The immune-suppressive landscape in lepromatous leprosy revealed by single-cell RNA sequencing

Lepromatous leprosy (L-LEP), caused by the massive proliferation of Mycobacterium leprae primarily in macrophages, is an ideal disease model for investigating the molecular mechanism of intracellular bacteria evading or modulating host immune response. Here, we performed single-cell RNA sequencing of both skin biopsies and peripheral blood mononuclear cells (PBMCs) of L-LEP patients and healthy controls. In L-LEP lesions, we revealed remarkable upregulation of APOE expression that showed a negative correlation with the major histocompatibility complex II gene HLA-DQB2 and MIF, which encodes a pro-inflammatory and anti-microbial cytokine, in the subset of macrophages exhibiting a high expression level of LIPA. The exhaustion of CD8+ T cells featured by the high expression of TIGIT and LAG3 in L-LEP lesions was demonstrated. Moreover, remarkable enhancement of inhibitory immune receptors mediated crosstalk between skin immune cells was observed in L-LEP lesions. For PBMCs, a high expression level of APOE in the HLA-DRhighFBP1high monocyte subset and the expansion of regulatory T cells were found to be associated with L-LEP. These findings revealed the primary suppressive landscape in the L-LEP patients, providing potential targets for the intervention of intracellular bacteria caused persistent infections.

A Case of Untreated Myeloid Sarcoma of the Pancreas Head Region: Diagnostic Process of AML Subtyping in an Autoptic Case

This study describes an autopsy case of pancreatic/peripancreatic myeloid sarcoma in a 70-year-old man, initially presenting with obstructive jaundice. Pathologically, diffuse infiltration of round cells containing atypical nuclei with marked cleavage was observed mainly in the pancreas head, peripancreatic lymph nodes, spleen, bilateral lung, and bone marrow. Immunohistochemically, the tumor cells were negative for CD20, CD79a, CD3, CD5, c-kit, CD34, and TdT and positive for myeloperoxidase, CD33, CD68, and CD163. Flow cytometry of the peripheral blood showed underexpression of CD11c and aberrant expression of CD56 in the monocyte subset. The peripheral blood smear showed an increase in monocytes and atypia in neutrophils and monocytes, as well as enlarged platelets and polychromatic erythroblasts. Hence, it was suggested that the myeloid sarcoma was derived from the acute transformation of chronic myelomonocytic leukemia. Myeloid sarcoma is an extramedullary-mass-forming hematologic malignancy that is difficult to diagnose, especially when the initial presentation is a pancreatic mass. However, early diagnosis is important for appropriate therapy. Although bone marrow examination could not be performed because of the patients’ severe condition, the pathological specimen obtained with autopsy helped subtype the patient’s leukemia. The immunohistochemical features of this case merit attention.

Phenotypic and Functional Heterogeneity of Monocyte Subsets in Chronic Heart Failure Patients

Chronic heart failure (CHF) results when heart cannot constantly supply the body tissues with oxygen and required nutrients, and it can be categorized as heart failure (HF) with preserved ejection fraction (HFpEF), and HF with reduced ejection fraction (HFrEF). There are different causes and mechanisms of the HF pathogenesis; however, the inflammation can be regarded as one of the factors promoting both HFrEF and HFpEF. Monocytes, a subgroup of leucocytes, are known as cellular mediators in response to cardiovascular injury and are closely related to inflammatory reactions. These cells are a vital component of the immune system and are the source of macrophages, which participate in cardiac tissue repair after injury. However, the monocytes are not homogenous as thought, and thus can present different functions under different cardiovascular disease conditions. In addition, there is still an open question whether the functions of monocytes and macrophages should be regarded as a cause or a consequence in CHF development. Therefore, our aim was to summarize the current studies on the function of various monocyte subsets in CHF with a focus on the role of a certain monocyte subset in HFpEF and HFrEF patients, and the relation to inflammatory markers.

162 Whole-body cryotherapy treatment modulates the innate immune response in non-professional football players

Aims Whole-body cryotherapy (WBC) is a recently widely strategy used for muscle recovery after injury that can to activate inflammatory response. WBC consists of short exposure, of about 2-3 minutes, to dry air at cryogenic temperatures up to -190 °C. The aim of our study is to analyze WBC effects on metabolic, hormonal, and immunological responses of non-professional football players (NPFPs). Methods and results Nine male NPFPs (age: 20 ± 2 years) on the same team are recruited and, in particular, they played and trained each day before, during, and after WBC treatment. We collected NPFPs blood samples immediately before WBC and after 5 once-day sessions, then we evaluated a set consisting of 50 analytes, including hormones profile, haematologic parameters, and serum chemistry. We proceeded with monocytes (Mo) phenotyping and then we investigated the concentration of some plasmatic markers with anti-inflammatory effects (IL2RA, IL1RN) or typically increased during inflammation [CCL2, IL-18, free mitochondrial DNA (mtDNA)]. WBC treatment (WBC-t) lead to a decrease not only in mean platelet volume, mean corpuscular haemoglobin, and ferritin levels, but also in testosterone and estradiol levels, even if they remain within the normal ranges. This treatment is also responsible for total Mo increased and, in particular, a reduction of classical Mo has been demonstrated in parallel with an increase of non-classical ones. Moreover, each Mo subset shows lower expression of CXCR4. Considering pro-inflammatory molecules, IL1RA showed a tendency to decrease, while IL1RN and IL18 decreased in plasma after WBC-t. Conversely, circulating mtDNA levels appeared unaltered by treatment. Conclusions The differences detected in monocyte subset after WBC-t suggest that, in this condition, Mo could be redistributed into the surrounding tissue. In addition, CXCR4 reduction in Mo subsets could be due to their differentiation process. Hence, WBC could promote Mo differentiation through a mechanism that is still unknown. Since WBC seems to regulate the innate immune system in the enrolled NPFPs, it could have a role in tissue repair beyond a beneficial anti-inflammatory effect.

Single-cell RNA-sequencing reveals distinct immune cell subsets and signaling pathways in IgA nephropathy

Background IgA nephropathy (IgAN) is the most common primary glomerulonephritis globally. Increasing evidence suggests the importance of host immunity in the development of IgAN, but its dynamics during the early stage of IgAN are still largely unclear. Results Here we successfully resolved the early transcriptomic changes in immune cells of IgAN by conducting single-cell RNA-sequencing (scRNA-seq) with peripheral blood mononuclear cells. The differentially expressed genes (DEGs) between control and IgAN were predominantly enriched in NK cell-mediated cytotoxicity and cell killing pathways. Interestingly, we discovered that the number and cytotoxicity of NK cells are significantly reduced in IgAN patients, where both the number and marker genes of NK cells were negatively associated with the clinical parameters, including the levels of urine protein creatinine ratio (UPCR), serum galactose-deficient IgA1 and IgA. A distinctive B cell subset, which had suppressed NFκB signaling was predominantly in IgAN and positively associated with disease progression. Moreover, the DEGs of B cells were enriched in different viral infection pathways. Classical monocytes also significantly changed in IgAN and a monocyte subset expressing interferon-induced genes was positively associated with the clinical severity of IgAN. Finally, we identified vast dynamics in intercellular communications in IgAN. Conclusions We dissected the immune landscape of IgAN at the single-cell resolution, which provides new insights in developing novel biomarkers and immunotherapy against glomerulonephritis.

Case Report: Evolution of a Severe Vascular Refractory Form of ECD Requiring Liver Transplantation Correlated With the Change in the Monocyte Subset Analysis

Erdheim–Chester disease is a rare histiocytosis characterized by iconic features associated with compatible histology. Most patients have somatic mutations in the MAP-kinase pathway gene, and the mutations occur in CD14+ monocytes. Differentiation of the myeloid lineage plays a central role in the pathogenesis of histiocytosis. Monocytes are myeloid-derived white blood cells, divided into three subsets, but only the CD14++CD16− “classical monocyte” can differentiate into dendritic cells and tissue macrophages. Since most mutations occur in CD14+ cells and since ECD patients have a particular monocytic phenotype resembling CMML, we studied the correlation between disease activity and monocytic subset distribution during the course of a severe vascular form of ECD requiring liver transplantation. During early follow-up, increased CD14++CD16− “classical monocyte” associated with decreased CD14lowCD16++ “non-classical monocyte” correlated with disease activity. Further studies are needed to confirm the use of monocyte as a marker of disease activity in patients with ECD.

Non-Classical Monocyte Abundance Is an Independent Adverse Risk Factor for Relapse in Pediatric B-ALL

Background Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer and while curable in the majority of cases, 15%-20% of children will relapse with only 50% surviving long-term. Treatment failures arise from the outgrowth of pre-existing or acquired sub-clones that are genetically or epigenetically primed to resist treatment. In addition, the bone marrow microenvironment is known to provide a protective niche. We performed the first mapping of the human B-cell ALL (B-ALL) immune bone marrow (BM) microenvironment at single cell resolution at diagnosis, remission and relapse (Witkowski, 2020). We uncovered a striking rewiring of the myeloid compartment during B-ALL progression with significant over-representation of a leukemia-associated monocyte subpopulation expressing high levels of the Macrophage Colony Stimulating Factor Receptor (M-CSFR/CSF1R). Using both peripheral blood (PB) complete blood count analysis and RNA-seq data, we demonstrated that high monocyte abundance at B-ALL diagnosis is predictive of inferior pediatric and adult overall survival in two large independent clinical cohorts. To determine the association of non-classical monocyte abundance in BM and PB with risk of relapse, we examined a cohort of clinical samples from children enrolled on Children's Oncology Group (COG) protocols. Methods Using an unmatched case-control design, we established a preliminary cohort of PB and BM samples collected at diagnosis from 24 B-ALL patients with eventual relapse and 24 patients in long-term remission. Four remission samples from an NYU Langone cohort were used to validate the expansion of this population in the presence of B-ALL. We applied a customized flow cytometry based assay to identify CD115-expressing human monocyte subsets: classical (CD45 +CD56 -CD14 +CD16 -), non-classical (CD45 +CD56 -CD14 -CD16 +), as well as B-cells (CD19, CD22, CD10) and T/NK cells (CD3, CD56). We then performed univariate and multivariable analysis of outcome (relapse versus long-term remission) compared to monocyte subset abundance, adjusting for potential confounding factors (age, gender, CNS status, NCI risk, genetic subtype, WBC at diagnosis, and end of induction minimal residual disease). Results We observed a significantly higher percentage of non-classical monocytes in the diagnostic BM from the COG cohort when compared to remission samples (COG diagnostic B-ALL BM non-classical percentage mean 52.19% vs NYU B-ALL remission BM non-classical percentage mean 1.775%, P = 0.0001). We also observed a strong correlation between the percentage of non-classical monocytes in the PB when compared to their matched BM specimens (r = 0.6, P = 0.0001). Multivariable analysis revealed a significant association between PB non-classical monocyte percentage at diagnosis and patient outcome (remission cohort non-classical monocyte percentage [mean, range]: 52.4%, 33.3-68.1%, n = 24, relapse cohort non-classical monocyte percentage: 65.9%, 56.4-84.7%, n = 24, P = 0.021). Similarly, a strong trend was observed in the BM, although it did not reach statistical significance. Flow cytometric analysis confirmed CD115 (M-CSFR/CSF1R) expression in this non-classical monocyte population, thereby validating a novel target for intervention. Conclusions These findings validate the presence of a unique monocyte subpopulation associated with childhood B-ALL and suggests that assessing this population in PB at diagnosis may be of prognostic significance. The availability of small molecule inhibitors and monoclonal antibodies targeting CSF1R-expressing monocytes may offer a novel approach to treating B-ALL. Figure 1 Figure 1. Disclosures Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Teachey: Sobi: Consultancy; NeoImmune Tech: Research Funding; Janssen: Consultancy; BEAM Therapeutics: Consultancy, Research Funding. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy.

ELN IMDS Flow Working Group Validation of the Monocyte Assay for Chronic Myelomonocytic Leukemia Diagnosis By Flow Cytometry

Introduction It was proposed that peripheral blood (PB) monocyte subset analysis evaluated by flow cytometry, hereafter referred to as "monocyte assay", could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay required a large multicenter validation. Methods PB and/or bone marrow (BM) samples from adult patients displaying monocytosis were assessed with the "monocyte assay" by ten ELN iMDS Flow working group centers (6 equipped with BD FACSCanto™ II (BD Biosciences), 3 with Navios™ (Beckman Coulter) and one with BD™ LSRII (BD Biosciences)) with harmonized protocols. The corresponding files were reanalyzed in a blind fashion by a skilled operator and the cMo (CD14 ++CD16 -) percentages obtained by both analyses were compared. Information regarding age, gender, complete blood count, marrow cytomorphology, cytogenetics and molecular analysis was collected. Confirmed diagnoses were collected when available as well as follow-up for CMML patients. Results The comparison between cMo percentages from 267 PB files provided by the 10 centers and the centralized cMo percentages showed a good global significant correlation (r=0.88; p<0.0001; FigA) with no bias (FigB). Confirmed diagnoses were available for 212 files, namely 101 CMML according to the WHO criteria, 99 reactive monocytosis, and 12 MPN with monocytosis. A phenotype in favor of CMML, either classical with accumulation of cMo ≥94% or a bulbous aspect (FigC), was observed respectively in 81 and 14 patients. Hence, a total of 95 out of the 101 CMML patients translated into a sensitivity of 94% (FigD). Assessment of C reactive protein counts were available in seven of the 14 patients with the characteristic bulbous profile and correlated with an inflammatory state, showing a median of 93.0 [7.0-157.4] mg/L. Conversely, a phenotype not in favor of CMML (FigC) was observed in 83 of the 99 patients with reactive monocytosis and in 10 of 12 patients with MPN with monocytosis, leading to a 84% specificity (FigD). We established a Receiver Operator Curve (ROC) and again obtained a 94% cut-off value of cMo with an area under the ROC curve (AUC) of 0.865 (FigE). The second aim of this multicenter study was to assess the feasibility of the monocyte assay on 117 BM samples provided by 7 out of the 10 ELN centers, 43 of which being paired to PB samples. The comparison between cMo percentages provided by the 7 centers and the centralized cMo percentages showed a lower global significant correlation compared to PB samples (r=0.74; p<0.0001; FigF) with a slight underestimation of cMo percentage by the participating centers (FigG). The comparison between PB and BM samples cMo% obtained by centralized reanalysis showed an excellent global correlation (r=0.93; p<0.0001; FigH) with a higher percentage in the marrow (FigI). Seventy-nine files were associated to a confirmed diagnosis, as expected mostly CMML (n=69), only seven reactive monocytosis and three MPN with monocytosis. Thus, we determined a sensitivity of the "monocyte assay" on BM samples of 87% (a phenotype in favor of CMML being observed in 60 out of the 69 CMML with 6 bulbous aspect profiles) and a specificity of 80% (a phenotype not in favor of CMML being observed in 5 of the 7 patients with reactive monocytosis and in 3 of the 3 patients with MPN with monocytosis). Conclusions This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples. Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

A Sex-Stratified Analysis of Monocyte Phenotypes Associated with HIV Infection in Uganda

Women with HIV may experience higher rates of non-AIDS comorbidities compared to men with HIV, but the underlying mechanisms are not well understood. We investigated sex-related differences in the effects of HIV on monocyte phenotypes within the Ugandan Study of HIV effects on the Myocardium and Atherosclerosis (mUTIMA). Of 133 participants who provided blood for flow cytometry assays, 86 (65%) were women and 91 (68%) were persons living with HIV (PLWH) on antiretroviral therapy. The median age was 57 (interquartile range, 52–63) years. PLWH exhibited a lower proportion of circulating CD14+CD16- classical monocytes (66.3% vs. 75.1%; p < 0.001), and higher proportion of CD14+CD16+ inflammatory monocytes (17% vs. 11.7%; p = 0.005) compared to HIV-uninfected participants. PLWH had an increased expression of the chemokine receptor CX3CR1 in total monocytes (CX3CR1+ monocytes, 24.5% vs. 4.7%; p < 0.001) and monocyte subsets. These findings were generally similar when analyzed by sex, with no significant interactions between sex and HIV status in adjusted models. Our data show that the inflammatory monocyte subset is expanded and monocyte CX3CR1 chemokine receptor expression is enhanced among PLWH, regardless of sex. Whether these parameters differentially affect risk for non-AIDS comorbidities and clinical outcomes in women with HIV requires additional investigation.