a large amount of inter-tumoral and intra-tumoral heterogeneity, with leukemic blasts from different patients and different populations within any one patient projecting to different cell states along normal development. Finally, we mapped engrafted leukemic cells from patient derived xenografts (PDX) back to our healthy reference. While we observed patient-specific transcriptomic shifts in engrafted versus primary leukemic blasts, we found that the overall transcriptomic hierarchy is maintained in the most PDX, with engrafted cells projecting to near-identical stages of arrest along our healthy hematopoietic trajectory. Interestingly, for PDX that projected to different areas in development compared to primary sample, we discovered alterations in expression of key transcription factors that regulate hematopoietic development.Our single cell multi-omic reference map of pediatric hematopoiesis serves as a valuable reference for mapping RNA-seq and ATAC-seq data back to nearest healthy precursors in normal hematopoietic development. On-going analysis is utilizing single cell transcriptomic, chromatin accessibility data from additional leukemic patients, including patients with B-ALL, to determined key genes and regulators that are altered in comparison to nearest healthy cell-types. In addition, population level signatures learned from healthy reference are being tested in bulk-transcriptomic ALL datasets. We are eager to present the results of these analyses at ASH.*CC and JX, as well as, DTT and KT contributed equally to this work [Display omitted] DisclosuresTeachey: Janssen: Consultancy; NeoImmune Tech: Research Funding; Sobi: Consultancy; BEAM Therapeutics: Consultancy, Research Funding.

Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β-selection.

Signals from the pre-T cell receptor and Notch coordinately instruct β-selection of CD4-CD8-double negative (DN) thymocytes to generate αβ T cells in the thymus. However, how these signals ensure a high-fidelity proteome and safeguard the clonal diversification of the pre-selection TCR repertoire given the considerable translational activity imposed by β-selection is largely unknown. Here, we identify the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery as a critical proteostasis checkpoint during β-selection. Expression of the SEL1L-HRD1 complex, the most conserved branch of ERAD, is directly regulated by the transcriptional activity of the Notch intracellular domain. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired mouse αβ T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. Accordingly, genetically inactivating PERK rescued T cell development from Sel1l-deficient thymocytes. In contrast, IRE1α/XBP1 pathway was induced as a compensatory adaptation to alleviate Sel1l-deficiency-induced ER stress. Dual loss of Sel1l and Xbp1 markedly exacerbated the thymic defect. Our study reveals a critical developmental signal controlled proteostasis mechanism that enforces T cell development to ensure a healthy adaptive immunity.

A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy versus infected or cancerous cells via T-cell receptor mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T cell receptors (preTCRs) on thymocytes foster development of αβT lymphocytes through their β chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αβTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRβ-pMHC interaction. The linkage model of preTCRβ-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography and single molecule electron microscopy studies.

Pre-T cell receptors topologically sample self-ligands during thymocyte β-selection.

Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre-T cell receptors (preTCRs) and αβTCRs. Using x-ray crystallography, we show how a preTCR applies the concave β-sheet surface of its single variable domain (Vβ) to "horizontally" grab the protruding MHC α2-helix. By contrast, αβTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVβ module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in "vertical" head-to-head binding. The preTCR topological fit ensures that CDR3β reaches the peptide's featured C-terminal segment for pMHC sampling, establishing the subsequent αβTCR canonical docking mode. "Horizontal" docking precludes germline CDR1β- and CDR2β-MHC binding to broaden β-chain repertoire diversification before αβTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.

Specific homeodomain-DNA interactions are required for HOX11-mediated transformation

AbstractHOX11 encodes a homeodomain protein that is aberrantly expressed in T-cell acute lymphoblastic leukemia as a consequence of the t(10;14) and t(7;10) chromosomal translocations. We previously reported that HOX11 immortalizes murine hematopoietic progenitors and induces pre–T-cell tumors in mice after long latency. It has been demonstrated in a number of studies that HOX11, similar to other homeodomain proteins, binds DNA and transactivates transcription. These findings suggest that translocation-activated HOX11 functions as an oncogenic transcription factor. Here we report that HOX11 represses transcription through both TATA-containing and TATA-less promoters. Interestingly, transcriptional repression by HOX11 is independent of its DNA binding capability. Moreover, a systematic mutational analysis indicated that repressor activity was separable from immortalizing function, which requires certain residues within the HOX11 homeodomain that make base-specific or phosphate-backbone contacts with DNA. We further showed that the pathologic action of HOX11 involves DNA binding-dependent transcriptional pathways that are distinct from those controlling expression of a chromosomal target gene (Aldh-1). We conclude that dysregulated expression of a particular set of downstream target genes by DNA binding via the homeodomain is of central importance for leukemia initiation mediated by HOX11.