Immune Destruction Research Articles

Abstract A030: Blood-based screening panel for lung cancer based on clonal hematopoietic mutations in tumor infiltrating immune cells

Abstract Early detection can significantly reduce mortality due to lung cancer. However, the high cost of the currently approved screening protocol has limited its uptake. Presented here is a blood-based screening panel based on clonal hematopoietic mutations. Mutations in tumor cells that inhibit immune destruction have been extensively studied. However, mutations in immune cells that may prevent an effective anti-tumor immune response remain relatively unstudied. Animal model studies suggest that clonal hematopoietic mutations in tumor infiltrating immune cells can modulate cancer progression, representing potential predictive biomarkers. The goal of this study was to determine if the clonal expansion of these mutations in blood samples could predict the occurrence of lung cancer. We identified a set of 98 potentially pathogenic clonal hematopoietic mutations in tumor infiltrating immune cells. A logistic regression machine learning model based on these mutations correctly classified lung cancer and non-cancer blood samples with 94.12% sensitivity (95% Confidence Interval: 92.20-96.04%) and 85.96% specificity (95% Confidence Interval: 82.98-88.95%) in a test set of 578 lung cancer and 545 non-cancer samples from 18 different cohorts. More importantly, 89.98% of the cancer cases were unambiguously predicted with probability of cancer >0.90 and 74.86% of the non-cancer cases with probability of cancer <0.10. These results suggest that it may be possible to develop an accurate blood-based lung cancer screening panel. Unlike most other “liquid biopsies” currently under development, the assay presented here is based on standard sequencing protocols and uses a relatively small number of rationally selected mutations as predictors. Citation Format: Ramu Anandakrishnan, Ryan Shahidi, Andrew Dai, Veneeth Antony. Blood-based screening panel for lung cancer based on clonal hematopoietic mutations in tumor infiltrating immune cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A030.

Abstract A076: Discovery and characterization of a novel, immunoproteasome activator that modulates the immunopeptidome, increases MHC class I antigenic presentation and enhances antitumor immunity

Abstract Evasion of immune destruction is a hallmark of cancer that thwarts the efficacy of immunotherapy and reduces patient benefit. The development of immunotherapies has sparked a revolution in oncology by releasing the power of the human immune system, but the identity of novel, actionable antigens exclusively presented on cancer cells remains elusive. Immune cells contain a highly specialized variant of proteasomes, known as immunoproteasomes, in which the three constitutive catalytic subunits are replaced by cytokine-inducible, homologous catalytic subunits. Immunoproteasomes are specially adapted for a role in MHC class I antigen processing and presentation to CD8+ T-cells. Here, we performed a high-through screen to detect novel molecular entities that activated proteasomal peptide-hydrolyzing activity. We identified a curated panel of molecules which not only enhanced proteasomal hydrolysis of the cell-permeable substrate LLVY-R110 but also enhanced the presentation of MHC class I antigens. E.G7-Ova is a mouse lymphoma cell line derived by electroporation of EL4 cells (C57BL/6, H-2b, T lymphoma) with chicken ovalbumin (OVA) cDNA. Proteasomes degrade OVA to generate the peptide SIINFEKL that is presented by MHC-class I molecules. Studies demonstrated that the hits identified in the screen increased SIINFEKL presentation on E.G7-Ova cells. The lead compound, compound A, dramatically enhanced the expression of the proteasome activator PA28a/b, expressed by PSME1/2. Treatment of multiple myeloma (MM) cells with compound A enhanced the hydrolysis of Ac-ANW-MCA, which is selectively cleaved by the immunoproteasome beta5-associated catalytic activity. Treatment of MM cells followed by mass spectrometry (MS) demonstrated that compound A modulated the relative presentation of individual antigenic peptides bound to MHC class I molecules on tumor cells. The identification of actionable tumor antigens is indispensable for the development of several cancer immunotherapies, including T cell receptor (TCR)–transduced T cells and patient-specific mRNA or peptide vaccines. MS-based immunopeptidomics enables the discovery of canonical and non-canonical antigens, i.e., antigens derived from sequences outside protein-coding regions or generated by non-canonical antigen-processing mechanisms. This method enables the rapid identification of non-canonical peptides, a fraction of which may be detected exclusively by cancer cells. Novel, immunoproteasome-selective therapeutics have the potential to contribute not only to CD8+ T-cell-mediated control of human cancers, but also intracellular infections, autoimmune diseases, neurodegenerative diseases and proteinopathies that are characterized by the pathologic accumulation of toxic, proteinaceous aggregates. Citation Format: James J. Driscoll, Priyanka S. Rana, James J. Ignatz-Hoover. Discovery and characterization of a novel, immunoproteasome activator that modulates the immunopeptidome, increases MHC class I antigenic presentation and enhances antitumor immunity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A076.

Dysregulation of Immune Regulators in AML Bone Marrow Promotes Dysfunction of CD8 + T Cells

Background: Acute myeloid leukemia (AML) originates in the bone marrow, evading immune recognition and destruction. The bone marrow microenvironment (BME), comprising diverse immune cells and regulators, profoundly influences AML development. However, the impact of immune regulators in the BME, particularly on T-cell anti-AML immunity, remains inadequately understood. Therefore, evaluating the impact of immune regulators in the AML bone marrow microenvironment on T cell dysfunction is of paramount importance and necessity. M ethod : Bone marrow blood samples were obtained from pediatric AML patients at diagnosis and age-matched healthy individuals considered as hematopoietic stem cell transplantation (HSCT) donors after written informed consent. By centrifuging at 2000-3000rpm for 10min, we obtained the supernatant as plasma and stored it in a -80°C refrigerator. After using proteomics to identify the expression of 180 immune regulatory factors in plasma samples, we performed GO and KEGG functional enrichment analysis. After density gradient centrifugation, we isolated bone marrow mononuclear cells (BMMNCs) and immediately identified frequencies of T cell populations and expression levels of functional markers by flow cytometry when they were fresh. Moreover, the co-culture model of CD8 +T cells and THP-1 cell line was used to evaluate the anti-AML effector function of T cells. CD8 +T cells in BMMNCs were sorted by FACS and then subjected to whole transcriptome sequencing and in vitro experiments. After using the TRRUST and KnockTF databases for transcription factor prediction analysis, we selected phospho-flow cytometry to detect and verify the association between the level of phosphorylation of transcription factors and the function of CD8 +T cells. The data were analyzed using R software. Results: Using proteomics techniques, we uncovered a substantial presence of immune-suppressive regulatory factors in AML bone marrow plasma. Interestingly, the canonical NF-κB pathway, mediated by immune regulators and vital for immune regulation in BME, displayed significant downregulation, implying T-cell dysfunction. In our investigation, we identified dysfunctional CD8 +T cells in the primary AML bone marrow, characterized by heightened expression of co-inhibitory receptors and pro-dysfunctional transcription factors, as well as significantly decreased anti-AML effector function. Transcriptome sequencing further linked AML CD8 +T cell dysfunction to reduced transcriptional activity of NF-κB p65 in the canonical NF-κB signaling pathway. Intriguingly, in vitro inhibition of NF-κB p65 and its phosphorylation led to elevated expression of exhaustion markers, such as PD-1, LAG-3, and TIM-3 on primary CD8 +T cells. Most notably, the stimulation of the canonical NF-κB signaling pathway effectively restored activation and effector function-related pathways in dysfunctional AML CD8 +T cells. Conclusion: Immune regulators in the bone marrow microenvironment play a vital role in maintaining dysfunctional AML CD8 +T cells. These findings provide a novel strategy to investigate the potential mechanisms and interventions for T-cell dysfunction.

Two Hit Theory for the Pathogenesis of Type 3 Congenital Amegakaryocytic Thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is an autosomal recessive disorder characterized by thrombocytopenia with an absence of megakaryocytes. It can lead to aplastic anemia or myelodysplastic syndrome (MDS). CAMT is linked to a mutation in the c-MPL gene on chromosome 1, which encodes the thrombopoietin receptor. In 2005, CAMT was categorized into three types based on the severity of c-MPL gene loss. Type 1 is the most severe, leading to early-onset pancytopenia and bone marrow abnormalities. Type 2 shows a temporary increase in platelets during infancy, followed by bone marrow failure at 3-6 years due to partial gene function. Type 3 has normal c-MPL function but produces ineffective megakaryocytes. We report a case of a patient with CAMT-like symptoms, without a c-MPL gene mutation, suggesting the presence of an alternative genetic defect. Methods Multi-institutional collaboration and review of medical records in addition to PubMed search for thrombocytopenia, bleeding complications, macrocytosis, trisomy 6, GATA2 variant, inherited bone marrow failure. Case Description A 12-year-old male with severe thrombocytopenia initially observed at 2 years of age. Bleeding complications included scalp hematoma, excessive bleeding during adenoidectomy, widespread petechiae, purpura, epistaxis, and gum bleeding. During this time platelet count ranged between 30-80,000 x10 3/uL. At 10 years of age, bleeding worsened with platelet counts of 6-20,000 x10 3/uL. Mean platelet volume (MPV) remained within the normal range. Simultaneously there was a decline in hemoglobin and the emergence of macrocytosis. Mother had moderate thrombocytopenia during pregnancy, older sibling with similar thrombocytopenia but asymptomatic. Clinical presentation made immune thrombocytopenia, autoimmune disorders, and immune dysregulation-induced platelet destruction unlikely. Inherited thrombocytopenia panel testing for 42 germline variants was negative. Bone marrow biopsy showed 60% cellularity and was remarkable for a decrease in megakaryocytes. Cytogenetic analysis revealed trisomy 6 in 7/20 metaphases. Molecular hematopathology identified a GATA2 variant in 50% of the alleles. Inherited BMF panel revealed heterozygosity for multiple variants of unknown significance (VUS), including c.5096A>G (p.Asp1699Gly) in BRCA2 gene, c.3898A>G (p.Ile300Val) in LYST gene, 1408A>G (p.Thr470Ala) in PALB2 gene, and c.7240G>A (p.Val2414Met) in tVPS13B gene. Another BMF panel revealed two additional heterozygous VUS - NBEAL2 and ADAMTS13. Testing for Fanconi anemia and dyskeratosis congenita were negative. Whole exome sequencing revealed heterozygosity for a 1.57 Mb duplication on chromosome 3q29. Additional molecular pathology panel testing for 468 genes, germline genetic testing for lysosomal disorders and mucopolysaccharidoses, were also negative. Repeat bone marrow testing demonstrated a progressive decrease in cellularity to 5-30% with trilineage hypoplasia. With extensive yet inconclusive diagnostic workup, worsening clinical presentation, and progressive hypocellularity of the bone marrow, he underwent an ABO incompatible stem cell transplantation (9:10, A HR) from a mismatched unrelated donor. The conditioning regimen was myeloablative with busulfan, cyclophosphamide, anti-thymocyte globulin, and post-transplant cyclophosphamide. Currently, he is 150 days post-transplant with peripheral blood chimerism demonstrating 97.7% donor on day 98. Platelet counts have normalized, and he has not required a transfusion for nearly 100 days. Discussion This case report highlights the concept of a “double hit” hypothesis. While homozygous mutations of PALB2 and BRCA2 are individually associated with Fanconi anemia and BMF syndromes, no hematological pathology has been documented in their heterozygous forms, raising the question of whether the co-expression of these variants in a heterozygous state could result in a similar disease phenotype as their homozygous counterparts. This finding prompts further exploration into the potential impact of combined variants on disease manifestation.

Predictors of Response to Rituximab in Chronic Immune Thrombocytopenia: Do Race, Gender and ABO Blood Type Matter?

Introduction: Immune thrombocytopenia (ITP)is characterized by immune mediated destruction of platelets causing isolated thrombocytopenia with a peripheral blood platelet count <100,000/microL in the absence of other causes that may be associated with thrombocytopenia. It remains a diagnosis of exclusion. Corticosteroids are the first-line treatment for ITP and more than 75% of adults with ITP will respond to them. However, approximately 70-80% of adult patients with ITP will experience treatment failure with corticosteroids or become dependent on them and require second-line therapy. Various options in the second line setting include thrombopoietin receptor agonists, rituximab, and splenectomy. In one study rituximab was noted to result in an overall platelet count response in 62.5% of adults. However not all patients remain in remission and clinical factors that are predictive of response are lacking. Racial disparities are known to predict response to rituximab in other hematologic disorders such as immune mediated thrombotic thrombocytopenic purpura (Mezapa et al, 2022). Herein, we are examining the effect of race, gender and ABO blood type on response to rituximab in patients diagnosed with ITP. Methods: This retrospective cohort study included adult patients, aged 18 and above, with a diagnosis of ITP who had received single agent rituximab within a multihospital academic healthcare system between January 2015 and December 2022. Initially 196 patients were identified but only 127 of those were included in the study. We excluded patients with missing response data, and those with combined rituximab and other therapies (stable or decreasing doses of steroids were allowed). Our primary end point was any response to rituximab by day 180 after starting rituximab. Any response was defined as complete response or partial response. Complete response was defined as platelet count ≥ 100,000 /microL and absence of bleeding. Partial response was defined as platelet count of at least 30,000/microL after week 4 from rituximab administration or doubling of platelet count from baseline without administration of any platelet increasing therapy. Our secondary end point was relapse rate which was defined as platelet count below 100,000/microL if complete response had been attained or below 30,000/microL or less than 2 fold increase of baseline platelet count. We also investigated clinical and socioeconomic predictors of response. Exploratory analysis was performed. Summary statistics are presented as percentages for categorical data and median with interquartile range for quantitative data. Chi square test was used to compare percentages and t-test was used to compare means. Univariate and multivariate regression models were used to analyze the predictors of any response to rituximab. Kaplan-Meier method was used to calculate estimates of relapse-free survival, and log-rank test was used to compare these estimates. Result: We identified 127 patients diagnosed with ITP who received rituximab. The median age was 64 years (IQR 48-74), there were 46.5% females. The majority of the patients were Caucasians (95.3%), and rituximab was most commonly received as a second line therapy (73.2%). Median initial platelet count was 28,000/microL. Complete response was observed in 50% of African American patients compared with 43.0% in Caucasian patients (p-value 0.63). No predictors for any response on univariate or multivariate analyses were found. Race (OR 0.30, 95CI 0.03-3.25, p-value 0.32) and female gender (OR 0.76, 95CI 0.33-1.76, p-value 0.51) did not affect response to rituximab in our patient cohort. Relapse rate was 60% in African American patients versus 67.5% in Caucasians (p-value 0.72), and 68.7% in males versus 64.9% in females (p-value 0.71). ABO blood type also did not predict a response to Rituximab in our patient population (Table 1) Conclusion: In our study, race and gender did not affect response or relapse rates after rituximab treatment in chronic ITP. Patient's ABO blood type also did not predict a difference in response to Rituximab. However, our study was limited by a small sample size and further multi-institutional analysis is warranted to confirm the findings of our study.

Targeting of CD25+ Activated T Cells in Gvhd-Associated Marrow Suppression and Cytopenias

Introduction: Secondary cytopenias occurring after hematopoietic cell transplant (HCT) are an observed complication of graft-versus-host disease (GVHD). Causes include peripheral immune mediated destruction (hemolytic anemia or thrombocytopenia), usually associated with autoantibody production, and peripheral destruction associated with microangiopathy (TMA). However, cases have been observed where marrow production of the affected lineage is abnormal. Immune-mediated cases are treated with increased immunosuppression, e.g., steroids or calcineurin inhibitors (CNI), and TMA with complement inhibition. To better understand the mechanisms and treatment of hypoproductive cytopenias in GVHD, we combined analyses of hematopoietic stem and progenitor cells (HSPC) and peripheral blood and marrow activated T cells. We describe 3 cases of GVHD-associated cytopenias marked by HSPC depletion and presence of CD25 (IL-2R alpha subunit)-positive activated T cells in the marrow that responded to combinations of immunosuppression containing basiliximab (basil) anti-CD25 monoclonal antibody blockade. Methods/Results: We describe combined HSPC and T cell analyses in 3 patients (P) with severe GVHD-associated cytopenias. P1 was at 5 years post-HCT for acute lymphoblastic leukemia, P2 at 2 years post-HCT for severe aplastic anemia (SAA), and P3 at 1.5 months post-liver transplant for alcohol use disorder-associated cirrhosis and hepatocellular carcinoma ( Table 1). Because of refractory cytopenias, all were being considered for HCT. However, marrow analyses revealed that each had normal frequencies of phenotypic HSC, with variable decreases in cellularity and frequencies of committed progenitors ( Table 2). Chimerism analyses showed that 100% of CD34+ cells in P1 and P2 were of donor origin, and in P3 of host origin. All 3 marrows had activated CD3+ T cells per expression of CD69 and/or CD38, and frequency of CD25 on >10% of T cells. In addition, P1 had evidence of IgG bound to committed progenitor populations. The treatment strategy was to block IL-2 signaling with basil (induction of 20 mg on day 1, 4, 7, 14, 21, 28, followed by alternate week and then monthly dosing for 2-9 months), combined with suppression of IL-2 production with steroid and/or CNI. After induction, repeat analyses of the blood and marrow T-cells confirmed complete blockade of CD25. Therapy was also individualized for specific clinical conditions or as noted on marrow evaluation. P1 received prednisone, CNI, and rituximab for a presumed anti-HSPC autoantibody. At 9 months he was weaned off all therapy and remains on no immunosuppressive treatment 2 years later. P2 did not receive CNI due to chronic renal failure but received steroids and later sirolimus. At ~18 months, his cytopenias recurred, but subsequently responded to basil re-induction and steroids. P3 has received prednisone, CNI, and sirolimus indefinitely for prevention of liver rejection and/or hepatocellular carcinoma relapse. P1 had no viral reactivation; P2 had CMV pneumonia with his second course responsive to antiviral therapy, completion of basil, and tapering of steroids; and P3 had CMV reactivation responsive to antiviral therapy and EBV viremia. P3 has a recent diagnosis of EBV+ intrahepatic B cell lymphoma. In all cases, blood counts normalized except P2 who has anemia due to CKD. Discussion: HSPC analyses in these patients with GVHD and cytopenias demonstrate that peripheral changes are not due to loss of HSC, but instead to quantitative and/or qualitative defects in committed progenitor populations, likely mediated by activated CD25+ T cells. In addition, the presence of IgG on HSPCs in P1 demonstrates a novel role for antibody-mediated humoral suppression in some cases. The presence of HSC indicates that there may be no need for re-transplant. Previous studies of SAA have shown activated T cells to be drivers of aplasia and is the basis for immune suppressive therapy (anti-thymocyte globulin and CNI). Our analyses of patients with GVHD-associated cytopenias demonstrates that targeting of IL-2 signaling via combined CD25 receptor blockade and pharmacologic inhibition of IL-2 production can correct the marrow defects and cytopenias, albeit with need for attention to viral infections. Targeting CD25 in patients with evidence of CD25+ activated T cells is a rational approach to the difficult problem of GVHD-associated marrow suppression and cytopenias.

Evaluation of ISTH Severe Bleeding Criteria and Development of a Mortality Prognosis Model for Hospitalized Immune Thrombocytopenia Patients: A Multicenter, Retrospective and Prospective Cohort Study

Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by the immune system's erroneous destruction of platelets, which play a crucial role in blood clot formation. This condition precipitates a decreased platelet count and consequently heightens the risk of bleeding events, ranging from mild petechiae to severe, potentially fatal occurrences such as intracranial hemorrhage. Therefore, accurate anticipation and management of these severe bleeding risks form an integral facet of clinical care for ITP patients. The International Society on Thrombosis and Hemostasis (ISTH) has instituted criteria designed to classify and forecast severe bleeding in patients with thrombotic disorders. Despite their widespread use in diverse clinical scenarios, the validity and efficacy of these criteria for ITP patients remain underexplored. Furthermore, disparities in the application and interpretation of these criteria among practitioners underscore the necessity for a comprehensive evaluation within the ITP context. Consequently, our study is poised to fill these crucial gaps. We intend to evaluate the performance and applicability of the ISTH severe bleeding criteria specifically within the ITP patient population. Concurrently, we aspire to develop and validate a robust, reliable mortality prediction model for this unique group of hospitalized patients. Methods: This study was a multicenter, retrospective cohort investigation conducted across 20 medical centers nationwide. A total of 1487 adult patients diagnosed with immune thrombocytopenia were enrolled. The study period spanned from Jan 2020 to Jan 2023, ensuring a comprehensive assessment of patient outcomes. To further evaluate the joint impact of multiple prognostic factors, a multivariate Cox regression model was constructed. Prospective observational cohorts were subsequently established with the objective of assessing the real-world performance of the mortality prognosis model. Results: In our study, out of the 1487 enrolled adult patients with immune thrombocytopenia (ITP), 420 patients (28.24%) experienced bleeding events. The incidence of major bleeding types among these patients was assessed based on the critical bleeding diagnostic criteria. Intracranial bleeding was found to be the most common type, occurring in 229 patients (54.46%). This was followed by respiratory compromise in 87 patients (20.66%) and hemodynamic instability bleeding in 76 patients (18.08%). Intramuscular bleeding was the least common, observed in only 3 patients (0.70%). Furthermore, we evaluated the efficacy of the ISTH severe bleeding criteria in predicting mortality among ITP patients. Our analysis unveiled a significant association between meeting the ISTH severe bleeding criteria and increased mortality rates. Patients who met the criteria demonstrated a higher incidence of death compared to those who did not meet the criteria. Additionally, we developed a mortality prognosis model for hospitalized ITP patients, incorporating various clinical and laboratory parameters. The model considered the presence of intracranial bleeding, admission platelet count, cardiovascular disease, diabetes mellitus, and admission creatinine levels as predictive factors. The mortality prognosis model demonstrated strong predictive power, with an area under the receiver operating characteristic curve of 0.82. This indicates its ability to distinguish between patients at high and low risk of mortality. Kaplan‒Meier survival analyses further confirmed significant survival differences between patients who met the ISTH severe bleeding criteria and those who did not. Conclusions: Our study reaffirms the substantial relevance of ISTH severe bleeding criteria in assessing bleeding profiles and prognosis among hospitalized ITP patients. The developed mortality prognosis model demonstrates excellent predictive capabilities for mortality in this patient population and is currently undergoing validation in a prospective cohort. These findings emphasize the importance of individualized management strategies to optimize outcomes in ITP patients. Further research and validation are warranted to refine and validate the mortality prognosis model for broader clinical application.

Gender Disparities in Outcomes of Autoimmune Hemolytic Anemia Admissions: An Analysis of the National Inpatient Sample

Introduction Autoimmune hemolytic anemia (AIHA) is a condition characterized by the immune destruction of red blood cells, which can necessitate hospitalization in severe cases. The purpose of this study is to compare outcomes of AIHA hospitalizations between genders. Methods We utilized the International Classification of Diseases (ICD) codes to identify all hospitalizations with the primary diagnosis of AIHA in the National Inpatient Sample (NIS) from 2002 to 2019. We excluded all pediatric (age < 18 years) and elective hospitalizations. We described the baseline characteristics of both groups including demographics and comorbidities. We fitted multivariable logistic regression models to compare outcomes between males and females while adjusting for age, race, and comorbidities. Results A total of 60,529 met the inclusion and exclusion criteria. The majority of these were females (59.1% vs 40.1%, p < 0.001). Males were older (median age 67 vs. 66 years, p < 0.001) and had more white race (73.4% vs. 67.9%, p < 0.001). The distribution of associated comorbidities also differed different between both groups ( (Table 1). On adjusted analysis, males had higher in-hospital mortality rates. They also had higher rates of most secondary outcomes; including rates of circulatory shock, need for mechanical ventilation, bacterial pneumonia, acute myocardial infarction, deep vein thrombosis, hemorrhagic stroke, acute kidney injury, and requiring new hemodialysis. Females had higher rates of urinary tract infections. No significant differences were found in the rates of requiring blood transfusions, sepsis, acute heart failure, pulmonary embolism, ischemic stroke, encephalopathy, and gastrointestinal bleeding (Figure 1). Furthermore, male hospitalizations were associated with higher costs (median [IQR] of $11,400 [6,700-20,500] vs. $10,000 [6,000- 17,200]) despite similar length of stay (median [IQR] of 4 days [3-7] vs. 4 days [3-7]). Conclusion Male patients hospitalized with AIHA experience poorer in-hospital compared to females. More research is essential to understand the underlying causes for these disparities and to tailor management to improve outcomes for both male and female AIHA patients.

Distinct Immune and Molecular Profiles of Hepatitis-Associated Aplastic Anemia

Background: Hepatitis-associated aplastic anemia (HAAA) is characterized by the development of hematopoietic failure within months following an acute episode of non-infectious hepatitis. Although disease presentation with pancytopenia and a hypocellular marrow in HAAA is similar to non-hepatitis immune AA (non-HAAA), the immune and molecular profile in HAAA has not been completely elucidated. Here, we aimed to assess HAAA patients for cytokines and presence of clonal events linked to AA, the presence of paroxysmal nocturnal hemoglobinuria (PNH) clones and somatic mutations in peripheral blood mononuclear cells (PBMC) subpopulations, and compared to a non-HAAA cohort. Methods: A cohort of HAAA patients (n=30), age and sex-matched non-HAAA patients (n=24) and healthy controls (n=23) seen at the National Institutes of Health since 2003 were retrospectively screened for a panel of inflammatory cytokines and growth factors by Luminex, and somatic mutations in sorted CD3-, CD3+CD4+, and CD3+CD8+ PBMC fractions by error-corrected DNA sequencing. A customized panel with 42 myeloid-related and 49 autoimmunity-related genes at minimum limited of detection of 0.1% were used. All samples were before any immunosuppressive treatment (IST). Cytokine levels and frequencies of PNH clones and clonal hematopoiesis (CH) in different fractions were compared among groups. Results: HAAA patients were in average young (median age=23); 37% were at age < 18 (Table 1). Although disease severity and blood counts were similar to the non-HAAA cohort, immune and molecular markers of immune AA were distinct among cohorts. The frequency of PNH clones was significantly lower in HAAA compared to the non-HAAA cohort (15% v 58%, p=0.0008). The median size of PNH clones in HAAA and non-HAAA was 1.4% and 7.4%, respectively (p =0.03). The cytokine profiles performed in the sera of 22 HAAA before IST were marked by significantly higher levels of IL-1ra (p=0.0008), CXCL-10 (p=0.0038), and VEGF (p=0.0023) in comparison to 31 non-HAAA assessed patients; IL-6 (p=0.0002) and CD40L (p=0.0001) levels were significantly lower than in non-HAAA. As reported in immune AA, HAAA patients had higher levels of TPO (p=0.0001), G-CSF (p=0.0001), and EPO (p=0.0001) than controls; lower levels of CCL-5 (p=0.0001) and CCL-11 (p=0.0025) were seen in the HAAA. FLT3-L levels were markedly elevated in both AA cohorts when compared to HC. The clonal landscape was also strikingly different between HAAA and non-HAAA in all fractions evaluated (Figure 1). In HAAA, 3 out of 21 assessed patients (14%) had CH in the CD3- fraction at maximum variant allele frequency (VAF) of 1.8%; one patient had two RUNX1 mutations while a JAK2 and DNMT3A mutation were found in the other two patients. No mutations were found in CD4+ and CD8+ compartments in subjects with HAAA. In contrast, 13 of 24 non-HAAA patients (54%) had CH in the CD3- populations, which was dominated by PIGA, BCOR, and DNMT3A clones, a pattern consistent with the known clonal landscape of immune AA. The frequency of non-HAAA with CH in the CD4+ and CD8+ compartments was 7/24 (29%) and 8/24 (30%) patients, respectively; CH in these fractions was similar to those found in CD3- dominated by mutations in PIGA, BCOR, and DNMT3A at VAFs ranging from 0.2% to 15% (Figure 1). CH in lymphoid-related genes was only found in a single patient with a STAT3 p.S614Rmutation at VAF of 3% exclusive to the CD8+ fraction. Conclusion: HAAA has a distinct immunologic and molecular profile than non-HAAA patients. PNH clones were less frequent in HAAA and patterns of cytokines were significantly different than the non-HAAA cohort. CH in PIGA and BCOR, highly associated with AA, were not present in our small HAAA cohort. Our results indicate a distinct underlying pathophysiology that initiates the immune destruction resulting in marrow failure in HAAA patients. Confirming these findings in a larger cohort and correlating with clinical outcomes is ongoing.

Immunoregulation: the interplay between metabolism and redox homeostasis.

Regulatory T cells are fundamental for the induction and maintenance of immune homeostasis, with their dysfunction resulting in uncontrolled immune responses and tissue destruction predisposing to autoimmunity, transplant rejection and several inflammatory and metabolic disorders. Recent discoveries have demonstrated that metabolic processes and mitochondrial function are critical for the appropriate functioning of these cells in health, with their metabolic adaptation, influenced by microenvironmental factors, seen in several pathological processes. Upon activation regulatory T cells rearrange their oxidation-reduction (redox) system, which in turn supports their metabolic reprogramming, adding a layer of complexity to our understanding of cellular metabolism. Here we review the literature surrounding redox homeostasis and metabolism of regulatory T cells to highlight new mechanistic insights of these interlinked pathways in immune regulation.

Immunomodulatory effect of Myrtenol on benzo (a) pyrene-induced lung cancer in Swiss albino mice via modulation of tumor markers, cytokines and inhibition of PCNA expression.

Lung cancer is one of the most common cancers in men. Although many diagnostic and treatment regimens have been followed in the treatment for lung cancer, increasing mortality rate due to lung cancer is depressing and hence requires alternative plant based therapeutics with with less side-effects. Myrtenol exhibits anti-inflammatory and antioxidant properties. Hence we intended to study the effect of Myrtenol on B(a)P-induced lung cancer. Our study showed that B(a)P lowered hematological count, decreased phagocyte and avidity indices, nitroblue tetrazolium (NBT) reduction, levels of immunoglubulins, antioxidant levels, whereas Myrtenol treatment restored them back to normal levels. On the other hand, xenobiotic and liver dysfunction marker enzymes and pro-inflammatory cytokines were elevated on B(a)P exposure, which retuned back to normal by Myrtenol. This study thus describes the immunomodulatory and antioxidant effects of Myrtenol on B[a]P-induced immune destruction.

Early Initiation of Oral Therapy with Cyclosporine and Eltrombopag for Treatment Naïve Severe Aplastic Anemia: Interim Results of a Phase II Trial

Introduction: Severe aplastic anemia (SAA), a life-threatening bone marrow failure disease, is treated with allogeneic bone marrow transplantation or immunosuppression (IST). Pancytopenia meeting “Camitta” criteria and an empty bone marrow (<30% cellularity) establish the diagnosis of SAA. However, neither diagnosis nor therapeutic interventions are straightforward, resulting in delays of therapy. SAA must be differentiated from hematologic malignancy, and constitutional forms should be excluded by functional or genetic testing. Administration of IST usually occurs at a tertiary care center, and in younger patients' evaluation for BMT is first required. Ongoing immune mediated destruction of stem cells further depletes marrow reserve and increases regenerative stress with such delays. In this phase II study, we tested the feasibility and safety of early initiation of cyclosporine (CSA) to block T cells and eltrombopag (EPAG) to stimulate hematopoiesis followed by standard of care therapies to expedite treatment (NCT04304820). Methods: Twenty-six patients with treatment-naive SAA were enrolled since 2020. Patients were 3 years or older and median age was 35 years. Exclusion criteria included a diagnosis or high clinical suspicion for inherited disease or evidence of myeloid neoplasm. To ensure rapid initiation of therapy, enrollment was not dependent on pending specialized tests (telomere length, chromosomal breakage, cytogenetics, or genomics). Lower dose CSA (2mg/kg) and full dose EPAG were initiated after confirming eligibility. Upon completion of evaluation and confirmation of acquired SAA diagnosis, all subjects received standard IST (horse ATG, CSA, and EPAG; Figure 1A). Primary feasibility endpoint assessed a composite measure of misdiagnosis, non-compliance, and treatment-related serious adverse events (TRSAE) during oral therapy. Secondary endpoints were efficacy at 6 months, relapse, clonal evolution, and overall survival. Results were compared to a historical cohort of 139 patients treated with hATG, CSA, and EPAG initiated the same day (NCT01623167). Results: All patients completed the protocol-defined treatment period, with median time on oral therapy of 17 days (range 0 - 86 days). All subjects ultimately were confirmed to have acquired SAA (no misdiagnoses), all tolerated oral treatment well without any TRSAE, and patients were compliant with local and study-site follow-up. Thus, there were zero events for the primary feasibility endpoint. A single SAE during oral treatment occurred in subject #1, enrolled at the height of the COVID pandemic and was severely ill with persistently positive COVID-19 PCR and recurrent ileitis requiring hospitalization care; his time to standard IST was the longest (86 days). There was significant increase in absolute neutrophil count (ANC; median 0.315x10 3/mL vs 0.570, p=0.041) in a brief interval of oral therapy prior to receiving hATG. There was also a trend towards improvement in absolute reticulocyte count (29x10 3/mL vs. 35.8) and a decrease in absolute lymphocyte count (1.5x10 3/mL vs. 1.3; Figure 1B). Compared to historical cohort, less patients (19% vs 52%) had very SAA (ANC <200 K/uL); age, sex, race, and pre-treatment blood counts were similar in two cohorts. Hematologic response occurred in 68% at 6 months with 45% complete responses, rates similar to the historical cohort (80% and 40%, respectively). Time to platelet and red blood cell transfusion independence was 43 and 51 days, respectively. Only age (18-40 years) was predictive for 6 month response. With median follow-up of 396 days, the cumulative incidence of relapse at 2 years was 17%, lower than in the historical cohort (27%). Clonal evolution to secondary myeloid malignancy was not increased compared to historical cohort; one subject had myelodysplastic syndrome with excess blasts 1 with deletion 20q, and two had transient trisomy 8 without dysplasia. Overall survival was 96.2%; no deaths occurred during study treatment period of oral medications only. Conclusion: Early initiation of CSA and EPAG is a feasible and safe approach to rapidly treat patients with SAA as a bridge to standard therapies and it may lead to improved blood counts, in turn, decreasing early infectious complications. Hematologic response was comparable to our historic cohort. Rates of long-term complications need to be confirmed in a larger cohort with ongoing study enrollment.

An Immunoproteasome Activator That Increases MHC Class I Antigen Presentation to Enhance Anti-Tumor Immunity

Evasion of immune destruction is a hallmark of cancer that thwarts the efficacy of immunotherapy and reduces patient benefit. The development of immunotherapies has sparked a revolution in oncology by releasing the power of the human immune system, but the identity of novel, actionable antigens exclusively presented on cancer cells remains elusive. Proteasomes are multi-subunit proteolytic complexes that selectively degrade intracellular proteins. Proteasomes play a critical in controlling myriad cellular processes including metabolism, cell cycle progression, signal transduction, protein quality control and differentiation. Importantly, proteasomes are also the source of most antigenic peptides presented to the immune system on major histocompatibility complex (MHC)-class I molecules. In this process, intracellular and viral proteins are degraded in the cytosol to 8 and 9-amino acid peptides, which are then transported into the endoplasmic reticulum, where they associate with MHC-class I molecules and are presented to the cell surface. Immune cells contain a highly specialized, cytokine-inducible proteasome variant, known as the immunoproteasome, in which the three constitutive catalytic subunits are replaced by homologous catalytic subunits. Immunoproteasomes are specially adapted for a role in MHC class I antigen processing and presentation to CD8 + T-cells. Here, we performed a high-through screen (HTS) to detect novel molecular entities that activated immuno-proteasomal catalytic activity. We identified a curated panel of molecules which not only enhanced hydrolysis of the cell-permeable substrate LLVY-R110 but also enhanced the presentation of MHC class I antigens. E.G7-Ova is a mouse lymphoma cell line derived by electroporation of EL4 cells (C57BL/6, H-2b, T lymphoma) with chicken ovalbumin ( OVA) cDNA. Proteasomes degrade OVA to generate the peptide SIINFEKL that is presented by murine MHC-class I H-2Kb molecules. E.G7-Ova cells were pretreated with compound A and co-cultured with the T-cell hybridoma B3Z that had been genetically-engineered to express a SIINFEKL-restricted t-cell receptor (TCR). Pretreatment of E.G7-Ova cells with compound A significantly increased tumor cell lysis by B3Z cells. The treatment of multiple myeloma (MM) cells with compound A also dramatically enhanced the expression of pan-MHC class I antigens. Treatment of MM cells with compound A increased the intracellular level of the proteasome activator PA28α/β, which is expressed by PSME1/2. Compound A treatment also enhanced hydrolysis of the fluorogenic peptide substrate Ac-ANW-AMC, which is selectively cleaved by the immunoproteasome β5-associated catalytic activity. Treatment of MM cells with compound A followed by mass spectrometry (MS) revealed that immunoproteasome activation modulated the relative presentation of individual antigenic peptides bound to MHC class I molecules. Pre-treatment of MM cells with compound A also significantly enhanced the anti-myeloma activity of human cytotoxic T-cells. Taken together, our results demonstrate that immunoproteasome activation has the potential to overcome immune escape and promote host immunity. In addition, our model systems now allows us to identify actionable tumor antigens on patient tumor cells as an indispensable tool for the development of cancer immunotherapies, e.g., T cell receptor (TCR)-transduced T-cells and patient-specific mRNA and peptide vaccines. Finally, immunoproteasome activators have the potential to contribute not only to cytotoxic T-cell mediated tumor lysis but also the treatment of intracellular infections and autoimmune and neurodegenerative proteinopathies characterized by the pathologic accumulation of toxic, proteinaceous aggregates.

Replication-incompetent influenza A viruses armed with IFN-γ effectively mediate immune modulation and tumor destruction in mice harboring lung cancer

Low pathogenic influenza A viruses (IAVs) have shown promising oncolytic potential in lung cancer-bearing mice. However, as replication-competent pathogens, they may cause side effects in immunocompromised cancer patients. To circumvent this problem, we genetically engineered nonreplicating IAVs lacking the hemagglutinin (HA) gene (ΔHA IAVs), but reconstituted the viral envelope with recombinant HA proteins to allow a single infection cycle. To optimize the therapeutic potential and improve immunomodulatory properties, these replication-incompetent IAVs were complemented with a murine interferon-gamma (mIFN-γ) gene. After intratracheal administration to transgenic mice that develop non-small cell lung cancer (NSCLC), the ΔHA IAVs induced potent tumor destruction. However, ΔHA IAVs armed with mIFN-γ exhibited an even stronger and more sustained effect, achieving 85% tumor reduction at day 12 postinfection. In addition, ΔHA-mIFN-γ viruses were proven to be efficient in recruiting and activating natural killer cells and macrophages from the periphery and in inducing cytotoxic T lymphocytes. Most important, both viruses, and particularly IFN-γ-encoding viruses, activated tumor-associated alveolar macrophages toward a proinflammatory M1-like phenotype. Therefore, replication-incompetent ΔHA-mIFN-γ-IAVs are safe and efficient oncolytic viruses that additionally exhibit immune cell activating properties and thus represent a promising innovative therapeutic option in the fight against NSCLC.

The alterations of innate immunity and enhanced severity of infections in diabetes mellitus.

Diabetes mellitus (DM) is a metabolic inflammatory disease with a high incidence worldwide. Patients with DM are at a high risk for all types of infections. Type 1 DM is characterised with immune destruction of pancreatic β cells, while type 2 diabetes is characterised with insulin resistance and β cell dysfunction, both of which result in disorders of glucose and lipid metabolism. This metabolic disorder causes functional defects of immune cells, aberrant production of inflammatory cytokines, dysregulated immune responses, advanced pathophysiological injury of the body, and increased mortality in populations with DM upon infections. Starting with the change of natural immune system in patients with DM, this paper focused on the enhanced severity of infections in DM and the underlying innate immune alterations in preclinical and clinical studies, aiming to better understand the influence of DM on the susceptibility, pathophysiology, and clinical outcomes in infections.

CMTM6 shapes antitumor Tcell response through modulating protein expression of CD58 and PD-L1.

The dysregulated expression of immune checkpoint molecules enables cancer cells to evade immune destruction. While blockade of inhibitory immune checkpoints like PD-L1 forms the basis of current cancer immunotherapies, a deficiency in costimulatory signals can render these therapies futile. CD58, a costimulatory ligand, plays a crucial role in antitumor immune responses, but the mechanisms controlling its expression remain unclear. Using two systematic approaches, we reveal that CMTM6 positively regulates CD58 expression. Notably, CMTM6 interacts with both CD58 and PD-L1, maintaining the expression of these two immune checkpoint ligands with opposing functions. Functionally, the presence of CMTM6 and CD58 on tumor cells significantly affects Tcell-tumor interactions and response to PD-L1-PD-1 blockade. Collectively, these findings provide fundamental insights into CD58 regulation, uncover a shared regulator of stimulatory and inhibitory immune checkpoints, and highlight the importance of tumor-intrinsic CMTM6 and CD58 expression in antitumor immune responses.

Sensing and signaling in periodontal inflammation via toll like receptors: A review

Periodontal disease is a chronic inflammatory condition that affects the supporting structures of teeth, including the gingiva, periodontal ligament, and alveolar bone. Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-associated molecular patterns (PAMPs) from bacteria that cause periodontal disease. TLR activation triggers an inflammatory response that aims to eliminate the bacteria and repair damaged tissue. However, if the inflammation persists, it can lead to further tissue destruction and bone loss. This review discusses the mechanisms of TLRs in periodontal disease progression, including the recognition of bacteria, activation of immune cells, tissue destruction, and inhibition of immune response. Strategies for down-regulating TLRs, such as small-molecule inhibitors, antibodies, natural products, micro RNA, and nutritional interventions, are also discussed. Targeting TLRs is a promising therapeutic approach for reducing chronic inflammation associated with periodontal disease. Further research is needed to develop safe and effective strategies for down-regulating TLRs in humans.

Beyond αβ T cells: NK, iNKT, and γδT cell biology in leukemic patients and potential for off-the-shelf adoptive cell therapies for AML.

Acute myeloid leukemia (AML) remains an elusive disease to treat, let alone cure, even after highly intensive therapies such as stem cell transplants. Adoptive cell therapeutic strategies based on conventional alpha beta (αβ)T cells are an active area of research in myeloid neoplasms given their remarkable success in other hematologic malignancies, particularly B-cell-derived acute lymphoid leukemia, myeloma, and lymphomas. Several limitations have hindered clinical application of adoptive cell therapies in AML including lack of leukemia-specific antigens, on-target-off-leukemic toxicity, immunosuppressive microenvironments, and leukemic stem cell populations elusive to immune recognition and destruction. While there are promising T cell-based therapies including chimeric antigen receptor (CAR)-T designs under development, other cytotoxic lymphocyte cell subsets have unique phenotypes and capabilities that might be of additional benefit in AML treatment. Of particular interest are the natural killer (NK) and unconventional T cells known as invariant natural killer T (iNKT) and gamma delta (γδ) T cells. NK, iNKT, and γδT cells exhibit intrinsic anti-malignant properties, potential for alloreactivity, and human leukocyte-antigen (HLA)-independent function. Here we review the biology of each of these unconventional cytotoxic lymphocyte cell types and compare and contrast their strengths and limitations as the basis for adoptive cell therapies for AML.

Potential immunosuppressive clonal hematopoietic mutations in tumor infiltrating immune cells in breast invasive carcinoma

A hallmark of cancer is a tumor cell’s ability to evade immune destruction. Somatic mutations in tumor cells that prevent immune destruction have been extensively studied. However, somatic mutations in tumor infiltrating immune (TII) cells, to our knowledge, have not been previously studied. Understandably so since normal hematopoiesis prevents the accumulation of somatic mutations in immune cells. However, clonal hematopoiesis does result in the accumulation of somatic mutations in immune cells. These mutations cannot “drive” tumor growth, however, they may “facilitate” it by inhibiting an effective anti-tumor immune response. To identify potential immunosuppressive clonal hematopoietic (CH) mutations in TII cells, we analyzed exome and RNA sequencing data from matched tumor and normal blood samples, and single-cell RNA sequencing data, from breast cancer patients. We selected mutations that were somatic, present in TII cells, clonally expanded, potentially pathogenic, expressed in TII cells, unlikely to be a passenger mutation, and in immune response associated genes. We identified eight potential immunosuppressive CH mutations in TII cells. This work is a first step towards determining if immunosuppressive CH mutations in TII cells can affect the progression of solid tumors. Subsequent experimental confirmation could represent a new paradigm in the etiology of cancer.

Supramolecular Modulation of Tumor Microenvironment through Pillar[5]arene-Based Host-Guest Recognition to Synergize Cancer Immunotherapy.

Despite the tremendous breakthrough of immunotherapy, the low response rate and resistance of immune checkpoint inhibitors (ICIs) toward solid tumors occur frequently. A highly hypoxic tumor microenvironment (TME) provides tumor cells with high concentrations of HIF-1α and polyamines to evade immune cell destruction. Reprogramming of an immunogenic TME has exhibited a brilliant future to boost immunotherapeutic performances. Herein, a supramolecular nanomedicine (TAPP) is developed on the basis of host-guest molecular recognition and metal coordination, showing the capability to remodel the immunosuppressive TME. Tamoxifen (Tmx) and Fe3+ are encapsulated into TAPP to achieve the combination of chemotherapy and chemodynamic therapy (CDT). Tmx directly downregulates HIF-1α, and a pillar[5]arene-based macrocyclic host successfully eliminates polyamines in tumors. Enhanced immunogenic cell death is achieved by Tmx and Fe3+, and the therapeutic efficacy is further synergized by immune checkpoint blockade (ICB) therapy. This supramolecular reprogramming modality encourages cytotoxic T lymphocyte infiltration, achieving pre-eminent immune response and long-term tumor suppression.