CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

IL-33 dysregulates regulatory T cells and impairs established immunologic tolerance in the lungs.

BACKGROUND: Airway exposure to environmental antigens generally leads to immunologic tolerance. A fundamental question remains: Why is airway tolerance compromised in patients with allergic airway diseases? IL-33 promotes innate and adaptive type 2 immunity and might provide the answer to this question. OBJECTIVE: The goal of this study was to investigate the roles played by IL-33 in altering regulatory T (Treg) cells in the lungs and in affecting previously established airway immunologic tolerance. METHODS: We analyzed CD4+ forkhead box P3 (Foxp3)+ Treg cells that were isolated from the lungs of naive BALB/c mice and those treated with IL-33. Airway tolerance and allergen-induced airway inflammation models in mice were used to investigate how IL-33 affects established immunologic tolerance in vivo. RESULTS: CD4+Foxp3+ Treg cells in the lungs expressed the IL-33 receptor ST2. When exposed to IL-33, Treg cells upregulated their expression of the canonical TH2 transcription factor GATA3, as well as ST2, and produced type 2 cytokines. Treg cells lost their ability to suppress effector T cells in the presence of IL-33. Airway administration of IL-33 with an antigen impaired immunologic tolerance in the lungs that had been established by prior exposure to the antigen. Dysregulated Foxp3+ Treg cells with distinct characteristics of TH2 cells increased in the lungs of mice undergoing IL-33-dependent allergen-driven airway inflammation. CONCLUSIONS: IL-33 dysregulated lung Treg cells and impaired immunologic tolerance to inhaled antigens. Established airway tolerance might not be sustained in the presence of an innate immunologic stimulus, such as IL-33.

T Cell Adolescence: Maturation Events Beyond Positive Selection.

Single-positive thymocytes that successfully complete positive and negative selection must still undergo one final step, generally termed T cell maturation, before they gain functional competency and enter the long-lived T cell pool. Maturation initiates after positive selection in single-positive thymocytes and continues in the periphery in recent thymic emigrants, before these newly produced T cells gain functional competency and are ready to participate in the immune response as peripheral naive T cells. Recent work using genetically altered mice demonstrates that T cell maturation is not a single process, but a series of steps that occur independently and sequentially after positive selection. This review focuses on the changes that occur during T cell maturation, as well as the molecules and pathways that are critical at each step.

Histone Deacetylase 3 Is Required for T Cell Maturation.

Recent thymic emigrants are newly generated T cells that need to undergo postthymic maturation to gain functional competency and enter the long-lived naive T cell pool. The mechanism of T cell maturation remains incompletely understood. Previously, we demonstrated that the transcriptional repressor NKAP is required for T cell maturation. Because NKAP associates with histone deacetylase 3 (HDAC3), we examined whether HDAC3 is also required for T cell maturation. Although thymic populations are similar in CD4-cre HDAC3 conditional knockout mice compared with wild-type mice, the peripheral numbers of CD4(+) and CD8(+) T cells are dramatically decreased. In the periphery, the majority of HDAC3-deficient naive T cells are recent thymic emigrants, indicating a block in T cell maturation. CD55 upregulation during T cell maturation is substantially decreased in HDAC3-deficient T cells. Consistent with a block in functional maturation, HDAC3-deficient peripheral T cells have a defect in TNF licensing after TCR/CD28 stimulation. CD4-cre HDAC3 conditional knockout mice do not have a defect in intrathymic migration, thymic egress, T cell survival, or homeostasis. In the periphery, similar to immature NKAP-deficient peripheral T cells, HDAC3-deficient peripheral T cells were bound by IgM and complement proteins, leading to the elimination of these cells. In addition, HDAC3-deficient T cells display decreases in the sialic acid modifications on the cell surface that recruit natural IgM to initiate the classical complement pathway. Therefore, HDAC3 is required for T cell maturation.

Immature recent thymic emigrants are eliminated by complement.

Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature naive T cells. In CD4-cre NKAP conditional knockout mice, NKAP-deficient RTEs fail to complete T cell maturation. In this study, we demonstrate that NKAP-deficient immature RTEs do not undergo apoptosis, but are eliminated by complement. C3, C4, and C1q are bound to NKAP-deficient peripheral T cells, demonstrating activation of the classical arm of the complement pathway. As thymocytes mature and exit to the periphery, they increase sialic acid incorporation into cell surface glycans. This is essential to peripheral lymphocyte survival, as stripping sialic acid with neuraminidase leads to the binding of natural IgM and complement fixation. NKAP-deficient T cells have a defect in sialylation on cell surface glycans, leading to IgM recruitment. We demonstrate that the defect in sialylation is due to aberrant α2,8-linked sialylation, and the expression of three genes (ST8sia1, ST8sia4, and ST8sia6) that mediate α2,8 sialylation are downregulated in NKAP-defcient RTEs. The maturation of peripheral NKAP-deficient T cells is partially rescued in a C3-deficient environment. Thus, sialylation during T cell maturation is critical to protect immature RTEs from complement in the periphery.

An RNA-Based Vaccine Platform for Use against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M.tb), a bacterial pathogen that causes tuberculosis disease (TB), exerts an extensive burden on global health. The complex nature of M.tb, coupled with different TB disease stages, has made identifying immune correlates of protection challenging and subsequently slowing vaccine candidate progress. In this work, we leveraged two delivery platforms as prophylactic vaccines to assess immunity and subsequent efficacy against low-dose and ultra-low-dose aerosol challenges with M.tb H37Rv in C57BL/6 mice. Our second-generation TB vaccine candidate ID91 was produced as a fusion protein formulated with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion) or as a novel replicating-RNA (repRNA) formulated in a nanostructured lipid carrier. Protein subunit- and RNA-based vaccines preferentially elicit cellular immune responses to different ID91 epitopes. In a single prophylactic immunization screen, both platforms reduced pulmonary bacterial burden compared to the controls. Excitingly, in prime-boost strategies, the groups that received heterologous RNA-prime, protein-boost or combination immunizations demonstrated the greatest reduction in bacterial burden and a unique humoral and cellular immune response profile. These data are the first to report that repRNA platforms are a viable system for TB vaccines and should be pursued with high-priority M.tb antigens containing CD4+ and CD8+ T-cell epitopes.

The impact of Down syndrome-specific non-malignant hematopoietic regeneration in the bone marrow on the detection of leukemic measurable residual disease.

BACKGROUND: Detection of measurable residual disease detection (MRD) by flow cytometry after the first course of chemotherapy is a standard measure of early response in patients with acute myeloid leukemia (AML). Myeloid leukemia associated with Down Syndrome (ML-DS) is a distinct form of AML. Differences in steady-state and regenerating hematopoiesis between patients with or without DS are not well understood. This understanding is essential to accurately determine the presence of residual leukemia in patients with ML-DS. METHODS: A standardized antibody panel defined quantitative antigen expression in 115 follow-up bone marrow (BM) aspirates from 45 patients following chemotherapy for ML-DS or DS precursor B-cell acute lymphoblastic leukemia (B-ALL-DS) with the "difference from normal (ΔN)" technique. When possible, FISH and SNP/CGH microarray studies were performed on sorted cell fractions. RESULTS: 93% of BM specimens submitted post chemotherapy had a clearly identifiable CD34+ CD56+ population present between 0.06% and 2.6% of total non-erythroid cells. An overlapping CD34+ HLA-DRheterogeneous population was observed among 92% of patients at a lower frequency (0.04%-0.8% of total non-erythroid cells). In B-ALL-DS patients, the same CD34+ CD56+ HLA-DRheterogeneous expression was observed. FACS-FISH/Array studies demonstrated no residual genetic clones in the DS-specific myeloid progenitor cells. CONCLUSIONS: Non-malignant myeloid progenitors in the regenerating BM of patients who have undergone chemotherapy for either ML-DS or B-ALL-DS express an immunophenotype that is different from normal BM of non-DS patients. Awareness of this DS-specific non-malignant myeloid progenitor is essential to the interpretation of MRD by flow cytometry in patients with ML-DS.

Morphologic remission status is limited compared to ΔN flow cytometry: a Children's Oncology Group AAML0531 report.

Risk stratification for acute myeloid leukemia (AML) uses molecular and cytogenetic abnormalities identified at diagnosis. Response to therapy informs risk, and morphology continues to be used more frequently than flow cytometry. Herein, the largest cohort of pediatric patients prospectively assessed for measurable residual disease (MRD) by flow cytometry (N = 784) is reported. The "difference from normal" (ΔN) technique was applied: 31% of all patients tested positive (AML range, 0.02% to 91%) after the first course of treatment on Children's Oncology Group study AAML0531. Detection of MRD following initial chemotherapy proved the strongest predicator of overall survival (OS) in univariable and multivariable analyses, and was predictive of relapse risk, disease-free survival, and treatment-related mortality. Clearance of MRD after a second round of chemotherapy did not improve survival. The morphologic definition of persistent disease (>15% AML) failed 27% of the time; those identified as MRD- had superior outcomes. Similarly, for patients not achieving morphologic remission (>5% blasts), 36% of patients were MRD- and had favorable outcomes compared with those who were MRD+ (P < .001); hence an increase in myeloid progenitor cells can be favorable when ΔN classifies them as phenotypically normal. Furthermore, ΔN reclassified 20% of patients in morphologic remission as having detectable MRD with comparable poor outcomes. Retrospective analysis using the relapse phenotype as a template demonstrated that 96% of MRD- patients had <0.02% of the relapse immunophenotype in their end of induction 1 marrow. Thus, the detection of abnormal myeloid progenitor cells by ΔN is both specific and sensitive, with a high predictive signal identifiable early in treatment. This trial was registered at www.clinicaltrials.gov as #NCT00372593.

Heterologous Immunization With Defined RNA and Subunit Vaccines Enhances T Cell Responses That Protect Against Leishmania donovani.

The rapid generation of strong T cell responses is highly desirable and viral vectors can have potent CD8+ T cell-inducing activity. Immunity to leishmaniasis requires selective T cell responses, with immunization schemes that raise either CD4 or CD8 T cell responses being protective in small animal models. We have defined the leishmaniasis vaccine candidate recombinant fusion antigens, LEISH-F2 and LEISH-F3+, that when formulated in a stable emulsion with a Toll-like receptor (TLR) 4 agonist, induce protective CD4+ T cell responses in animal models as well as providing therapeutic efficacy in canine leishmaniasis and in clinical trials in leishmaniasis patients. We used the genetic sequences of these validated vaccine antigens to design RNA vaccine constructs. Immunization of mice with the RNA replicons induced potent, local innate responses that were surprisingly independent of TLR7 and activated antigen-presenting cells (APC) to prime for extremely potent antigen-specific T helper 1 type responses upon heterologous boosting with either of the subunit vaccines (recombinant antigen with second generation glucopyranosyl lipid A in stable oil-in-water emulsion; SLA-SE). Inclusion of RNA in the immunization schedule also generated MHCI-restricted T cell responses. Immunization with LEISH-F2-expressing RNA vaccine followed later by subunit vaccine afforded protection against challenge with Leishmania donovani. Together, these data indicate the utility of heterologous prime-boost immunization schemes for the induction of potent antigen-specific CD4 and CD8 T cell responses for protection against intracellular pathogens.

Improved Immune Responses in Young and Aged Mice with Adjuvanted Vaccines against H1N1 Influenza Infection.

Elderly people are at high risk for influenza-related morbidity and mortality due to progressive immunosenescence. While toll-like receptor (TLR) agonist containing adjuvants, and other adjuvants, have been shown to enhance influenza vaccine-induced protective responses, the mechanisms underlying how these adjuvanted vaccines could benefit the elderly remain elusive. Here, we show that a split H1N1 influenza vaccine (sH1N1) combined with a TLR4 agonist, glucopyranosyl lipid adjuvant formulated in a stable oil-in-water emulsion (GLA-SE), boosts IgG2c:IgG1 ratios, enhances hemagglutination inhibition (HAI) titers, and increases protection in aged mice. We find that all adjuvanted sH1N1 vaccines tested were able to protect both young and aged mice from lethal A/H1N1/California/4/2009 virus challenge after two immunizations compared to vaccine alone. We show that GLA-SE combined with sH1N1, however, also provides enhanced protection from morbidity in aged mice given one immunization (based on change in weight percentage). While the GLA-SE-adjuvanted sH1N1 vaccine promotes the generation of cytokine-producing T helper 1 cells, germinal center B cells, and long-lived bone marrow plasma cells in young mice, these responses were muted in aged mice. Differential in vitro responses, dependent on age, were also observed from mouse-derived bone marrow-derived dendritic cells and lung homogenates following stimulation with adjuvants, including GLA-SE. Besides enhanced HAI titers, additional protective factors elicited with sH1N1 + GLA-SE in young mice were observed, including (a) rapid reduction of viral titers in the lung, (b) prevention of excessive lung inflammation, and (c) homeostatic maintenance of alveolar macrophages (AMs) following H1N1 infection. Collectively, our results provide insight into mechanisms of adjuvant-mediated immune protection in the young and elderly.

The science of vaccine adjuvants: advances in TLR4 ligand adjuvants.

TLR ligands are used in modern vaccine adjuvants, TLR4 ligand-based adjuvants are the most advanced in commercial vaccines. Increased understanding of TLR4 receptor-ligand interactions enables chemical synthesis and modification of new leads and our understanding of the biological/immunological mechanisms of combination adjuvants enables formulation of potent and safe vaccine compositions. Characterization of non-glycolipid TLR4 ligands provided new mechanistic information that could lead to new formulations. This review discusses advances in TLR4 agonist design-both glycolipid and non-glycolipid based TLR4 ligands-as well as CD14 activation as options to activate or synergize with TLR4 signaling. Finally, we review the molecular and cellular mechanisms that are elicited by formulated TLR4 targeted combination adjuvants during the initiation of innate immune responses leading to quality adaptive responses.

An Essential Role for the Transcription Factor Runx1 in T Cell Maturation.

The transcription factor Runx1 has essential roles throughout hematopoiesis. Here, we demonstrate that Runx1 is critical for T cell maturation. Peripheral naïve CD4(+) T cells from CD4-cre Runx1 cKO mice are phenotypically and functionally immature as shown by decreased production of TNF-α upon TCR stimulation. The loss of peripheral CD4(+) T cells in CD4-cre Runx1 cKO mice is not due to defects in homeostasis or decreased expression of IL-7Rα, as transgenic expression of IL-7Rα does not rescue the loss of CD4(+) T cells. Rather, immature Runx1-deficient CD4(+) T cells are eliminated in the periphery by the activation and fixation of the classical complement pathway. In the thymus, there is a severe block in all aspects of intrathymic T cell maturation, although both positive and negative selection are unaltered. Thus, loss of Runx1 leads to the earliest characterized block in post-positive selection intrathymic maturation of CD4 T cells.

Cell extrinsic alterations in splenic B cell maturation in Flt3-ligand knockout mice.

B lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3-ligand (FL-/-) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL-/- mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL-/- mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF-R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL-/- mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL-/- mice. A Bcl2 transgene did not rescue TS cells in FL-/- mice, uncoupling FL-deficiency to Bcl2-dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL-/- mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.

NKAP is required for T cell maturation and acquisition of functional competency.

Newly generated T cells are unable to respond to antigen/MHC. Rather, post-selection single-positive thymocytes must undergo T cell maturation to gain functional competency and enter the long-lived naive peripheral T cell pool. This process is poorly understood, as no gene specifically required for T cell maturation has been identified. Here, we demonstrate that loss of the transcriptional repressor NKAP results in a complete block in T cell maturation. In CD4-cre NKAP conditional knockout mice, thymic development including positive selection occurs normally, but there is a cell-intrinsic defect in the peripheral T cell pool. All peripheral naive CD4-cre NKAP conditional knockout T cells were found to be functionally immature recent thymic emigrants. This defect is not simply in cell survival, as the T cell maturation defect was not rescued by a Bcl-2 transgene. Thus, NKAP is required for T cell maturation and the acquisition of functional competency.