Characteristics of isatuximab-derived interference in serum protein electrophoresis and immunofixation, and an absence of sustained in vivo interference due to belantamab mafodotin and denosumab.

OBJECTIVES: Some therapeutic monoclonal antibodies, like daratumumab and elotuzumab, produce interfering monoclonal bands on serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE). Whether other common therapeutic antibodies also produce interference has not been systematically evaluated. DESIGN AND METHODS: SPEP/IFE from patients receiving isatuximab (48 patients), belantamab mafodotin (BM; 41), and denosumab (41) were retrospectively reviewed for therapeutic antibody interference. Cases exhibiting isatuximab interference were quantified and the maximum duration of isatuximab effect was evaluated. To characterize band position, neat human serum was spiked with BM or denosumab at supratherapeutic concentrations. Band migration patterns were compared on SPEP and IFE, with band position expressed relative to other constant protein fractions. RESULTS: Isatuximab-induced IFE interference was common (81.3 % of evaluated patients) with a maximum observed duration of 8 weeks. 10.4 % of isatuximab patients had IgG kappa monoclonal gammopathies that co-migrated with the drug; this subset could benefit from HYDRASHIFT 2/4 isatuximab testing. 8.3 % of IFE cases were negative for an isatuximab band but showed large, endogenous M-spikes migrating elsewhere. All patients in this group expired within 1 year of this finding. We hypothesize that an inability to detect isatuximab in this setting corresponds to a large residual myeloma burden that reduces isatuximab serum concentration. This observation may serve as a negative prognostic factor. Spiking studies demonstrated that BM and denosumab produce interference in vitro, but sustained interference was not observed in >40 treated patients. CONCLUSIONS: Therapeutic antibody interference in patients receiving isatuximab is common, and can persist for at least 8 weeks after administration. >10 % of patients receiving isatuximab may benefit from HYDRASHIFT testing post-therapy. In contrast, BM and denosumab fail to produce sustained interference in treated patients.

Flow cytometry quantification of tumor-infiltrating lymphocytes to predict the survival of patients with diffuse large B-cell lymphoma.

Introduction: Our previous studies have demonstrated that tumor-infiltrating lymphocytes (TILs), including normal B cells, T cells, and natural killer (NK) cells, in diffuse large B-cell lymphoma (DLBCL) have a significantly favorable impact on the clinical outcomes of patients treated with standard chemoimmunotherapy. In this study, to gain a full overview of the tumor immune microenvironment (TIME), we assembled a flow cytometry cohort of 102 patients diagnosed with DLBCL at the Duke University Medical Center. Methods: We collected diagnostic flow cytometry data, including the proportion of T cells, abnormal B cells, normal B cells, plasma cells, NK cells, monocytes, and granulocytes in fresh biopsy tissues at clinical presentation, and analyzed the correlations with patient survival and between different cell populations. Results: We found that low T cell percentages in all viable cells and low ratios of T cells to abnormal B cells correlated with significantly poorer survival, whereas higher percentages of normal B cells among total B cells (or high ratios of normal B cells to abnormal B cells) and high percentages of NK cells among all viable cells correlated with significantly better survival in patients with DLBCL. After excluding a small number of patients with low T cell percentages, the normal B cell percentage among all B cells, but not T cell percentage among all cells, continued to show a remarkable prognostic effect. Data showed significant positive correlations between T cells and normal B cells, and between granulocytes and monocytes. Furthermore, we constructed a prognostic model based on clinical and flow cytometry factors, which divided the DLBCL cohort into two equal groups with remarkable differences in patient survival and treatment response. Summary: TILs, including normal B cells, T cells, and NK cells, are associated with favorable clinical outcomes in DLBCL, and flow cytometry capable of quantifying the TIME may have additional clinical utility for prognostication.

The Distinctive Nature of Thyroid MALT Lymphomas Including IRTA1 Expression.

Mucosa-associated lymphoid tissue (MALT) lymphomas often express IgM and IRTA1 with only a minority demonstrating plasmacytic differentiation. However, like primary cutaneous marginal zone lymphoproliferative disorders (PCMZLPD), thyroid MALT lymphomas (T-MALT-L) frequently show plasmacytic differentiation and IgG positivity. Whether T-MALT-L share other features with PCMZLPD, including frequent IgG4 positivity and infrequent IRTA1 expression, and how IRTA1 staining compares to that in Hashimoto thyroiditis (HT) are unknown. Therefore, the clinicopathologic features of 18 T-MALT-L were assessed, and their IRTA1 expression compared with that in 5 HT cases. All T-MALT-L cases included a B-lymphoid component. Plasmacytic differentiation was present in 15 cases and was extensive in 12. Fourteen cases were IgG+ including 2 IgG4+ (12 κ+, 2 κ-/λ-). One case was IgAλ+. Plasmacytic cells were uniformly CD19+/CD56- but CD138- in 7/15 cases. IRTA1+ cells were present in 16/16 cases, ranging from scattered cells to >50%. They were often concentrated in "MALT ball"-type lymphoepithelial lesions, perifollicular regions, and sometimes in germinal centers. IRTA1 positivity was also present in all HT cases, although it was never very extensive and often had a perifollicular distribution, occasionally with sparse aggregates and positive cells within rare thyroid follicles. Thus, T-MALT-L share some features with PCMZLPD but are more similar to noncutaneous MALT lymphomas, with prominent lymphoepithelial lesions, ubiquitous although variable IRTA1 positivity, and infrequent IgG4 positivity. Plasmacytic differentiation is also common although CD138 loss is frequent and light chain staining may be absent. IRTA1 staining may help in the differential diagnosis with HT, although there is some overlap in staining patterns.

Characterization of Casirivimab Plus Imdevimab, Sotrovimab, and Bamlanivimab Plus Etesevimab-Derived Interference in Serum Protein Electrophoresis and Immunofixation Electrophoresis.

BACKGROUND: Therapeutic monoclonal antibodies can be a source of assay interference in clinical serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE), producing monoclonal bands that can be misinterpreted as a monoclonal gammopathy related to a B-cell or plasma cell neoplasm. The extent to which new anti-COVID-19 monoclonal antibodies produce this interference is unknown. METHODS: Casirivimab plus imdevimab, sotrovimab, and bamlanivimab plus etesevimab were spiked into patient serum samples to evaluate for SPEP/IFE interference, to characterize the position of therapy-derived bands relative to a reference band (either combined beta band or beta 1 band, depending on instrument platform), and to confirm heavy and light chain utilization of each medication. Serum samples from patients who had recently received casirivimab plus imdevimab or sotrovimab were also evaluated for comparison. RESULTS: When spiked into serum samples, all tested anti-COVID-19 monoclonal antibodies generated interference in SPEP/IFE. Importantly, the patterns of interference differed between spiked serum samples and serum from patients who had recently received casirivimab plus imdevimab or sotrovimab. CONCLUSIONS: Imdevimab can be added to the growing list of therapeutic monoclonal antibodies that produce sustained interference in SPEP/IFE. Although casirivimab and sotrovimab also produce assay interference in vitro, these antibodies are not reliably detected in serum from recently infused patients. The value of relative band position in recognizing bands that may represent therapeutic monoclonal antibodies is also emphasized. Clinicians and laboratorians should consider therapeutic monoclonal antibody interference in diagnostic SPEP/IFE and review a patient's medication list when new or transient monoclonal bands are identified.

IRTA1 positivity helps identify a MALT-lymphoma-like subset of primary cutaneous marginal zone lymphomas, largely but not exclusively defined by IgM expression.

BACKGROUND: It has been proposed that primary cutaneous marginal zone lymphomas (PCMZLs) include a MALT-lymphoma-like IgM+ subset and a class-switched subset, which is unlike most other MALT lymphomas. Whether expression of the MALT lymphoma-associated biomarkers IRTA1 and MNDA would support this concept and whether they might help explain why some patients have both subtypes is uncertain. METHODS: Twenty-five PCMZLs from 21 patients were stained for IRTA1 by in situ hybridization and for MNDA by immunohistochemistry. In two patients, polymerase chain reaction (PCR)-based B-cell clonality studies were performed on biopsy specimens of metachronous lesions, which expressed different heavy chains. All results were correlated with the histopathologic and clinical findings. RESULTS: Five of six IgM+ PCMZLs were IRTA1+ vs three of 18 evaluable class-switched cases (P = 0.0069). Two of the class-switched IRTA1+ cases were in patients with clonally-related IRTA1+ IgM+ PCMZLs. IRTA1 positivity showed a statistically significant correlation with several MALT-lymphoma-associated histopathologic findings. In contrast, all PCMZL cases showed at least some MNDA expression with no differences between IgM+ and class-switched cases. CONCLUSIONS: IRTA1 identifies MALT-lymphoma-like PCMZLs that are largely but not exclusively IgM+. This supports the concept of two PCMZL subsets but suggests their distinction should not be based solely on their heavy chain expression.

Class-switched Primary Cutaneous Marginal Zone Lymphomas Are Frequently IgG4-positive and Have Features Distinct From IgM-positive Cases.

Primary cutaneous marginal zone lymphoma (PCMZL) can be subdivided into 2 groups based on immunoglobulin (Ig) heavy chain usage: IgM-positive cases that constitute a less common and more T-helper type 1-driven process, and more common heavy chain class-switched cases that are predominantly T-helper type 2-driven. Although some report a significant IgG4-positive subset, others have found a much smaller proportion. To further evaluate the proportion of IgG4-positive PCMZL, to address whether IgG4-positive cases have any distinctive characteristics, and to assess whether additional features separating IgM-positive and class-switched cases could be identified, the clinicopathologic features of 26 PCMZL obtained from 19 patients were investigated. Twenty of 26 (77%) PCMZL were heavy chain class-switched (19 IgG-positive, 1 IgA-positive), including 9 that were IgG4-positive (35%). IgG4-positive and other class-switched PCMZL were morphologically similar. IgM-positive cases occurred in older individuals (median: 69 vs. 46.5 y; P=0.0001), more often involved the subcutis (P=0.002), demonstrated plasma cells diffusely scattered versus at the periphery of the lymphoid infiltrate (P=0.005), uniformly showed follicular colonization (P=0.0001), contained more numerous B cells (P=0.0004), and were more likely to have a T-cell CD4:CD8 ratio of <3:1 (P=0.03). None of the IgM-positive PCMZL harbored a MYD88 L265P mutation. No significant differences in clinical outcome were documented. These results highlight the high frequency of IgG4-positive PCMZL, which are otherwise similar to other class-switched cases, provide additional evidence supporting the distinction between class-switched and IgM-positive cases, and emphasize the indolent nature of at least the class-switched PCMZL, which may warrant their categorization as a clonal chronic lymphoproliferative disorder.

NF2 and ATRX gene copy number losses on a case of ovarian ependymoma.

Ovarian ependymomas are rare glial neoplasms that typically occur in women on their third to fourth decades of life. They are histologically similar to ependymomas of the central nervous system but may have a broader immunophenotype. We describe a 27-year-old woman who presented to the emergency department with a 3-week history of cough and shortness of breath. Further workup disclosed a left pelvic mass and extensive intra-abdominal metastases. Pathology revealed sheets of monomorphic cells within a fibrillary stroma, papillary projections, true ependymal rosettes, and pseudorosettes consistent with an ependymoma of ovarian origin. Next-generation sequencing showed ATRX and NF2 copy number losses. Fluorescence in situ hybridization for EWSR1 demonstrated monosomy of 22q in greater than 90% of cells. These molecular alterations have not been previously reported in ovarian or extra-central nervous system ependymomas.

mTOR Mediates IL-23 Induction of Neutrophil IL-17 and IL-22 Production.

It has been shown recently that neutrophils are able to produce IL-22 and IL-17, which differentially regulate the pathogenesis of inflammatory bowel disease. However, it is still largely unknown how the neutrophil production of IL-22 and IL-17 is regulated, and their role in the pathogenesis of inflammatory bowel disease. In this study, we found that IL-23 promoted neutrophil production of IL-17 and IL-22. IL-23 stimulated the neutrophil expression of IL-23R as well as rorc and ahr. Retinoid acid receptor-related orphan receptor γ t and aryl-hydrocarbon receptor differentially regulated IL-23 induction of neutrophil IL-17 and IL-22. In addition, IL-23 induced the activation of mTOR in neutrophils. Blockade of the mTOR pathway inhibited IL-23-induced expression of rorc and ahr, as well as IL-17 and IL-22 production. By using a microbiota Ag-specific T cell-mediated colitis model, we demonstrated that depletion of neutrophils, as well as blockade of IL-22, resulted in a significant increase in the severity of colitis, thereby indicating a protective role of neutrophils and IL-22 in chronic colitis. Collectively, our data revealed that neutrophils negatively regulate microbiota Ag-specific T cell induction of colitis, and IL-23 induces neutrophil production of IL-22 and IL-17 through induction of rorc and ahr, which is mediated by the mTOR pathway.

Interactions between Neutrophils and Leishmania braziliensis Amastigotes Facilitate Cell Activation and Parasite Clearance.

Leishmania braziliensis and Leishmania amazonensis are both causative agents of cutaneous leishmaniasis in South America. However, patient prognosis and the host immune response differ considerably depending on the infecting parasite species. The mechanisms underlying these differences appear to be multifactorial, with both host and parasite components contributing to disease outcome. As neutrophils are a prominent component of the inflammatory infiltrate in chronic cutaneous, diffuse cutaneous and mucocutaneous lesions, we examined neutrophil activation and microbicidal activity against amastigotes of L. amazonensis and L. braziliensis. We found that murine neutrophils internalized L. braziliensis amastigotes with greater efficiency than did L. amazonensis amastigotes. Additionally, L. braziliensis infection was a potent trigger for neutrophil activation, oxidative burst, degranulation and the production of interleukin (IL)-22 and IL-10, while L. amazonensis amastigotes poorly induced these responses. Finally, neutrophils were able to kill L. braziliensis amastigotes, especially when cells were activated with phorbol myristate acetate. L. amazonensis amastigotes, however, were highly resistant to neutrophil microbicidal mechanisms. This study reveals, for the first time, differential neutrophil responsiveness to distinct species of Leishmania amastigotes and highlights the complexity of neutrophil-amastigote interactions during chronic leishmaniasis.

Leishmania amazonensis amastigotes highly express a tryparedoxin peroxidase isoform that increases parasite resistance to macrophage antimicrobial defenses and fosters parasite virulence.

Professional phagocytes generate a myriad of antimicrobial molecules to kill invading microorganisms, of which nitrogen oxides are integral in controlling the obligate intracellular pathogen Leishmania. Although reactive nitrogen species produced by the inducible nitric oxide synthase (iNOS) can promote the clearance of intracellular parasites, some Leishmania species/stages are relatively resistant to iNOS-mediated antimicrobial activity. The underlying mechanism for this resistance remains largely uncharacterized. Here, we show that the amastigote form of L. amazonensis is hyper-resistant to the antimicrobial actions of cytokine-activated murine and human macrophages as compared to its promastigote counterpart. Amastigotes exhibit a marked ability to directly counter the cytotoxicity of peroxynitrite (ONOO-), a leishmanicidal oxidant that is generated during infection through the combined enzymatic activities of NADPH oxidase and iNOS. The enhanced antinitrosative defense of amastigotes correlates with the increased expression of a tryparedoxin peroxidase (TXNPx) isoform that is also upregulated in response to iNOS enzymatic activity within infected macrophages. Accordingly, ectopic over-expression of the TXNPx isoform by L. amazonensis promastigotes significantly enhances parasite resistance against ONOO- cytotoxicity. Moreover, TXNPx-overexpressing parasites exhibit greater intra-macrophage survival, and increased parasite growth and lesion development in a murine model of leishmaniasis. Our investigations indicate that TXNPx isoforms contribute to Leishmania's ability to adapt to and antagonize the hostile microenvironment of cytokine-activated macrophages, and provide a mechanistic explanation for persistent infection in experimental and human leishmaniasis.

Hepatocyte growth factor in dampening liver immune-mediated pathology in acute viral hepatitis without compromising antiviral activity.

BACKGROUND AND AIM: Hepatocyte growth factor (HGF) is a pleiotropic cytokine related with cell proliferation and survival; however, its role in viral hepatitis is not elucidated. In this study, we studied HGF immune role in viral hepatitis. METHODS: Mice received hydrodynamically delivered HGF plasmid or control plasmid and then infected with adenovirus, and parameters of immune-mediated liver damage were evaluated. We studied dendritic cell (DC) activation in the presence of HGF. T cells collected from infected mice were restimulated with virally infected DC to measure cytokine production in vitro. RESULTS: HGF ameliorated the liver inflammation during viral hepatitis as alanine transferase, intrahepatic lymphocytes, and splenocyte counts were diminished by HGF. Lower histological scores of liver pathology were observed in the HGF group. DC from the HGF group expressed reduced CD40. The hepatic expression and serum concentration of IL-12p40 were diminished in HGF-transfected mice. In vitro experiments with DC confirmed that HGF diminished CD40 expression and IL-12p40 production. The expression and serum levels of IFN-γ, IL-6 and CXCL9 were significantly decreased in the HGF group. HGF overexpression diminished the expression and concentration of IL-10 and TGF-ß. The frequency of PD-1(+) Tim-3(+) in CD8 T cells was decreased by HGF overexpression. Moreover, T cells in the HGF group at day 14 secreted more IFN-γ and TNF-α than those in the control group when restimulated with virally infected DC. CONCLUSION: HGF modulated DC activation and T cell priming, thereby limiting the immune-mediated damage in the liver. However, viral clearance was not compromised by HGF.

Downregulation of microRNA-107 in intestinal CD11c(+) myeloid cells in response to microbiota and proinflammatory cytokines increases IL-23p19 expression.

Commensal flora plays an important role in the development of the mucosal immune system and in maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host-microbiota interaction are still not completely understood. In this study, we examined how microbiota and intestinal inflammatory conditions regulate host microRNA expression and observed lower microRNA-107 (miR-107) expression in the inflamed intestines of colitic mice, compared with that in normal control mice. miR-107 was predominantly reduced in epithelial cells and CD11c(+) myeloid cells including dendritic cells and macrophages in the inflamed intestines. We demonstrate that IL-6, IFN-γ, and TNF-α downregulated, whereas TGF-ß promoted, miR-107 expression. In addition, miR-107 expression was higher in the intestines of germ-free mice than in mice housed under specific pathogen-free conditions, and the presence of microbiota downregulated miR-107 expression in DCs and macrophages in a MyD88- and NF-κB-dependent manner. We determined that the ectopic expression of miR-107 specifically repressed the expression of IL-23p19, a key molecule in innate immune responses to commensal bacteria. We concluded that regulation of miR-107 by intestinal microbiota and proinflammatory cytokine serve as an important pathway for maintaining intestinal homeostasis.

Leishmania amazonensis amastigotes trigger neutrophil activation but resist neutrophil microbicidal mechanisms.

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.

Immunomodulatory and antibacterial effects of cystatin 9 against Francisella tularensis.

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.