Abstract: Despite a recent increase in therapeutic options, patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) eventually require novel therapies. We conducted a phase 1 trial of blinatumomab and lenalidomide in R/R B-NHL. Three dose levels representing 2 schedules were explored. The primary end points were adverse events (AEs) and determining the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D). Thirty-five patients were enrolled, and 34 patients initiated treatment with a median number of prior regimens of 3 (range, 2-8). There were no dose-limiting toxicities (DLTs) in the first 2 dose levels. Dose level 3, 20 mg of lenalidomide daily on days 1 to 21 and days 29 to 49 of a 56-day induction cycle plus blinatumomab 9 μg/d continuous IV infusion (CIVI) on days 1 to 7, 28 μg/d CIVI on days 8 to 14, and 112 μg/d CIVI on days 15 to 56 was determined to be the MTD/RP2D. The most common grade ≥2 AE was neurotoxicity in 11 of 34 patients (32%), with 4 of 16 patients (25%) at the RP2D. At the RP2D, there was 1 DLT, a patient with grade 2 tremor and word-finding difficulty. For all patients completing induction, the overall response rate was 80% (95% confidence interval, 56-94) with a complete response rate of 70%, and 8 of 34 patients (24%) had durable remissions lasting >2 years. GranB+ CD56bright CD16dim CD11b+ natural killer cells and memory regulatory T cells in the peripheral blood at baseline were predictive of response. Concomitant administration of lenalidomide appeared to reduce blinatumomab-mediated T-cell exhaustion. In conclusion, encouraging activity was seen with blinatumomab and lenalidomide in heavily pretreated R/R B-NHL (NCI Protocol no. 9924).

Abstract Introduction Immune checkpoint blockade (ICB) has transformed melanoma care, yet resistance is common and often driven by tumor-associated macrophages (TAMs). AXL, a Gas6-activated tyrosine kinase, is classically a tumor-intrinsic driver of invasion but is predominantly expressed by TAMs in melanoma, suggesting a role in regulating the tumor-immune microenvironment. We tested if AXL inhibition could reprogram macrophage function, disrupt PD-1:PD-L1 interactions, and restore responsiveness to PD-1 blockade. Methods AXL expression was analyzed using TCGA, serum ELISA, and single-cell RNA-seq datasets. ICB-resistant melanoma models (Yumm1.7, B16F10) were treated with warfarin or bemcentinib (AXL inhibitors) alone or with anti-PD-1, with or without macrophage depletion. PD-1:PD-L1 interactions were quantified by immune Förster resonance energy transfer (iFRET). In vitro, polarized macrophages were analyzed for AXL-dependent efferocytosis, T cell crosstalk, and cytokine secretion. Results AXL was enriched in TAMs across cohorts, with soluble AXL highest in stage IV disease. AXL inhibition reduced tumor burden and synergized with anti-PD-1, efficacy was lost after CSF1R depletion and enhanced by F4/80 depletion. iFRET showed restoration of PD-1:PD-L1 blockade despite unchanged PD-L1 expression. In vitro, AXL function was context-dependent: M1-like macrophages upregulated AXL with immunostimulatory outputs, while M2-like macrophages became more AXL-driven under anti-PD-1. Combination therapy reprogrammed the secretome toward a Th1/Th17, chemokine-rich milieu ↑CXCL9/10, IL-12, IL-23; ↓IL-10, MCP-1). Conclusion AXL is a dominant TAM-driven regulator of ICB resistance in melanoma. AXL inhibition restores functional PD-1:PD-L1 blockade and rebalances macrophage signaling toward immunostimulation in a microenvironment-specific manner. These findings support AXL as both a biomarker and therapeutic target to overcome ICB resistance, with implications for tailoring macrophage-targeted strategies in refractory melanoma. Funding Source N/A Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Introduction Advanced age is associated with increased morbidity and mortality following acute viral infections, including SARS-CoV-2. Despite this, most preclinical models rely on young animals and fail to account for age-related immune remodeling. How advanced aging alters antiviral immune responses and contributes to immune-mediated pathology remains incompletely understood.Methods Young (2-6 months), aged (15-18 months), and advanced aged (20-29 months) mice were infected with murine cytomegalovirus (MCMV) or influenza virus. Survival, viral burden, cytokine production, immune cell phenotypes, and tissue pathology were assessed using flow cytometry, histology, serum cytokine analysis, and RNA sequencing. Mouse findings were compared with publicly available transcriptomic datasets from SARS-CoV-2-infected human cohorts across age groups.Results Advanced aged mice exhibited markedly increased mortality and organ pathology following viral infection despite maintaining viral loads comparable to younger mice. These outcomes were associated with heightened systemic and tissue inflammatory cytokine production, reduced antigen-specific T cell responses, and increased frequencies of NK cells and non-antigen-specific bystander T cell activation. Coagulopathy with thrombolytic clot formation was observed exclusively in advanced aged mice. Transcriptomic analysis revealed enrichment of inflammatory and coagulation pathways in influenza-infected advanced aged mice, paralleling findings in elderly humans with SARS-CoV-2 infection, who also displayed reduced expression of T cell-associated genes.Discussion These findings demonstrate that advanced age profoundly alters antiviral immune responses, shifting immunity away from effective antigen-specific T cell responses toward inflammatory and innate pathways that contribute to immune-mediated pathology. The results highlight the importance of modeling advanced aging in preclinical studies and suggest that age-dependent immune imbalance may underlie increased inflammation, coagulopathy, and mortality during viral infection in both mice and humans.

Lupus panniculitis (LP) is a neglected, often treatment-refractory subtype of cutaneous lupus erythematosus. It is characterized by inflammation of subcutaneous adipose tissue, and the pathomechanisms are poorly understood. We sought to explore the cellular and molecular signatures of LP and their relationship to systemic lupus erythematosus. We performed imaging mass cytometry (IMC; LP n = 8, Healthy Control (HC) n = 6) and targeted transcript profiling by NanoString nCounter (Human Immunology Panel, 579 genes; LP n = 9, HC n = 9). LP lesions showed extensive septal leukocyte infiltration. The infiltrate consisted predominantly of T cells (48%), followed by B cells (14%) and antigen-presenting cells (APCs). T cells exhibited a cytotoxic (CD8+, GranzymeB+) and skin-homing (CLA+) phenotype, with upregulation of Th1 markers. They closely interacted with M1 macrophages. B cells displayed strong spatial interactions with naïve T cells. NanoString analyses revealed upregulation of T- and B-cell receptor signaling pathway genes, antigen presentation-related genes (MHC-I/II), and Th1 associated programs (STAT1/IRF1), together with activation of innate immune, complement, and pattern-recognition receptor pathways. This was paralleled by strong upregulation of IDO1, a key enzyme in tryptophan metabolism, and inversely correlated, reduced AHR expression. Our data provide new pathomechanistic insights into LP, highlighting overlaps with systemic lupus, but also LP-specific features. This may pave the way for adopting more targeted treatment approaches for LP.

Langerhans cells express the nonpolymorphic antigen-presenting molecule CD1a, positioning them as contributors to host immunity against Mycobacterium leprae in human leprosy. CD1a was originally shown to present non-canonical lipopeptide antigens such as dideoxymycobactin and chemically diverse hydrophobic ligands. Here, we generated CD4⁺ T cell lines from leprosy lesions that recognized M. leprae in a CD1a-restricted manner. Unexpectedly, antigen recognition was protease-sensitive, prompting biochemical purification that identified two microbial protein antigens: LppX, a 25-kDa lipoglycoprotein, and Ag85A, a 30-kDa secreted protein with no known lipid modification. Recombinant proteins activated the corresponding T cell lines in a CD1a-dependent manner. Epitope mapping identified 12-mer peptides that fully reconstituted antigenicity, were conserved between M. leprae and M. tuberculosis , and elicited robust, dose-dependent IFN-γ production and T cell proliferation, establishing that DNA-encoded, ribosomally translated peptides serve as CD1a-restricted cognate antigens. Biochemical analyses showed peptide binding to CD1a, supported by isoelectric focusing and surface plasmon resonance ( K D ∼75 μM for Ag85A). CD1a-peptide tetramers specifically stained cognate T cells, soluble CD1a was sufficient to present peptide antigen, and transfer of the LppX-specific TCR into naïve T cells restored antigen responsiveness. Using CD1a-peptide tetramers, we identified antigen-specific T cells enriched in patients undergoing reversal reactions compared with patients with lepromatous leprosy and healthy donors. The CD1a-restricted T cell lines secreted IFN-γ and IL-26, cytokines with established antimicrobial activity. Together, these findings demonstrate that CD1a can present canonical microbial peptides as part of a cell-mediated immune response in leprosy, extending the known spectrum of CD1a ligands. Because CD1a is nonpolymorphic and presents antigens to antimicrobial T cells, CD1a-peptide complexes may provide a broadly applicable platform for studying, detecting, and potentially targeting mycobacterial immunity.

Abstract Lymphodepleting preconditioning (LD) is essential for the efficacy of chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies. However, in the setting of autoimmune diseases (ADs), the contribution of LD for efficacy is unclear. Here, we report on the early safety, efficacy, and correlative data of the first 4 patients with pemphigus vulgaris (PV) who received resecabtagene autoleucel (rese-cel), a fully human CD19 CAR T-cell therapy, without LD in the RESET-PV trial, a substudy of the DesCAARTes trial. Following infusion, pemphigus disease area index scores improved significantly in all patients. A favorable safety profile was observed, with a single episode of grade 1 cytokine release syndrome. Immune effector cell–associated neurotoxicity was not observed. Rese-cel expansion was similar in patients with PV compared with other rese-cel–treated patients with AD who received LD. B-cell depletion was observed in all patients with PV, with 3 of 4 patients achieving B-cell aplasia. Elevations in serum B-cell activating factor (BAFF) level were observed, with 3 of 4 patients achieving levels within the lowest end of the range exhibited in rese-cel–treated patients with AD who received LD. PV autoantibodies decreased in 2 of 4 patients. These preliminary data suggest that LD may be dispensable for humanized CAR T-cell efficacy in patients with AD. This trial was registered at www.clinicaltrials.gov as NCT04422912.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease with heterogeneous and poorly understood mechanisms. We perform single-cell RNA sequencing of lesional (LAD) and non-lesional (NAD) skin from 42 AD patients and 23 healthy controls (HC). Keratinocytes (KCs) are the predominant disease-relevant cell type. In healthy skin, KC differentiation follows a linear trajectory from basal KCs through seven differentiated stages to terminal keratinized cells. In LAD, this process is disrupted, showing a reversed terminal transition mainly driven by DK7 cells. APOD, LYZ and SERPINB4 emerge as LAD-specific regulators linked to IL-13/IL-22 responses, ER stress and oxidative damage. Mitochondrial and ER dysfunction are specifically enriched in LAD DK6 cells, indicating a key pathogenic compartment. Spatial transcriptomics and cell-cell interaction analysis further identify LAD-specific TWEAK signaling from IL-13+ Th2/cycling T cells to Fn14+ basal/differentiated KCs. Here, we reveal disrupted KC differentiation and immune signaling circuits in AD, highlighting potential therapeutic targets.

Importance Defining on-treatment remission in plaque psoriasis is important for benchmarking patient responses to therapies. This also helps to facilitate shared understanding, align treatment expectations, and enable more effective psoriasis management. Objective To establish a consensus-based definition of on-treatment remission for plaque psoriasis through a multistage Delphi initiative. Evidence Review The Remission Workgroup from the medical board and scientific advisory board of the National Psoriasis Foundation engaged various stakeholders, both US based and international, to participate in the consensus process. Following a working group meeting to determine the overall consensus approach, a systematic review of remission definitions in the current literature was performed. This review helped to inform the content of consensus materials. The consensus effort involved 2 stages: pre-Delphi interviews and surveys to inform the Delphi questions, followed by a Delphi exercise with physicians to define on-treatment remission for plaque psoriasis. Outcome measures considered included body surface area (BSA), Investigator Global Assessment (IGA), the product of the Physician Global Assessment and body surface area (PGA x BSA), and Psoriasis Area and Severity Index (PASI) score at various cutoff levels and time points. Findings The consensus process involved 92 stakeholders, including dermatologists, rheumatologists, researchers, patients, payers, and life sciences professionals. In the pre-Delphi interviews and surveys, patients emphasized that on-treatment remission meant the absence of psoriasis signs and symptoms while recieving therapy. Payers expressed that defining remission is important for long-term treatment coverage. Following the Delphi exercise and discussion with participating physicians specializing in psoriatic disease management, on-treatment remission in plaque psoriasis was defined as patients maintaining a BSA of 0% or IGA of 0 for at least 6 months while on treatment. Conclusions and Relevance Through a Delphi consensus process, on-treatment remission for plaque psoriasis was defined as patients maintaining a BSA involvement of 0% or IGA of 0 for at least 6 months while on treatment. This clear and standardized benchmark is applicable to both research and practice settings.

MEK inhibition (MEKi) combined with programmed death ligand 1 inhibition (immune checkpoint inhibitor [ICI]) modulates the tumor immune microenvironment. This phase 1 study evaluated sequencing schemes of MEKi and ICI with trametinib and pembrolizumab in NSCLC. In this 3+3 dose escalation study, patients with advanced NSCLC were treated with lead-in trametinib (arm A) or lead-in pembrolizumab (arm B) for cycle 1, followed by a 1.5 to 2 mg oral daily dose of trametinib (d 1-10) with pembrolizumab 200 mg intravenously every 21 days. Eligible patients with progressive disease on or after platinum-based chemotherapy were enrolled. Prior ICI was allowed. Tumor tissue was analyzed with quantitative immunofluorescence. High-parameter flow cytometry was performed on blood. Adverse events were graded using the Common Terminology Criteria for Adverse Events version 4 and efficacy was evaluated by Response Evaluation Criteria in Solid Tumors version 1.1. Fifteen patients enrolled (nine arm A and six arm B) with 13 (86%) harboring KRAS mutations and 10 (66%) receiving prior ICI. Five patients (33%) experienced at least one grade greater than or equal to 3 treatment-related adverse event including one dose-limiting toxicity (grade 3 esophagitis). Two patients had a partial response (ORR = 14%). Trametinib lead-in was associated with decreased T-regulatory cells and myeloid-derived suppressor cells (p = 0.002 and p = 0.05, respectively). The activity of trametinib and pembrolizumab is modest in NSCLC with increased toxicity compared with programmed death ligand 1 blockade alone. The recommended phase 2 dose for the combination is 2 mg of oral trametinib (d 1-10) and 200 mg of intravenous pembrolizumab every 21 days, with lead-in trametinib. Adverse events were comparable with other MEKi and ICI combination studies. Though limited clinical activity was observed, lead-in MEKi may induce favorable immune cell alterations.

Necrobiosis is a histologic term used to describe abnormal deposits of "degenerating" collagen within the skin. It can be found as an incidental finding in various granulomatous conditions, but is a hallmark of necrobiosis lipoidica (NL) and necrobiotic xanthogranuloma (NXG). There is limited prior research on necrobiosis. Here, we employed single-cell analysis of lesional and nonlesional skin to study the pathophysiology of necrobiosis. Our findings demonstrate that necrobiotic lesional skin is characterized by SPP1hi macrophages expressing MARCO; NKG7-expressing effector CD8+ T cells coexpressing CCL5, IFNG, GZMs, and PRF1; CCL5hi fibroblasts coexpressing CXCL9, diverse collagens (e.g., COL4A4, COL11A1, COL8A1), and TIMP1; and IGHM-expressing plasma cells. Integrative analysis of signaling ligands and receptor expression identified strong cell-cell communication between NKG7+ T cells, CCL5hi fibroblasts, and SPP1-expressing macrophages. In contrast, these cell populations were not dominant features of systemic sclerosis, another collagen deposition disease. Furthermore, although SPP1-expressing macrophages were detectable in sarcoidosis, IFNG-expressing T cells were a more defining feature of sarcoidosis compared with NL and NXG. From these findings, we speculate that necrobiosis results from the deposition of diverse collagens and ECM proteins through a process driven by CCL5-expressing fibroblasts and SPP1-expressing macrophages.

PURPOSE:Sulfur mustard (SM) is an alkylating agent used in warfare and terrorism that inflicts devastating ocular injuries. Although the clinical symptoms are well described, the underlying mechanisms are not fully understood, hindering the development of effective treatments. One major roadblock is the lack of a suitable model due to the extremely hazardous nature of SM, which requires strict safety measures. As a safer and practical alternative, we report a novel model that uses mechlorethamine (nitrogen mustard) gel, an FDA-approved topical chemotherapeutic administered by patients at home. Here we demonstrate its suitability to induce mustard corneal injury in any laboratory. METHODS:Ex vivo porcine corneas were injured with mechlorethamine gel. Hematoxylin-eosin staining and immunohistochemistry were performed to evaluate histopathology of SM-like corneal injuries: epithelium thickness and stromal separation, keratocyte and inflammatory cell counts, and expression of inflammation and fibrosis markers. RESULTS:This model showed the characteristic histopathology and expression of cyclooxygenase-2 (inflammation) and fibronectin-1 (fibrosis), which were consistent with other well-established SM-like corneal injury models. CONCLUSION:Given its ease of implementation and safety, this mechlorethamine model could be used to study the full course of mustard corneal injuries. This model is expected to facilitate the understanding of mustard ocular injuries and the development of novel therapeutics. TRANSLATIONAL RELEVANCE:This model will allow safe evaluation of SM-like corneal injuries within 24 hours, facilitating the identification of early/new molecules that might help to develop novel treatments.

Myeloid derived cells and tumor associated macrophages (TAMs) are a nascent field in melanoma. Single-cell sequencing technology improves the resolution of the tumor microenvironment (TiME) interactions. Although lacking consensus, several sub-types of TAMs have been associated with solid tumors using single-cell RNA (scRNA) sequencing data. Due to a lack of consensus across models and tumors, we sought out to define the myeloid sub-compartment in melanoma using public scRNA sequencing data. Here we show, 6 myeloid cell clusters termed M\_C1 to M\_C6 with gene expression showing differences in structure and function. The M\_C1 subtype was prognostic in brain metastatic melanoma outcome for overall survival (OS) based on the overall frequency. This finding was externally validated using publicly available bulk RNA sequencing data from a clinical trial (advanced melanoma treated with nivolumab) and the tumor cancer genome atlas (TCGA) for Stage I-IV. The M\_C1 cell type appears to be a hybrid dendritic cell and macrophage that shows prognostic ability across all stages of melanoma and response prediction with immunotherapy. Due to the nature of the findings, there may be implications for this as a biomarker of prognosis in melanoma and cell therapy in the future. ### Competing Interest Statement The gene signature developed in this work has been submitted under provisional patent

Defining on-treatment remission in plaque psoriasis is important for benchmarking patient responses to therapies. This also helps to facilitate shared understanding, align treatment expectations, and enable more effective psoriasis management. To establish a consensus-based definition of on-treatment remission for plaque psoriasis through a multistage Delphi initiative. The Remission Workgroup from the medical board and scientific advisory board of the National Psoriasis Foundation engaged various stakeholders, both US based and international, to participate in the consensus process. Following a working group meeting to determine the overall consensus approach, a systematic review of remission definitions in the current literature was performed. This review helped to inform the content of consensus materials. The consensus effort involved 2 stages: pre-Delphi interviews and surveys to inform the Delphi questions, followed by a Delphi exercise with physicians to define on-treatment remission for plaque psoriasis. Outcome measures considered included body surface area (BSA), Investigator Global Assessment (IGA), the product of the Physician Global Assessment and body surface area (PGA × BSA), and Psoriasis Area and Severity Index (PASI) score at various cutoff levels and time points. The consensus process involved 92 stakeholders, including dermatologists, rheumatologists, researchers, patients, payers, and life sciences professionals. In the pre-Delphi interviews and surveys, patients emphasized that on-treatment remission meant the absence of psoriasis signs and symptoms while recieving therapy. Payers expressed that defining remission is important for long-term treatment coverage. Following the Delphi exercise and discussion with participating physicians specializing in psoriatic disease management, on-treatment remission in plaque psoriasis was defined as patients maintaining a BSA of 0% or IGA of 0 for at least 6 months while on treatment. Through a Delphi consensus process, on-treatment remission for plaque psoriasis was defined as patients maintaining a BSA involvement of 0% or IGA of 0 for at least 6 months while on treatment. This clear and standardized benchmark is applicable to both research and practice settings.