Iberdomide increases innate and adaptive immune cell subsets in the bone marrow of patients with relapsed/refractory multiple myeloma.

Iberdomide is a potent cereblon E3 ligase modulator (CELMoD agent) with promising efficacy and safety as a monotherapy or in combination with other therapies in patients with relapsed/refractory multiple myeloma (RRMM). Using a custom mass cytometry panel designed for large-scale immunophenotyping of the bone marrow tumor microenvironment (TME), we demonstrate significant increases of effector T and natural killer (NK) cells in a cohort of 93 patients with multiple myeloma (MM) treated with iberdomide, correlating findings to disease characteristics, prior therapy, and a peripheral blood immune phenotype. Notably, changes are dose dependent, associated with objective response, and independent of prior refractoriness to MM therapies. This suggests that iberdomide broadly induces innate and adaptive immune activation in the TME, contributing to its antitumor efficacy. Our approach establishes a strategy to study treatment-induced changes in the TME of patients with MM and, more broadly, patients with cancer and establishes rational combination strategies for iberdomide with immune-enhancing therapies to treat MM.

Cellular mechanisms associated with sub-optimal immune responses to SARS-CoV-2 bivalent booster vaccination in patients with Multiple Myeloma.

BACKGROUND: The real-world impact of bivalent vaccines for wild type (WA.1) and Omicron variant (BA.5) is largely unknown in immunocompromised patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster, including neutralizing assays to identify patterns associated with continuing vulnerability to current variants (XBB1.16, EG5) in the current post-pandemic era. METHODS: We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48 MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assays to assess inhibition of live virus. We measured spike specific T-cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow-cytometry based T-cell. In a subset of 38 patients, high-dimensional flow cytometry was performed to identify immune cell subsets associated with lack of humoral antibodies. FINDINGS: We find that bivalent vaccination provides significant boost in protection to the omicron variant in our MM patients, in a treatment specific manner. MM patients remain vulnerable to newer variants with mutations in the spike portion. Anti-CD38 and anti-BCMA therapies affect the immune machinery needed to produce antibodies. INTERPRETATION: Our study highlights varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairment in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate humoral and cellular responses to vaccination. FUNDING: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), NCI Serological Sciences Network for COVID-19 (SeroNet) and The Icahn School of Medicine at Mount Sinai.

Neurocognitive and hypokinetic movement disorder with features of parkinsonism after BCMA-targeting CAR-T cell therapy.

B-cell maturation antigen (BCMA) is a prominent tumor-associated target for chimeric antigen receptor (CAR)-T cell therapy in multiple myeloma (MM). Here, we describe the case of a patient with MM who was enrolled in the CARTITUDE-1 trial ( NCT03548207 ) and who developed a progressive movement disorder with features of parkinsonism approximately 3 months after ciltacabtagene autoleucel BCMA-targeted CAR-T cell infusion, associated with CAR-T cell persistence in the blood and cerebrospinal fluid, and basal ganglia lymphocytic infiltration. We show BCMA expression on neurons and astrocytes in the patient's basal ganglia. Public transcriptomic datasets further confirm BCMA RNA expression in the caudate of normal human brains, suggesting that this might be an on-target effect of anti-BCMA therapy. Given reports of three patients with grade 3 or higher parkinsonism on the phase 2 ciltacabtagene autoleucel trial and of grade 3 parkinsonism in the idecabtagene vicleucel package insert, our findings support close neurological monitoring of patients on BCMA-targeted T cell therapies.

TGF-ß1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis.

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-ß plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-ß1 than TGF-ß2 and TGF-ß3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-ß1/TGF-ß3 protein trap, to block the excessive TGF-ß signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-ß1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-ß1-induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-ß1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

A streamlined whole blood CyTOF workflow defines a circulating immune cell signature of COVID-19.

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining, and data acquisition protocols can all introduce technical variation that can confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We apply this workflow to characterize 184 whole blood samples collected longitudinally from a cohort of 72 hospitalized COVID-19 patients and healthy controls, highlighting dynamic disease-associated changes in circulating immune cell frequency and phenotype.

A Streamlined CyTOF Workflow To Facilitate Standardized Multi-Site Immune Profiling of COVID-19 Patients.

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 single parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining and data acquisition protocols can all introduce technical variation that can potentially confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We further demonstrate the application of this workflow to characterize immune responses in a cohort of hospitalized COVID-19 patients, highlighting key disease-associated changes in immune cell frequency and phenotype.

Vagaries of the ELISpot assay: specific detection of antigen responsive cells requires purified CD8(+) T cells and MHC class I expressing antigen presenting cell lines.

Quantification of antigen-specific CD8(+) T cells is important for monitoring infection, vaccination, and response to therapy in cancer and immune-mediated diseases. Cytokine enzyme-linked-immunospot (ELISpot) assays are often used for this purpose. We found that substantial spot formation in IFNγ ELISpot assays occurred independently of CD8(+) T cells even when classical MHC class I restricted peptides are used for stimulation. Using fractionated cells and intracellular cytokine staining, the non-CD8(+) T cell IFNγ production was attributed to the CD4(+) T cell fraction. We therefore refined a cell line-based ELISpot assay combining HLA-A*0201 expressing K562 cells for antigen presentation with purified CD8(+) T cells and demonstrated that it specifically detected CD8(+) T cell responses with detection limits comparable to traditional ELISpot assays and dextramer-based quantification. The assay was further adapted to whole antigen responses with antigen (pre-proinsulin)-expressing HLA-A*0201K562 cells. Thus, we revealed and corrected a weak spot of the CD8(+) ELISpot assay.

CD40 agonist antibody mediated improvement of chronic Cryptosporidium infection in patients with X-linked hyper IgM syndrome.

X-linked hyper-IgM syndrome (XHM) is a combined immune deficiency disorder caused by mutations in CD40 ligand. We tested CP-870,893, a human CD40 agonist monoclonal antibody, in the treatment of two XHM patients with biliary Cryptosporidiosis. CP-870,893 activated B cells and APCs in vitro, restoring class switch recombination in XHM B cells and inducing cytokine secretion by monocytes. CP-870,893 infusions were well tolerated and showed significant activity in vivo, decreasing leukocyte concentration in peripheral blood. Although specific antibody responses were lacking, frequent dosing in one subject primed T cells to secrete IFN-g and suppressed oocyst shedding in the stool. Nevertheless, relapse occurred after discontinuation of therapy. The CD40 receptor was rapidly internalized following binding with CP-870,893, potentially explaining the limited capacity of CP-870,893 to mediate immune reconstitution. This study demonstrates that CP-870,893 suppressed oocysts shedding in XHM patients with biliary cryptosporidiosis. The continued study of CD40 agonists in XHM is warranted.

Hierarchical IL-5 expression defines a subpopulation of highly differentiated human Th2 cells.

Each of the three Th2 cytokine genes, IL-4, IL-5, and IL-13, has different functions. We hypothesized that Th2 heterogeneity could yield Th2 subpopulations with different cytokine expression and effector functions. Using multiple approaches, we demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5(+) (IL-5(+), IL-4(+), IL-13(+)) and majority IL-5(-) Th2 (IL-5(-), IL-4(+), IL-13(+)) population. IL-5(+) Th2 cells comprised only 20% of all Th2 cells. Serial rounds of in vitro differentiation initially yielded IL-5(-) Th2, but required multiple rounds of differentiation to generate IL-5(+) Th2 cells. IL-5(+) Th2 cells expressed less CD27 and greater programmed cell death-1 than IL-5(-) Th2 cells, consistent with their being more highly differentiated, Ag-exposed memory cells. IL-5(+) Th2 cells expressed greater IL-4, IL-13, and GATA-3 relative to IL-5(-) Th2 cells. GATA-3 and H3K4me(3) binding to the IL5 promoter (IL5p) was greater in IL-5(+) relative to IL-5(-) Th2 cells, whereas there was no difference in their binding to the IL4p and IL13p. Conversely, H3K27me(3) binding to the IL5p was greater in IL-5(-) Th2 cells. These findings demonstrate Th2 lineage heterogeneity, in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. IL-5(+) Th2 cells are phenotypically distinct and have epigenetic changes consistent with greater IL5p accessibility. Recurrent antigenic exposure preferentially drives the differentiation of IL-5(+) Th2 cells. These results demonstrate that IL-5(+) and IL-5(-) Th2 cells, respectively, represent more and less highly differentiated Th2 cell subpopulations. Such Th2 subpopulations may differentially contribute to Th2-driven pathology.

T(H)2 heterogeneity: Does function follow form?

T(H)2 immune responses are required for the 2 fundamental pathological processes characteristic of allergic disease: IgE-mediated hypersensitivity and eosinophilic inflammation. The 3 established T(H)2 cytokines, IL-4, IL-5, and IL-13, each play a nonredundant role in allergic disease pathology. The recent explosion of T(H) subpopulations combined with the wide availability of polychromatic cytokine staining has facilitated the discovery of T(H)2 lineage heterogeneity. In this article we review T(H)2 heterogeneity and ask the following question: At what point do these subpopulations graduate from in vitro curiosities to immunologically robust therapeutic targets? We propose criteria to establish a T-cell subset as a biologically relevant entity and address the evidence to support these T(H)2 subpopulations having a unique function or specific contribution to allergic pathology or host defense.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response.

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a model antigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection. Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.