Immune Aplastic Anemia Research Articles

HLA-lacking clones in aplastic anaemia: Adaptive or maladaptive?

HLA loss represents the result of immune forces shaping bone marrow clonal dynamics in immune aplastic anaemia. Human leukocyte antigen (HLA)-deficient clones may rescue haematopoiesis by evading immune attacks, potentially guiding treatment strategies. Commentary on: Zaimoku etal. Haematopoietic regeneration by HLA-A*0206-deficient clones in severe aplastic anaemia without definitive immunosuppressive treatment. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19712.

Haematopoietic regeneration by HLA-A*0206-deficient clones in severe aplastic anaemia without definitive immunosuppressive treatment.

We describe the case of a 74-year-old man with severe aplastic anaemia who experienced persistent remission attributed to proliferation of HLA allele-deficient clones. Despite an initial worsening of pancytopenia with eltrombopag and ciclosporin treatment, gradual trilineage haematopoietic recovery occurred, with blood counts normalizing over 3 years. Flow cytometry and deep nucleotide sequencing revealed that haematopoiesis was primarily supported by several clones with somatic mutations that inactivated antigen presentation via HLA-A*0206. This suggests that monitoring haematopoietic regeneration by immune escape clones could be an alternative approach for immune aplastic anaemia patients who possess HLA allele-deficient clones and cannot tolerate standard therapy.

A Case of Immune Aplastic Anemia during Combined Treatment with Atezolizumab and Chemotherapy for Non-Small Cell Lung Cancer.

Immune check point inhibitors (ICIs) have durable antitumor effects. However, autoimmune toxicities, termed immune-related adverse events, occur in some patients. We report a case of severe immune aplastic anemia (AA) in a patient with non-small cell lung cancer who was receiving atezolizumab with bevacizumab/carboplatin/paclitaxel. Although the cancer has not recurred, his bone marrow is depleted and he did not respond to immunosuppressive therapy. He has survived for 1.5 years with blood transfusions and infection control. Immune AA associated with ICIs is rare, and a treatment has not yet been established. This case report provides information on the management and treatment response of patients with AA caused by ICIs. Further studies should investigate the mechanism and pathogenesis of immune AA caused by ICIs.

Changes in the Immunogenetic Landscape with the Age of Disease Onset in Acquired Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria: An Analysis of a Cohort of Adult and Pediatric Patients from the French National Reference Center for Aplastic Anemia

Over the past decade, the role of HLA molecules in the pathophysiology of immune aplastic anemia (IAA) has been highlighted by the identification of somatic clonal losses of HLA molecules on leukocytes and the enrichment of certain HLA alleles in patients with AA compared to the general population. More recently, the analysis of the HLA Evolutionary Divergence (HED) score in a large US adult cohort showed that IAA patients were carrying class II HLA molecules whose antigen-binding sites were characterized by a high degree of structural homology and that low class II HED score was associated with a higher risk of malignant progression and poorer survival (Pagliuca et al., Blood, 2021). To evaluate these findings in an independent cohort, we conducted a retrospective study of a large cohort of adult and pediatric patients diagnosed with IAA and/or PNH included in 2 different centers in Paris from the French National Center for Aplastic Anemia (RIME observatory). Patients' 2-field HLA genotyping for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci and HLA-DQA1 genotype imputations were compared with those of a control cohort of 593 allogeneic donors in the context of hematologic malignancies. The MidasHLA R package was used to assess HLA-disease associations using logistic regression analysis with a dominant inheritance model (Benjamini-Hochberg adjusted p-values). HED metrics, used as a surrogate for the width of the immunopeptidome presented by the patient's HLA molecules (Pierini and Lenz, Mol. Biol. Evol., 2018) were calculated for the different HLA loci and for the entire HLA class I and HLA class II genes (mean of HED scores for the corresponding loci). The cohort consisted of 449 patients who were children (<15 years of age, n=130), adolescents and young adults (15-25 years of age, n= 141), and adults (> 25 years of age, n= 178) at the time of diagnosis. Fifty-five percent had IAA, 28% had AA with a PNH clone, 8% had hepatitis-associated AA and 9% had hemolytic PNH. The distribution of diagnoses differed across age groups, with a decreasing incidence of hepatitis-associated AA and an increasing incidence of both AA/PNH and PNH with age. We first confirmed a strong association between patients and the DRB1*15:01, DQB1*06:02 and DQA1*01:02 alleles (these 3 alleles being in strong linkage disequilibrium) in addition to a few additional alleles at the DPB1 and B loci (Figure 1). This finding was consistent with several published studies in populations of different ethnicities. Using stepwise conditional testing, we then showed that only DQB1*06:02 (and DPB1*01:01 to a much smaller extent) remained associated with the disease (OR=2.49, adjusted p-value = 7.55E-09). Interestingly, the DQB1*06:02 association was lost when analyzing the subset of pediatric patients. Among patients, we then demonstrated a strong correlation between the age at disease onset and the presence of the DQB1*06:02 (Figure 2, exact Fisher's test, p=0.0005), DQA1*01:02 (p= 0.006) and DRB1*15:01 alleles (p< 0.0001). Intriguingly, we additionally found an overrepresentation of the ancestral A*01:01 B*08:01 DRB1*03:01 haplotype among patients with primary PNH (14.6%) compared to both patients with AA/PNH (2.4%, p=0.009) and controls (6.3%, p=0.083). Next, we investigated the HED metrics at the different HLA loci. There was no significant decrease in HED scores in patients compared to control, either at the DQB1, DQA1 and DRB1 loci or for the overall HED Class II score (mean value of HED-DRB1, -DQB1 and -DPB1), even in the subsets of DRB1*15:01 or DQB1*06:02 non-carriers. This result differed from the aforementioned US study, likely due to differences in geographic inclusion areas between the 2 cohorts. To rule out bias due to the inclusion of pediatric patients in our cohort, we then focused on the adult-onset subgroup and observed similar results. However, considering the entire cohort and despite no differences between patients and controls, we identified a strong association between age at onset and HED-B and HED-Class I (mean value of HED-A, -B and -C), with the divergence increasing with age (Kendall correlation test, p=0.001 and p=0.007, respectively). Taken together, these results highlight different immunogenetic landscapes according to the age of disease onset. We can hypothesize that the immune-mediated pathophysiological mechanisms may differ with age with a preponderant role of DQB1*06:02 appearing at later age of disease onset.

Single-Cell Dissection Reveals a Distinct Origin of Small Paroxysmal Nocturnal Hemoglobinuria Clones in Immune Aplastic Anemia and Healthy Individuals

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) clones are characterized by the absence of cell surface glycosylphosphatidylinositol (GPI)-anchors owing to somatic mutations in the PIGA gene on the X chromosome. Large PNH clones derived from PIGA mutant multipotent progenitors (MPPs) result in clinically significant hemolysis. In immune aplastic anemia (AA), the PNH clone represents immune escape from T cell-mediated pathogenesis and is usually small (<1%), sometimes likely derived from lineage-committed progenitors (LCPs), with detectable GPI-anchor deficient (PNH-type) cell populations only in granulocytes and monocytes. Small PNH-type cells are also present in healthy individuals at a much lower frequency (<0.003%) than in AA patients. The nature of the small PNH-type cells is not fully understood because of the difficulties in analyzing rare cell populations. Methods In this study, we developed a method to detect PIGA mutations within a small population of cells at single-cell resolution. PNH-type cells were enriched from peripheral blood leukocytes using magnetic microbeads and monoclonal antibodies specific for GPI-anchored proteins. The enriched cell fraction was then subjected to fluorescence-activated cell sorting after staining with lineage-specific markers and fluorescein-labelled proaerolysin. We sorted a limited number (100 cells in males and 50 cells in females) of PNH-type granulocytes (PNH-Gs) and T cells (PNH-Ts) as well as wild-type cells into separate tubes, and their PIGA genes were directly amplified by multiplex PCR without a DNA extraction procedure for subsequent deep nucleotide sequencing. To ensure the specificity of PIGA mutation detection, we set cut-off thresholds of 2% for single-nucleotide substitutions and 1% for indels, in addition to the thresholds based on base-position-specific error rates in 10 sets of 2000 wild-type cells, as previously reported (Zaimoku et al. Blood 2021). Results In 19 sets of 50-100 wild-type cells from 13 individuals, we detected only 7 mutations with variant allele frequencies (VAFs) ranging from 2.1% to 4.2% (Figure 1). Three of these were synonymous mutations, and the remaining 4 were missense mutations, suggesting that these mutations may be true passenger mutations rather than sequencing errors. In all 6 healthy individuals, small PNH-type cells exhibited a polyclonal pattern. We detected numerous small, likely inactivating, PIGA mutations in PNH-Gs, with a median of 11 (range, 6-18) mutations per 100 cells. In 3 individuals assessed twice, approximately half of the PIGA mutations were detected in both sets of PNH-Gs, whereas the remaining mutations were unique to each set. Similarly, PNH-Ts from 4 healthy individuals showed polyclonality, with a median of 14 (range, 6-22) mutations per 100 cells. Interestingly, PIGA mutations were not shared between PNH-Gs and PNH-Ts, except for 2 splice site mutations with VAFs <10%. The polyclonal pattern was further confirmed by Sanger sequencing, which successfully detected 7 unique PIGA mutations in 5 sets of 5 PNH-type cells (instead of 100 cells) in a healthy donor, whereas no mutations were detected in 3 sets of 5 wild-type cells, except for 1 synonymous mutation. In contrast, small PNH-Gs of 5 AA patients, with frequencies of 0.02%, 0.08%, 0.09%, 0.2%, and 0.5% of total granulocytes, were primarily derived from single PIGA mutant clones, which accounted for 90%-99% of PNH-G populations. In another patient with AA, PNH-Gs with a frequency of 3.3% were derived from 2 dominant clones. Notably, the dominant PIGA mutations in the PNH-Gs of AA patients were all detectable in PNH-Ts, even in 2 patients whose PNH-Ts were not detectable (<0.003%) and in 3 patients whose percentages of PNH-Ts were 0.004%, 0.02%, and 0.08% before microbead-based enrichment. Discussion Our novel sequencing approach provides insight into the clonality and lineage distribution of small PNH-type cells without in vitro culture manipulation. In healthy individuals, most PNH clones originate from LCPs, where many cell divisions occur, thereby providing opportunities for the acquisition of PIGA mutations. In immune AA, only PIGA mutations acquired at the MPP level lead to clonal proliferation, strongly suggesting that the immune pathogenesis of AA targets MPPs, rather than LCPs. A clonality analysis of small PNH-type cells thus helps diagnose the immune pathogenesis of AA.

HLA Class I Allele Loss and Bone Marrow Transplantation Outcomes in Immune Aplastic Anemia

In patients with immune-mediated acquired aplastic anemia (AA), HLA class I alleles often disappear from the surface of hematopoietic progenitor cells, potentially enabling evasion from cytotoxic T lymphocyte-mediated pathogenesis. Although HLA class I allele loss has been studied in AA patients treated with immunosuppressive therapy (IST), its impact on allogeneic bone marrow transplantation (BMT) has not been thoroughly investigated. The purpose of this study was to evaluate the clinical implications of HLA class I allele loss in patients with acquired AA undergoing allogeneic BMT. The study enrolled acquired AA patients who underwent initial BMT from unrelated donors through the Japan Marrow Donor Program between 1993 and 2011. The presence of HLA class I allele loss due to loss of heterozygosity (HLA-LOH) was assessed using pretransplantation blood DNA and correlated with clinical data obtained from the Japanese Transplant Registry Unified Management Program. A total of 432 patients with acquired AA were included in the study, and HLA-LOH was detected in 20 of the 178 patients (11%) available for analysis. Patients with HLA-LOH typically presented with more severe AA at diagnosis (P=.017) and underwent BMT earlier (P < .0001) compared to those without HLA-LOH. They also showed a slight but significant recovery in platelet count from the time of diagnosis to BMT (P=.00085). However, HLA-LOH status had no significant effect on survival, engraftment, graft failure, chimerism status, graft-versus-host disease, or other complications following BMT, even when the 20 HLA-LOH+ patients were compared with the 40 propensity score-matched HLA-LOH- patients. Nevertheless, patients lacking HLA-A*02:06 or HLA-B*40:02, the alleles most frequently lost and associated with a better IST response, showed higher survival rates compared to those lacking other alleles, with estimated 5-year overall survival (OS) rates of 100% and 44%, respectively (P=.0042). In addition, in a specific subset of HLA-LOH- patients showing clinical features similar to HLA-LOH+ patients, the HLA-A*02:06 and HLA-B*40:02 allele genotypes correlated with better survival rates compared with other allele genotypes, with estimated 5-year OS rates of 100% and 43%, respectively (P=.0096). However, this genotype correlation did not extend to all patients, suggesting that immunopathogenic mechanisms linked to the loss of certain HLA alleles, rather than the HLA genotypes themselves, influence survival outcomes. The survival benefit associated with the loss of these two alleles was confirmed in a multivariable Cox regression model. The observed correlations between HLA loss and the pretransplantation clinical manifestations and between loss of specific HLA class I alleles and survival outcomes in AA patients may improve patient selection for unrelated BMT and facilitate further investigations into the immune pathophysiology of the disease.

Megakaryocytes Have Immune Characteristics in Murine Immune Bone Marrow Failure

Introduction Megakaryocytes (MKs) have familiar roles in the bone marrow (BM) microenvironment, including platelet production, maintenance of hematopoietic stem and progenitor cells (HSPCs) function, and more recent evidence suggests MK engagement in immune responses. Immune aplastic anemia (AA) is characterized by oligoclonal expansion of T-cells and death of HSPCs leading to bone marrow failure (BMF). As MKs demonstrate niche maintenance and immune effector function, we explored the potential roles of MKs in the development of AA in a murine model of immune BMF. Methods BMF was induced in CByB6F1 mice by intravenous infusion of lymph node (LN) cells from major histocompatibility complex mismatched C57BL/B6 (B6) donor mice at a dose of 30-40x10 6 cells/recipient. No irradiation was given to recipients to avoid extra damage to MKs and CByB6F1 recipient mice were sacrificed at D7, D10, D14, and D17 following LN cell infusion (Chen J, et al. Blood. 2004). Peripheral blood (PB) samples, tibias, femurs, and sterna were harvested at each timepoint. Immunophenotyping of MKs and T cells was performed by flow cytometry on flushed BM cells and two-photon excitation microscopy (2P) imaging was performed on fixed sternal samples, using fluorescence-labelled CD41 antibody to visualize the physical location of MKs in BM. To enrich for MKs, BM cells were filtered through a 10 µm pluriStrainer™ through which most lymphocytes, red blood cells, and other small cells were eluted while MKs and other large size cells remained on the strainer. Large cells and residual small cells were subsequently assessed by single cell RNA sequencing (sc-RNAseq) on the Parse Bio Evercode™ platform based on combinatorial barcoding technology, which was able to accommodate the large size of MKs, as no partitioning step is needed in the analysis workflow. Results BMF was successfully induced as measured by decreasing PB counts from point of lymphocyte infusion accompanied by increasing expansion of CD4 and CD8 T-cells and marked reduction of total BM cells. MKs persisted throughout the course of BMF as measured by both flow cytometry and 2P (Figure 1A) despite PB thrombocytopenia and progressive marrow hypocellularity. Concomitantly, MKs identified by large size on forward scatter versus side scatter and CD41+ expression had progressive upregulation of immune effector markers IA-IE and CD53 over the course of BMF. Following sc-RNAseq and bioinformatics analysis of enriched BM cells at D14, cell clustering identified a distinct cell cluster representing MKs (Figure 1B). MK identity was supported by co-expression of transcripts encoding platelet factor 4, TUBB1, Glycoprotein 5, and Itga2b (CD41). This MK cell population was distinct from macrophages which highly expressed Fcgr4, Cd5l, Ccr5, Cd74 and Gbp5 genes. Gene expression profiling demonstrated that MKs in BMF mice showed upregulation of immune pathways such as interferon gamma and interferon alpha response and downregulation of pathways related to platelet activation and platelet homeostasis, as compared with normal mice. Conclusions In a murine model of BMF, using multiple modalities, we demonstrate that MKs cease platelet production, manifested by reduction in peripheral blood platelet count and downregulation of gene expression pathways related to platelet production, while concurrently acquiring characteristics of immune effector cells. This suggests that MKs are active participants in immune BMF.

Human Adult Long-Term Repopulating Hematopoietic Stem Cells Can be Purified By Surface cMPL Expression

Simple combinations of markers that reliably demarcate human long-term repopulating hematopoietic stem cells (LT-HSCs) would facilitate our understanding of their unique molecular state and the development of stem cell-based therapeutics. The complex CD34+CD38-CD45RA-CD90+CD49f+ phenotype is now routinely accepted to define a subset of cord blood CD34+ cells enriched in LT-HSCs (Notta, Science 2011). The same phenotype with the addition of rhodamine can enrich human LT-HSCs within mobilized peripheral blood (MPB) CD34+ cells, but lower stem cell enrichment is observed (Huntsman, Blood 2015). The hematopoietic cytokine thrombopoietin (TPO) and its receptor cMPL act as primary regulators of HSC function. In recent clinical studies, the synthetic TPO agonist eltrombopag was shown to improve long-term trilineage hematopoiesis in patients with immune aplastic anemia, providing evidence that activation of cMPL receptor stimulates residual multipotent LT-HSCs within the bone marrow (BM). Building on these observations, we hypothesized that cMPL might also be a relevant marker to delineate LT-HSC activity within human adult CD34+ cell populations and provide the basis for a novel LT-HSC purification scheme. To assess the utility of surface cMPL expression for purification of human adult LT-HSCs, we first performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) of human MPB CD34+ cells from a healthy volunteer. CITE-seq was conducted with antibodies designed to react with human cMPL receptor and previously described HSC epitopes (CD34, CD38, CD90, CD45RA, and CD49f). We identified 7 distinct clusters in dimension-reduction (UMAP) analysis. By confirming expression levels of HSPC gene signatures and HSC surface markers, such as CD38, we assigned each CD34+ cell cluster to a distinct HSPC subpopulation, including a single LT-HSC population (cluster 2) (Fig A). Notably, CD34+ cell subsets expressing higher levels of surface cMPL expression were highly enriched in transcriptionally defined HSC population (cluster 2) and consistently co-expressing the known human LT-HSC phenotypic markers, including lower levels of surface CD38 and CD45RA expression and higher levels of surface CD90 and CD49f expression (Fig B). To determine whether surface cMPL expression can enrich functional LT-HSCs, we next performed serial xenotransplantation assays. We used fluorescence-activated cell sorting to partition human MPB CD34+ cells into cMPL high (top 10%) and cMPL low (bottom 10%) populations, and transplanted these cells into immunodeficient NBSGW mice. Serial peripheral blood (PB) sampling of primary xenografted mice revealed a striking difference in patterns of hematopoietic reconstitution over time between the two groups. In mice transplanted with cMPL high cells, human cell chimerism increased gradually throughout the 16-week engraftment period, while a progressive decline in engraftment was observed in the cMPL low group. Notably, mean human cell engraftment within the PB, BM and spleen of mice transplanted with cMPL high cells was 209-fold (p &amp;lt; 0.01), 37-fold (p &amp;lt; 0.0001) and 283-fold (p &amp;lt; 0.001) higher than mean human cell chimerism in the cMPL low group at 16 weeks post-transplantation, respectively. These data suggest that cMPL high and cMPL low populations are distinctly enriched in LT-HSCs and hematopoietic progenitors, respectively. Next, to quantitatively compare the frequency of self-renewing LT-HSCs within the cMPL high and cMPL low HSPC subsets, human CD45+ cells obtained from the BM of primary mice were injected into secondary NBSGW mice at limiting dilution and BM engraftment was measured at 16 weeks post-transplantation (total period of engraftment: 32 weeks). After accounting for engraftment parameters (i.e., cell dose and engraftment levels) in both primary and secondary mice, extreme limiting dilution analysis computed self-renewing LT-HSC frequencies of 1 in 1,267 within the cMPL high CD34+ cell population, and 1 in 149,010 in the cMPL low CD34+ cell fraction, representing a 116-fold enrichment of LT-HSCs using cMPL as a single marker purification strategy. Collectively, these data suggest that cMPL expression can effectively delineate LT-HSCs in human adult CD34+ HSPCs and thus serve as an additional relevant marker for the development of human adult HSC targeting therapeutics.

HLA Class I Genotypes Predict the Survival after Hematopoietic Stem Cell Transplantation in Immune Aplastic Anemia

Introduction Immune aplastic anemia (AA) is a bone marrow failure syndrome mediated by cytotoxic T cells, leading to the loss of HLA class I allele expression in hematopoietic stem and progenitor cells. In unrelated hematopoietic stem cell transplantation (HSCT), we recently reported an association between HLA loss and a short time to transplantation (TTT), consistent with an abrupt onset of immune AA. In addition, patients who lost HLA-A*02:06 or HLA-B*40:02 showed better survival outcomes after HSCT than those with other HLA allele losses. To clarify the clinical significance of the loss of specific HLA class I alleles in AA, we correlated loss-prone HLA class I allele genotypes with HSCT outcomes in patients with a short TTT, assuming the frequent absence of these alleles in this subgroup. Methods We conducted a nationwide retrospective analysis of patients with acquired AA ≥16 years old who underwent their first allogeneic HSCT in Japan between 2006 and 2020. Patient data were obtained from the Transplant Registry Unified Management Program (TRUMP ®), a comprehensive Japanese HSCT registry. Results From 1025 patients with acquired AA, we excluded patients with drug-induced AA and those missing 2-field HLA allele information and TTT information, leaving 874 patients for the analysis. The median patient age at HSCT was 34 years old, and the median TTT was 14 months. When focusing on the most frequently absent HLA class I alleles in Japanese patients with AA, including HLA-B*40:02, HLA-A*02:06, HLA-A*02:01, HLA-B*54:01, and HLA-A*31:01, all 5 alleles were associated with higher survival rates than in 220 patients without these alleles (Figure 1A). As expected, this association was more pronounced with a shorter TTT. For instance, among the 424 patients with a TTT &amp;lt;14 months, the 5-year survival rates were 79%, 78%, 85%, 78%, and 81% in patient groups with the respective HLA class I alleles above, while it was 55% in the other 101 patients (Figure 1B). Of note, having any of the 5 alleles (5-allele +) proved to be the best predictor of a survival out of 242792 combinations of 2 to 5 HLA-A, HLA-B, and HLA-DRB1 alleles. To adjust for potential confounders, such as age and graft sources, we performed propensity score matching and confirmed that the overall survival was significantly lower in patients without any of the 5 alleles (5-allele -) than in 5-allele + patients ( P = .00018). A multivariable Cox regression analysis including age, AA severity, prior IST, graft sources, HLA disparity, and HLA-B leader peptide dimorphism as covariates further demonstrated that the 5-allele genotype was an independent predictor of the survival. The consistent effect of the 5 alleles on the survival has been internally validated across various subgroups. In contrast, the 5 HLA class I alleles did not affect the survival in patients with hematological malignancies, including acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome. Regarding other post-HSCT complications, 5-allele - AA patients had a higher incidence of primary graft failure (GF) than 5-allele + AA patients, even when deaths before engraftment were censored and when compared to the propensity score-matched patients. Nevertheless, the 5 HLA alleles continued to stratify the survival among patients who achieved engraftment ( P &amp;lt; .0001). There were no significant differences in the incidence of acute and chronic graft-versus-host disease, late GF, or cause of death between 5-allele -and 5-allele + AA patients. To further stratify the survival rate in 5-allele - AA patients with a short TTT, we examined the impact of other HLA class I alleles and found that patients carrying HLA-B*07:02 or HLA-B*48:01, in which HLA loss had been previously detected in some AA patients, showed a particularly poor survival (Figure 1C; P = .0082). This association remained significant even after propensity score matching ( P = .031). Conclusions Significant associations between specific loss-prone HLA class I alleles and diverse survival outcomes after HSCT in AA patients with short TTT indicate the relevance of HLA information in patient selection for allogeneic HSCT. These findings also support the existence of a distinct immune pathogenesis affecting the clinical manifestations of AA differently, as observed in IST-treated patients. Further investigations in other ethnic groups are required to validate these observations.

Blocking common γ chain cytokine signaling ameliorates T cell-mediated pathogenesis in disease models.

The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.

Blockade of Common Gamma Chain Cytokine Signaling with REGN7257, an Interleukin 2 Receptor Gamma (IL2RG) Monoclonal Antibody, Protected Mice from Inflammatory and Autoimmune Diseases

Pathogenic immune cell responses in inflammatory and autoimmune diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells, that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. To understand the roles of γc cytokines in driving inflammatory and autoimmune diseases, we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic immune cell responses in mouse models of graft-versus-host disease (GVHD), immune aplastic anemia and multiple sclerosis (MS). We first evaluated the efficacy of REGN7257 in xenogeneic and allogeneic mouse models of GVHD. In the xenogeneic model, immunodeficient NOD-scid-IL2RGnull mice were engrafted with human peripheral blood mononuclear cells (huPBMC). In the allogeneic GVHD model, irradiated BALB/c mice were engrafted with bone marrow cells and splenocytes from Il2rghu/hu mice. In the xenogeneic GVHD mouse model, we also analyzed the effects of γc cytokine signaling blockade with REGN7257 on inflammation and immune cell infiltration into tissues (e.g. liver, bone marrow), as well as liver fibrosis. Finally, we assessed the effects of IL2RG blockade on pathogenic immune cell responses in an experimental autoimmune encephalomyelitis (EAE) model of MS, looking at immune cell infiltration into the central nervous system (CNS) and associated demyelination. In both xenogeneic and allogeneic models of GVHD, prophylactic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Similarly, in the xenogeneic GVHD model, dosing with REGN7257 starting 3 weeks after huPBMC administration effectively protected mice from weight loss and death. Consistent with the classic pathology of GVHD, tissues of control mice were highly infiltrated by immune cells, including CD4+ and CD8+ T-cells, while γc cytokine signaling blockade strongly reduced immune cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling. Finally, in an EAE mouse model of MS, inhibition of γc cytokine signaling with REGN7257 alleviated disease progression and significantly prolonged survival. This was associated with reduced inflammation and infiltration by immune cells (CD4+ T-cells, CD8+ T-cells, B-cells and monocytic cells) in the CNS, as well as decreases in myelin oligodendrocyte glycoprotein-specific antibody production. We showed that REGN7257 treatment efficiently protected the spinal cord from demyelination, which was associated with decreased oligodendrocyte injury and neuron protection. Blockade of γc cytokine signaling with REGN7257 protected mice against immune cell-mediated pathology in multiple disease models: an EAE model of MS, an allogeneic GVHD model, and a xenogeneic GVHD model that uniquely presents hallmarks of both acute and chronic GVHD as well as immune aplastic anemia. These data provide evidence of γc cytokines as key drivers of pathogenic immune cell responses, offering a potentially novel strategy for the management of inflammatory and autoimmune diseases, such as GVHD, immune aplastic anemia and MS.

HLA Class I Allele-Specific Pathology Defines Clinical Manifestations of Immune Aplastic Anemia

Leukocytes that have lost the cell surface expression of an HLA class I allele due to somatic HLA gene mutations or 6p loss of heterozygosity (LOH) are often present in patients with immune aplastic anemia (AA), consistent with T cell-mediated bone marrow cell destruction. A recent NIH study suggested that the clinical significance and underlying pathology of HLA loss may differ according to the type of HLA allele affected (Zaimoku et al. Blood 2021) because (1) the significant correlation of HLA loss with clonal evolution was primarily attributed to patients who lost HLA-B*14:02 and A*02:01; (2) all patients who lost B*40:02 and A*02:06 achieved a hematologic response to immunosuppressive therapy (IST), though HLA loss in general was not correlated with the IST response; (3) loss of B*07:02 and B*40:01 was associated with older age of onset; (4) patients who retained B*14:02, B*07:02, and B*40:01 also showed clinical features of patients who lost respective HLA alleles. We aimed to confirm the HLA class I allele-related clinical manifestations in Japanese AA patients. HLA loss was assessed by SNP array, HLA flow cytometry, digital PCR, or deep nucleotide sequencing. An HLA allele was classified as "affected by loss" in individual patients when the single allele was lost or inactivated by somatic mutations or when both HLA-A and HLA-B alleles in a haplotype were lost due to 6p LOH with no evidence of loss or inactivation of a single HLA allele. Clinical characteristics were compared according to the affected HLA alleles. In 496 Japanese patients with AA (severe, n = 368; non-severe, n = 128; median age, 59 [range, 1-89] years), there were 146 (29%) patients with HLA loss. HLA-B*40:02, A*02:06, A*31:01, and B*54:01 were more frequently affected in our cohort than in the NIH cohort, where B*14:02, A*02:01, B*08:01, and B*07:02 held the majority of lost alleles (Figure 1). The median age of patients with B*40:01 loss (75 years) was highest among the groups of patients with HLA allele loss, consistent with the NIH study. The prevalence of ≥0.003% paroxysmal nocturnal hemoglobinuria (PNH) clones was significantly reduced in patients with B*40:02 loss but not in others. Sex and severity of AA were not markedly different across the groups of patients with HLA allele loss. Outcomes after anti-thymocyte globulin (ATG)-based IST were evaluable in 263 of the 496 patients. Although HLA loss was correlated with the IST response at 6 months (as reported by our group), loss of HLA-A*02:01 did not contribute to the high response, consistent with the NIH study. Loss of frequently affected HLA alleles in Japanese patients (B*40:02, A*02:06, B*54:01, etc.) was generally correlated with the IST-response. Further, patients who carried 5 HLA-B alleles (B*40:01, B*40:02, B*44:03, B*54:01, and B*56:01) without loss still displayed higher response rates than other patients. In contrast, the favorable effect of A*02:06 loss was not seen in patients who carried A*02:06 without loss. A*02:06 loss, the presence of the 5 HLA-B alleles (irrespective of loss), and previously reported HLA class II alleles (DRB1*13 and DRB1*15) were independently correlated with the IST response. The incidence of clonal evolution to myeloid neoplasms after ATG-based IST was significantly higher in patients with HLA-A*02:01 loss than other patients (Figure 2), as observed in the NIH study; A*02:01 was affected in 4 of 7 patients showing both HLA loss and clonal evolution in our cohort; B*40:02, B*44:02, and B*52:01 were affected in other 3 patients. In summary, our study in Japanese AA patients broadly confirmed the associations of loss of specific HLA alleles with clinical manifestations observed in American patients, including older age of onset in patients with HLA-B*40:01 loss, high IST response rates in patients who lost B*40:02 and A*02:06 but not in patients with A*02:01 loss, and frequent clonal evolution in patients with A*02:01 loss. We also revealed the correlation of 5 HLA-B alleles with IST response, irrespective of loss, similar to the relationship of B*14:02 to clonal evolution in the United States, and the reduced prevalence of PNH clones in patients with B*40:02 loss. These findings strongly suggest that the heterogeneous pathology of AA can be classified by affected HLA alleles. This should be useful for studies to unravel the pathophysiology and improve the outcomes of patients with immune AA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK 1/2 Inhibition Preserves Hematopoietic Progenitor and Stem Cells, Prevents Aplasia, Inhibits Pro-Inflammatory Cytokines, and Prolongs Survival in Murine Immune Bone Marrow Failure

Background: Immune aplastic anemia (AA) is clinically characterized by marked pancytopenia due to cytotoxic T-lymphocyte mediated hematopoietic stem and progenitor cell (HSPC) destruction. Though hematopoietic stem cell transplant and immunosuppressive therapies are effective, both are onerous to administer and a fully oral regimen is desirable. Ruxolitinib (RUX) is a Janus Kinase (JAK) 1/2 inhibitor which suppresses cytotoxic T cell activation and inhibits production of inflammatory cytokines; RUX is FDA-approved for myeloproliferative neoplasms and graft versus host disease. Methods: RUX was administered as a previously validated food additive (Rux-chow) in an immune bone marrow failure (BMF) murine model (major histocompatibility complex mismatched C57BL/6 (B6)ÞCByB6F1 lymph node (LN) cell infusion following sublethal total body irradiation (TBI)). Animals were fed Rux-chow starting at two, four, and six days after BMF induction (D+2, D+4, D+6) until day 28; a control group ate chow without RUX for the duration. Survival was assessed for 56 (D+2) or 70 (D+4, D+6) days. Blood counts, T-cells, apoptosis, and gene expression were assessed from peripheral blood, spleen or bone marrow (BM). Inflammatory cytokines implicated in AA including IFN-g, TNF-a, IL-5, IL-1a, CCL2, FasL, CCL5, and IL-10 in the plasma were measured by Luminex. Colony-forming unit (CFU) assays were performed on BM mononuclear cells mixed in semisolid methylcellulose media containing interleukin (IL)-3, IL-6, stem cell factor and erythropoietin with colonies counted at day 10; results were compared with both untreated BMF and TBI controls. Results: All RUX-treated BMF mice (days +2, +4, and +6) achieved long term survival and all untreated BMF mice died (Figure 1A). RUX rescued blood counts in BMF mice, even with delayed treatment, that were sustained with drug discontinuation. Improvement in bodyweight (BW) was seen in Rux-fed mice. Infiltration and activation of BM T-cells were suppressed with a reduction in CD4+ and CD8+ T cells, decrease in CD25+ T-cells, and an increase in splenic FOXP3+ Tregs. Gene expression in RUX treated mice showed downregulation of immune pathways including cytokine signaling in the immune system, the JAK/STAT signaling pathway, and the IFN-g pathway, indicating inhibition of T cell function. RUX therapy preserved HSPCs phenotypically and functionally: hematopoietic progenitors were significantly greater among BM cells from BMF+RUX mice, at 2 weeks and at 8 weeks following BM failure induction, compared to both untreated BMF mice and TBI controls (Figure 1B). Inflammatory cytokines IFN-g, TNF-a, IL-5, CCL-2, CCL-5, FasL, and IL-10 were suppressed on RUX at two weeks but gradually increased when drug was discontinued (except IFN-g which remained at low levels long term). Conclusion: RUX resulted in prolonged overall survival achieved by rescue of BMF as demonstrated by a sustained improvement in peripheral blood counts; this occurred even with delayed administration up to 6 days after BMF induction. RUX alleviated T-cell mediated immune destruction by suppression of T cell infiltration and activation, expansion of Tregs, as well as functional preservation of HSPCs. RUX, an orally administered medication with a favorable toxicity profile, is a potential new therapy for aplastic anemia and related syndromes. A clinical trial using RUX in patients with immune BMF is currently under development. Figures:1A: Survival with delayed RUX therapy. 1B: CFU assays in RUX treated mice, TBI controls, and BMF controls Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Alemtuzumab and Eltrombopag Combination for Patients with Refractory or Relapsed Severe Aplastic Anemia

Background: Immune aplastic anemia (AA) results from cytotoxic T-lymphocyte-mediated destruction of hematopoietic stem cells. Although upfront immunosuppressive therapy (IST) is effective, patients, with relapsed and refractory disease need further treatment with a second round of IST or bone marrow transplantation (BMT). In this retrospective study, we assess the safety and efficacy of alemtuzumab combined with eltrombopag (EPAG) as second-line IST. Methods: Severe AA patients who relapsed after or were refractory to IST (horse anti-thymocyte globulin, cyclosporine and EPAG) were treated with alemtuzumab and EPAG in combination at the Clinical Center between 2017-2022. Alemtuzumab was given as previously reported in SAA: 10 mg/dose/day for 10 days as an intravenous infusion after a 1 mg test dose in adults and in children < 50Kg, 0.2mg/kg/day for 10 days (not to exceed 10 mg/day). EPAG was administered at 150 mg daily in adults and weight based for children as previously reported. Periodic blood counts and bone marrow evaluations were performed to assess for response and progression to myeloid malignancy. Toxicity was assessed based on the previous profile of the two drugs. Results: Six patients were included with a median age of 16 years (range 13- 39 years); half were children. Four of six had very severe disease at SAA presentation and the majority were refractory to initial IST. Two patients with relapsed SAA had achieved a response to IST at 6-month evaluation but then had declining blood counts at 60 and 49 days, respectively. All patients received alemtuzumab as second-line IST (Table 1). EPAG was subsequently added at a median of 60 days from alemtuzumab administration (range 26 days - 199 days) when there was no signs of response or when the counts declined further, .was performed at 3-months and 6-months from EPAG addition. Both patients with relapsed disease achieved a response at 6-month evaluationUPN3 showed early partial response (PR) at 3-months and then ultimately achieved a complete response (CR); at last follow-up, EPAG was being down-titrated with stable blood counts and no signs of evolution to myeloid malignancy. UPN4 achieved a PR at 6 months and at last follow-up maintained stable counts despite discontinuation of EPAG. Of the four refractory patients, only one (UPN1), responded to the addition of EPAG and continued treatment for 2 years. While the EPAG was reduced in dosage, evaluation for declining blood counts with a bone marrow biopsy revealed progression to acute myeloid leukemia, with monosomy 7 and an ASXL1 clone at 24% variant allele frequency. The patient achieved normal blood counts post BMT. Two of the non-responders (UPN2 and UPN5) died from SAA complications; UPN2 died shortly after BMT from refractory infection. UPN6 also had refractory disease; she is 5 months from alemtuzumab and 3 months from EPAG treatment without a response undergoing evaluation for BMT. Patients that responded (UPN1, UPN3, and UPN4) had a median hemoglobin baseline of 7 g/DL (range 6-7.6 g/DL) and increased to a median of 9.3 g/DL (range 8.4-11.6 g/DL), 6 months after EPAG. Responders had a median ANC baseline of 0.69 k/mcL (range 0.46-1.58 k/mcL) and increased to a median of 1.5 k/mcL(range 1.5-2.11). At baseline they also presented with a median platelet of 17 k/mcL at baseline (range 5-18 k/mcL) and increased to 23 k/mcL (range 23-37 k/mcL), 6 month,s after EPAG. None of the patients had toxicity, specifically liver function abnormalities, with the addition of EPAG. The median AST and ALT were 36 U/L (range 20-66 U/L) and 93 U/L (range 13-170 U/L) 6 months after EPAG, respectively. Patients had a median total bilirubin of 0.4 mg/DL (range 0.3-0.9 mg/DL). Conclusion: In absence of BMT, second-line IST is administered for relapsed and refractory SAA patients. The addition of EPAG to alemtuzumab after 1-3 months resulted in responses in relapsed patients but not in refractory cases. The combination was safe without increased toxicities, but prolonged use of EPAG in a single refractory patient associated with development of myeloid malignancy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

UM171 EXPANDS IMMATURE BONE MARROW CD34+ CELLS FROM PATIENTS WITH TELOMEROPATHIES

Telomeropathies, also referred to as telomere biology disorders (TBD), correspond to a spectrum of diseases characterized by genetic defects in the maintenance mechanisms of telomeres and telomerase. Individuals carrying variants in the genes involved in telomeres machinery may have critically short or dysfunctional telomeres, which leads to poor cell regeneration and predisposition to cancer. The commonly affected tissues are those that renew rapidly, such as the bone marrow, lungs, and skin. The most effective therapeutical approach to treat bone marrow failure in patients with TBD is allogeneic bone marrow transplantation, restricted by the low availability of compatible donors and complications of the conditioning regimen. Fares et al. (2013) demonstrated that the small molecule UM171 expands the CD34 + cell subpopulations from cord blood with superior engraftment potential. We demonstrated that UM171 expands primitive CD34 + cells from patients with immune aplastic anemia. The expanded hematopoietic progenitors showed no observable chromosomal, genetic, or telomeric changes. Here we assessed the potential of the HSC agonist UM171 to expand the HSPC compartment of patients with telomeropathies. Bone marrow aspirate samples were collected from five patients, and CD34 + cells were enriched using immunomagnetic labeling with human CD34 MicroBeads and a magnetic separator. Cells were cultured for 7 days in ACF medium supplemented with cytokines that support expansion of HSPC and UM171 or DMSO (negative control). To assess the capacity of the cells cultured either with DMSO or UM171 to generate hematopoietic progenitors, 1,000 cells/mL were resuspended in Methocult and seeded onto 35 mm dishes in triplicate. After 14 days, the colonies were counted and classified according to their morphology. After a 7-day expansion, the percentage of CD34 + cells was higher with UM171 in comparison to control (UM171, 48 ±5.1% vs. DMSO, 25.3% ±6.2% [mean ±standard error] n = 5; p = 0.003). The cell surface EPCR is a reliable marker for the purification of the HSPC compartment. Therefore, we evaluated the CD34 + EPCR + subpopulation, which was increased after the UM171 treatment (UM171, 14.4 ±4.1% vs. DMSO, 1.8% ± 0.6%; n = 5; p = 0.03). Additionally, cells expanded with UM171 gave rise to more progenitor cells, as observed by the CFU assay (UM171, 142 ± 18 vs. DMSO, 94% ± 9; n = 4; p = 0.01). No significant telomere attrition was observed with the expansion. Bone marrow failure is a common manifestation in patients with telomere disorders since the telomerase complex is essential for maintaining the self-renewal capacity of the HSPC pool. Our data suggest the possible future use of UM171 to enhance ex vivo production of HSPC from TBD patients, making it feasible to use these cells in autologous transplants. These findings should improve our understanding of the effect of UM171 on these cells and help to develop protocols for scaling up its production for future therapies. Our studies reveal UM171 to be a potent molecule to expand HSPC in patients with telomeropathies. Further experiments are required to confirm these results besides the assessment of the engraftment potential of the expanded cells in a murine model.

Anti-COX-2 autoantibody is a novel biomarker of immune aplastic anemia

In immune aplastic anemia (IAA), severe pancytopenia results from the immune-mediated destruction of hematopoietic stem cells. Several autoantibodies have been reported, but no clinically applicable autoantibody tests are available for IAA. We screened autoantibodies using a microarray containing >9000 proteins and validated the findings in a large international cohort of IAA patients (n = 405) and controls (n = 815). We identified a novel autoantibody that binds to the C-terminal end of cyclooxygenase 2 (COX-2, aCOX-2 Ab). In total, 37% of all adult IAA patients tested positive for aCOX-2 Ab, while only 1.7% of the controls were aCOX-2 Ab positive. Sporadic non-IAA aCOX-2 Ab positive cases were observed among patients with related bone marrow failure diseases, multiple sclerosis, and type I diabetes, whereas no aCOX-2 Ab seropositivity was detected in the healthy controls, in patients with non-autoinflammatory diseases or rheumatoid arthritis. In IAA, anti-COX-2 Ab positivity correlated with age and the HLA-DRB1*15:01 genotype. 83% of the >40 years old IAA patients with HLA-DRB1*15:01 were anti-COX-2 Ab positive, indicating an excellent sensitivity in this group. aCOX-2 Ab positive IAA patients also presented lower platelet counts. Our results suggest that aCOX-2 Ab defines a distinct subgroup of IAA and may serve as a valuable disease biomarker.

Granulocytic myeloid-derived suppressor cells to prevent and treat murine immune-mediated bone marrow failure

AbstractMyeloid-derived suppressor cells (MDSCs) are immature myeloid cells that originate in the bone marrow (BM) and have immunoregulatory functions. MDSCs have been implicated in the pathogenesis of several autoimmune diseases but have not been investigated in immune aplastic anemia (AA). We examined the roles of granulocytic-MDSCs (G-MDSCs) in murine models of human AA and BM failure (BMF). As both prophylaxis and therapy, BM-derived G-MDSCs improved pancytopenia and BM cellularity and suppressed BM T-cell infiltration in major histocompatibility complex (MHC)-matched C.B10 BMF mice. These effects were not obtained in the MHC-mismatched CByB6F1 AA model, likely because of MHC disparity between G-MDSCs and donor T cells. Single-cell RNA sequencing demonstrated that G-MDSCs downregulated cell cycle–related genes in BM-infiltrated T cells, consistent with suppression of T-cell proliferation by G-MDSCs through reactive oxygen species pathways. Clearance of G-MDSCs in the MHC-mismatched CByB6F1 model using anti-Ly6G antibody facilitated T cell–mediated BM destruction, suggesting an intrinsic immunosuppressive property of G-MDSCs. However, the same anti-Ly6G antibody in the MHC-matched C.B10 AA model mildly mitigated BMF, associated with expansion of an intermediate Ly6G population. Our results demonstrate that G-MDSC eradication and therapeutic efficacy are immune context-dependent.

Clonality in immune aplastic anemia: Mechanisms of immune escape or malignant transformation

Aplastic anemia (AA) is the prototypic bone marrow failure syndrome and can be classified as either acquired or inherited. Inherited forms are due to the effects of germline mutations, while acquired AA is suspected to result from cytotoxic T-cell mediated immune attack on hematopoietic stem and progenitor cells. Once thought to be a purely “benign” condition, clonality in the form of chromosomal abnormalities and single nucleotide variants is now well recognized in AA. Mechanisms underpinning this clonality likely relate to selection of clones that allow immune evasion or increased cell survival the marrow environment under immune attack. Widespread use and availability of next generation and other genetic sequencing techniques has enabled us to better understand the genomic landscape of aplastic anemia. This review focuses on the current concepts associated with clonality, in particular somatic mutations and their impact on diagnosis and clinical outcomes in immune aplastic anemia.

S163: EFFICACY OF JAK 1/2 INHIBITION IN MURINE IMMUNE BONE MARROW FAILURE

Background: Immune aplastic anemia (AA) is a severe blood disorder characterized by cytotoxic T-lymphocyte mediated stem cell destruction. Therapies including hematopoietic stem cell transplant and immunosuppression are effective but entail costs and risks, and are not effective or possible in all patients. The Janus Kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T cell activation and inhibits production of interferon gamma and tumor necrosis factor alpha in models of graft-versus-host disease. Aims: Assess RUX in murine immune AA for potential therapeutic benefit. Methods: In a murine major histocompatibility complex mismatched C67BL/6(B6) to CByB6F1 lymph node (LN) cell infusion AA model, and a C.B10 minor histocompatibility antigen mismatched B6 to C.B10 LN cell infusion AA model, RUX was administered as a food additive (Rux-chow), which achieves therapeutic levels in 48-72 hours after administration. Control BMF mice received chow without RUX. Animals were fed with Rux-chow two days before LN cell infusion as a prophylaxis (BMF+RUXD-2), or two days after LN cell infusion as therapy (BMF+RUXD+2). Recipient mice were either bled and euthanized at day 14 following LN infusion to collect tissues or were kept for 56 days to record animal survival. Blood counts were measured biweekly. Samples for flow cytometry, histology, and gene expression assays were collected. Results: In both AA murine models, RUX attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, and prevented mortality when used either prophylactically or therapeutically. Treated mice had significantly higher blood counts (neutrophils, red blood cells, hemoglobin, platelets) as well as bone marrow (BM) and RBM (residual BM, excluding T-cells), and cellularity relative to control BMF mice. RUX suppressed infiltration, proliferation and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. All mice who received RUX were alive at 56 days while control BMF mice all died (Figure 1). With the exception of two mice with low RBC at day 56, discontinuing Rux-chow at day 28 or day 42 did not affect animal blood counts measurements, nor survival. Gene expression in mice who received RUX revealed downregulated T cell function and JAK/STAT pathway-related genes (Stat1, Stat3, Stat4, Fas, Ly6a, Infg, Gzmb, Gzma, Gzmk, Infgr1, Il2rb, Il2rg, and Lag3). On network analysis of differentially expressed genes in downregulated pathways, Stat1 and Ifn-g genes were at the center of the network, connecting immune responses and cell cycle pathways. When toxicity was assessed, RUX exerted modest suppression of lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice, but the drug showed impressive clinical efficacy despite this. Image:Summary/Conclusion: RUX showed striking therapeutic efficacy, improving blood counts and prolonging survival in two different BMF murine models. In patients, clonal T cell expansion and activation, IFN-g and TNF-α upregulation, and Fas mediated cell death, lead to severe BM destruction and the development of AA. Our study demonstrated that JAK 1/2 inhibition with RUX produced its suppressive effects by inhibiting T cell activation, reducing inflammatory cytokine secretion, and limiting FasL/Fas-mediated BM destruction. Given these results, JAK 1/2 inhibition with RUX is an exciting and novel potential therapy for BMF patients based on a well characterized mechanism of action.

P800: BLOCKADE OF COMMON GAMMA CHAIN CYTOKINE SIGNALING WITH REGN7257, AN INTERLEUKIN 2 RECEPTOR GAMMA (IL2RG) MONOCLONAL ANTIBODY, PROTECTED MICE FROM GRAFT-VERSUS-HOST DISEASE AND IMMUNE APLASTIC ANEMIA

Background: Pathogenic T-cell responses in T cell-mediated diseases can be driven by cytokines of the common gamma chain (γc) cytokine family (IL2, IL4, IL7, IL9, IL15, and IL21). γc cytokines signal through their corresponding receptors, expressed primarily on immune cells (including T cells), that share a common coreceptor, interleukin 2 receptor subunit gamma (IL2RG) that is required for signaling. Aims: To understand the roles of γc cytokines in driving graft-versus-host disease (GVHD) and bone marrow failure disorders such as immune aplastic anemia (AA), we generated REGN7257, a fully human IL2RG monoclonal antibody that inhibits γc cytokine-induced signaling, and we tested its ability to suppress pathogenic T-cell responses in mouse models of T cell-mediated disease. Methods: We evaluated the efficacy of REGN7257 in a xenogeneic mouse model of GVHD, where immunodeficient NOD-scid-IL2RGnull mice were engrafted with human peripheral blood mononuclear cells (huPBMC). Mice were treated with REGN7257 either prophylactically or therapeutically, and monitored for weight loss and survival, peripheral human T-cell engraftment as well as serum pro-inflammatory cytokine levels over time. In a separate experiment, mice were sacrificed at day 49 post-huPBMC injection (after 4 weeks of antibody treatment) for tissue analysis (liver, lung, skin and bone marrow) of immune cell infiltration (T cells and macrophages), inflammation and/or fibrosis. Results: In a xenogeneic model of GVHD, both prophylactic and therapeutic γc cytokine signaling blockade with REGN7257 effectively protected mice from weight loss and resulted in improved survival, by reducing T-cell expansion in blood and production of pro-inflammatory cytokines in serum. Consistent with the classic pathology of GVHD, lungs, liver and skin of control mice were highly infiltrated by T cells, while γc cytokine signaling blockade strongly reduced T-cell infiltration, with reduced tissue levels of pro-inflammatory cytokines. Importantly, therapeutic γc cytokine signaling blockade in established GVHD (i.e. when mice already showed weight loss) provided similar benefit. Blockade of γc cytokine signaling also led to a reduction in the severity of chronic GVHD, with decreased macrophage infiltration in liver and associated hepatic fibrosis. In this xenogeneic model of GVHD, hemoglobin levels and platelet numbers in blood were both reduced, indicating anemia and thrombocytopenia, respectively, which are two complications associated with aplastic anemia. In addition to peripheral pancytopenia, recipient mice that were engrafted with huPBMC also showed severe marrow aplasia. Importantly, this phenotype of aplastic anemia was prevented by blockade of γc cytokine signaling. Summary/Conclusion: Blockade of γc cytokine signaling with REGN7257 protected against T cell-mediated pathology in a xenogeneic GVHD mouse model that uniquely presents hallmarks of both acute and chronic GVHD, with T-cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. These data provide evidence of γc cytokines as key drivers of T cell-mediated responses, offering a potentially novel strategy for the management of T cell-mediated diseases, such as GVHD and immune AA.