Mice (Mus musculus) used in biomedical research are commonly housed at 19°C-23°C, below their thermoneutral zone (29°C-34°C), where metabolic homeostasis occurs. Although traditional housing exposes mice to chronic cold stress and modulates their immune response, the cellular mechanisms by which thermoneutrality shapes immune responses remain underdefined. CD4+ T cells are major contributors to host immunity through T cell receptor (TCR) signaling activated via recognition of peptide-major histocompatibility complex class II (pMHCII). We demonstrate that thermoneutral housing, compared to traditional housing, enhances tonic TCR signaling, upregulates genes associated with endogenous TCR stimulation, and increases TCR-driven TNF expression in CD4+ T cells. Mechanistically, these effects are in part dependent on tonic TCR engagement with self-pMHCII. In inflammatory disease models, thermoneutrality-driven increased CD4+ T cell TNF production correlates with amplified tissue inflammation. Together, these findings reveal how housing temperature may shape inflammatory responses through self-pMHCII-dependent TCR signaling in CD4+ T cells.

Objectives/Goals: Kidney transplant rejection is a leading cause of graft failure. Diagnosis is dependent on allograft biopsy that 1: focuses on immune infiltrate and 2: does not inform treatment or recovery potential. Using single-cell transcriptomic technologies, we assess rejection’s impact on kidney cells and begin to define outcome-affecting interactions. Methods/Study Population: Single-cell RNA sequencing was performed on 25 kidney biopsies from 15 separate patients who had undergone acute cellular rejection. Cell typing was performed using canonical genes, and differential gene expression profiling was performed in R with Seurat. The proportion of cell types present at different time points and grades of rejection was determined. We focused first on renal parenchymal cell types and their gene expression changes with rejection grade and treatment of rejection. The spatial localization of these cell states was determined with spatial transcriptomics using the Xenium Prime 5K pan-human tissue panel on 11 kidney biopsy sections. After annotation, spatial-informed niches were identified to investigate cellular changes associated with injury. Results/Anticipated Results: From the single-cell data, we were able to identify several renal parenchymal cell states. Focusing on the proximal tubule (PT) cells as the most numerous cell type in the renal cortex, we identified healthy, injured, and damaged cell states based on expression of tubular injury markers, genes associated with epithelial to mesenchymal transition, and expression of interferon-response genes. The proportion of PT cells in an injured state increased with rejection severity and decreased with rejection treatment. Utilizing the spatial transcriptomic data, we were able to identify both healthy and injured PT states and define their localization with immune cell types and histologic injury patterns. We found varied transcriptomic niches and that niches with injured PT cells were enriched with immune cells. Discussion/Significance of Impact: PT cells exhibit patterns of injury in response to rejection and when injured cells exist in spatial niches containing immune cells that are contributing or responding to injury. Understanding these interactions will further define the injury process caused by rejection and guide development of personalized rejection treatment.

Lupus nephritis (LN), a severe manifestation of systemic lupus erythematosus (SLE), is a heterogeneous disease driven by diverse immune and tissue cell types. We obtained 538,194 single-cell and 142,881 single-nuclear profiles from kidney biopsies of 155 patients with LN and 30 preimplantation transplant biopsy controls, along with 327,326 single-cell blood profiles. We characterized key stromal and immune cell types and cell states; moreover, we distinguished cell states that were tissue specific from those that were also present in the blood. We observed that LN pathological features were associated with particular cell states. For example, after controlling for the effects of chronic tissue damage, we observed that expansion of glomerular and scar-associated macrophage populations correlated with increasing inflammatory disease activity. Scar-associated macrophages appear to drive LN fibrosis and, in active disease, infiltrate the glomeruli more than other myeloid cells. These observations support that therapeutic targeting of myeloid populations may offer a strategy to prevent renal inflammation and ongoing kidney damage in LN.

Objectives Lupus nephritis (LN) is a common, potentially fatal manifestation of systemic lupus erythematosus. We aimed to gain new insights into the immune responses underlying LN and their relation to the histologic heterogeneity observed in this disease, focusing on myeloid cells. Methods We used single-cell RNA-sequencing (scRNA-seq) data of dissociated kidney samples from 156 patients with LN and 30 healthy individuals. We applied spatial transcriptomics (ST), utilising a gene panel designed to capture all myeloid subsets identified in the scRNA-seq data, to profile kidney samples acquired from 6 patients with LN and 2 controls. Results We generated a catalogue of the myeloid subsets found in LN kidneys. Our analyses indicated that an increase in irreversible tissue damage, as measured by the National Institutes of Health chronicity index (CI), is associated with a gradual switch of the local immune response from one dominated by monocytes and macrophages to one featuring expanded CD4 T, GZMK+ CD8 T, B, and dendritic cells, with a parallel decrease in the interferon response. In proliferative/mixed LN only, the degree of active inflammation correlates with the expansion of disease-specific macrophage (DMac) subsets, which later contract as the CI increases. Trajectory analysis of the scRNA-seq data suggested that DMacs arise from both infiltrating monocytes and tissue-resident macrophages; this was supported by the ST data, as well as cell cultures. DMacs are implied to interact with parietal epithelial cells, promoting the development of glomerulosclerosis. Conclusions We suggest a detailed picture of the changes in the kidney immune mechanisms in LN as this disease progresses.

We and others have described homeostatic dysregulation of the CD4+ memory T-cell compartment with age. To gain greater insights into this dysregulation, we performed comprehensive single-cell genomic analysis of endogenous memory CD4+ T cells from young and aged mice. This analysis revealed 16 populations, composed of Th1, Th17, several subsets of regulatory T (Treg) cells, memory T cells, CD4+ CTLs, and T follicular helper (Tfh) cells. One of the most highly expressed genes in aged Tfh cells was Tnfsf8 (CD153 or CD30L), and flow cytometric analysis confirmed age-increased expression of CD153 on endogenous Tfh subsets and memory cells, but not Treg cells. At steady state, the absence of IL-6 significantly reduced CD153 expression on Tfh cells, and pharmacologic inhibition of c-MAF prevented IL-6-driven increase in CD153 expression. After immunization, expression of CD153 on Ag-specific CD4+ Tfh cells persisted significantly longer in aged mice, which required IL-6. Blockade of CD153 significantly reduced Tfh cell expression of ICOS and Ag-specific B-cell responses. Thus, although Tfh-mediated B cell responses, overall, normally decline with age, our data suggest that elevated expression of CD153, driven by an IL-6/c-MAF circuit, potentiates remaining Tfh function in aged mice.

Lupus nephritis (LN), a severe manifestation of Systemic Lupus Erythematosus (SLE), is a heterogeneous disease driven by diverse immune and tissue cell types. We obtained 538K single-cell and 140K single-nuclear profiles from kidney biopsies of 155 LN patients and 30 pre-implantation transplant biopsy controls, along with 325K single-cell blood profiles overlapping many of these patients. We identified key tissue cell types and cell states, and immune cell states; we were able to determine cell states that were tissue specific, and those that were present in the blood. We observed that LN pathological features are significantly associated with cell states using differential gene expression and Covarying Neighborhood Analysis (CNA). These analyses revealed broad changes in cell states associated with irreversible chronic tissue damage. After controlling for the effects of ongoing tissue damage, we observed that expansion of key glomerular and Scar Associated Macrophages (SAMs) populations tracked with increasing inflammatory disease activity. SAMs appear to drive LN fibrosis and, in active disease, infiltrate the glomeruli more than other myeloid cells. These observations strongly support that therapeutic targeting of myeloid populations may offer an as-of-yet unproven strategy to prevent renal inflammation and ongoing kidney damage in LN.

Reliable, minimally invasive biomarkers for predicting immunotherapy response in head and neck squamous cell carcinoma (HNSCC) remain an unmet clinical need. Here, using patients from a prospective, multi-institutional phase II clinical trial (NCT02641093), we performed whole-genome sequencing of 185 plasma cell-free DNA (cfDNA) samples collected longitudinally from 68 patients with locally advanced, surgically resectable HNSCC undergoing neoadjuvant and adjuvant pembrolizumab treatment. We developed the regional motif diversity score (rMDS), a novel fragmentomic metric quantifying the entropy of cfDNA 5' end motifs across genomic regions. Remarkably, unsupervised analysis revealed that rMDS robustly distinguished immunotherapy responders from non-responders, outperforming established cfDNA fragmentomic metrics and copy number alterations, while demonstrating independence from technical confounders. Longitudinal analysis revealed dynamic rMDS changes in genomic regions enriched for immune-, lectin-, and keratinization-related genes-hallmarks of squamous cell carcinoma-reflecting the interplay between tumor and peripheral immunity during the immunotherapy treatment. Interestingly, the regions with the most dynamic rMDS changes were highly enriched in telomere-proximal loci, suggesting a novel link between telomere biology and cfDNA fragmentation. A machine learning classifier based on rMDS achieved robust predictive performance across multiple validation settings (AUC 0.89-0.99), with the highest accuracy at post-treatment timepoints and superior to PD-L1 expression and tumor fraction in the same sample. Predicted responders demonstrated significant trends toward improved disease-free survival (log rank test p=0.035, hazard ratio: 2.67, 95% confidence interval: 1.03-6.92), underscoring the clinical utility of rMDS-based stratification. These findings position rMDS as a biologically meaningful and clinically actionable biomarker for immunotherapy response in HNSCC, supporting its integration into future risk assessment frameworks and broader cancer care.

BACKGROUND Minimally invasive biomarkers predicting the immunotherapy response in head and neck squamous cell carcinoma (HNSCC) remain an unmet clinical need. METHODS In a prospective, multi-institutional phase II trial, we performed whole-genome sequencing of 185 longitudinal plasma cell-free DNA (cfDNA) samples from 68 patients with locally advanced, surgically resectable HNSCC who received neoadjuvant and adjuvant pembrolizumab. We developed the regional motif diversity score (rMDS), a fragmentomic metric that quantifies the entropy of cfDNA 5′-end motifs across genomic regions. RESULTS Unsupervised analysis showed that rMDS robustly distinguished responders from nonresponders, outperforming established fragmentomic metrics and copy number alterations while remaining independent of technical confounders. Longitudinal rMDS changes localized to regions enriched for immune-, lectin-, and keratinization-related genes — hallmarks of squamous cell carcinoma — reflecting tumor–peripheral immunity interplay during treatment. The most dynamic regions clustered at telomere-proximal loci, suggesting a link between telomere biology and cfDNA fragmentation. An rMDS-based machine learning classifier achieved AUC 0.89–0.99 across validation settings, with the highest accuracy after treatment, outperforming PD-L1 expression and tumor fraction in matched samples. Predicted responders showed improved disease-free survival (log-rank P = 0.035; HR 2.67, 95% CI 1.03–6.92). CONCLUSION rMDS represents a biologically meaningful and clinically actionable biomarker for the immunotherapy response in HNSCC, and merits integration into future risk assessment frameworks. TRIAL REGISTRATION ClinicalTrials.gov NCT02641093. FUNDING National Human Genome Research Institute (NHGRI), NIH grant R56HG012360; startup funds from Cincinnati Children’s Hospital Medical Center, Northwestern University, and Robert H. Lurie Comprehensive Cancer Center; Science Olympiad Alumni Research Grant, Science Olympiad USA Foundation; Merck Sharp & Dohme Corp.

While significant progress has been made in defining subsets among antigen-experienced CD8 T cells, the heterogeneity of naïve CD8 T cells remains poorly understood. Here, we identify naïve CD8 T cell subsets with superior persistence and an enhanced capacity to generate effector and memory cells, leading to more effective protection. These functionally superior naïve CD8 T cells are marked by IL-18Rα, CD73, and CXCR3, and functionally less potent naïve CD8 T cells can convert into these superior subsets through tonic TCR signaling. Their enhanced response to infections is driven by better survival of the progeny effector cells during the T cell expansion phase. This improved survival is mediated by increased Ly6C expression on effector cells derived from these functionally superior naïve cells. Collectively, our findings reveal functional heterogeneity and plasticity among naïve CD8 T cells and uncover a mechanism by which functionally superior naïve subsets drive robust CD8 T cell responses, providing a previously unrecognized layer of immune regulation.

T cell-mediated rejection (TCMR) affects ∼50% of pediatric liver transplant recipients within 5 years, and late TCMR is associated with graft failure due to ineffective treatment. Although CD8+ T cells promote late TCMR, their clonal expansion, intragraft persistence, localization, and gene expression remain largely undefined. Here, single-cell RNA sequencing and immune repertoire profiling of 30 cryopreserved liver biopsies from rejecting and nonrejecting pediatric patients identified expanded intragraft CD8+ T cell clonotypes (CD8EXP) and their gene expression profiles. Expanded CD8+ clonotypes (CD8EXP) bore markers of effector and CD56hiCD161- "natural killer-like" T cells, retaining their clonotype identity and phenotype in serial biopsies despite histologic TCMR resolution. CD8EXP clonotypes localized to portal infiltrates during active TCMR and persisted in the lobule after histologic TCMR resolution. MultiNicheNet analysis revealed differential crosstalk between Kupffer cells and CD8EXP, with activation of transforming growth factor ß and downregulation of CD47 immune checkpoint signaling. Therefore, persisting intragraft CD8EXP clones remain active despite TCMR treatment and may contribute to long-term allograft fibrosis and failure of operational tolerance.

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying noninvasive, blood-based immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation and identify correlates of renal parameters. Unbiased analysis identified 3 immunologically distinct groups of patients that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at 1 year. Patients with enriched circulating granzyme B+ T cells showed more active disease and increased numbers of activated CD8+ T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive but with chronic renal injuries. The major immunologic axes of variation could be distilled down to 5 simple cytometric parameters that recapitulate several clinical associations, highlighting the potential for blood immunoprofiling to translate to clinically useful noninvasive metrics to assess immune-mediated disease in LN.

DDX3 and other DEAD-box RNA helicases regulate nuclear export, translation, splicing, and metabolism of RNA. Perturbation of Ddx3x on the mouse X-chromosome in all hematopoietic cells resulted in a loss of natural killer (NK) cells, yet whether DDX3X is important only in progenitors or within NK cells remained unexplored. Herein, we deleted Ddx3x from committed NK cells by crossing Ddx3x-floxed mice to Ncr1-iCre mice. The resulting Cre+ offspring exhibited a profound deficiency of NK cells in the spleen and bone marrow. Ncr1-iCre-mediated deletion of Ddx3x also blocked in vitro generation of NK cells. CRISPR-mediated deletion of Ddx3x or pharmacological inhibition of DDX3 helicase activity in mature mouse NK cells resulted in rapid loss of cell viability, consistent with a role for DDX3X in NK cell survival. Indeed, perturbation of DDX3X in NK cells caused a substantial decrease in protein expression levels of the prosurvival mediator MCL1 but did not affect expression of the related prosurvival proteins BCL-2 or BCL-xL. Genetic deletion of the pro-apoptotic targets of MCL1, Bak and Bax, rescued the survival of NK cells following inhibition of DDX3. Mechanistically, expression levels of Mcl1 mRNA and proteasomal degradation of MCL1 protein were independent of DDX3. Instead, DDX3 bolstered MCL1 expression by supporting de novo translation of MCL1 protein. Collectively, these findings highlight a crucial role for the RNA helicase DDX3X in maintaining the NK cell compartment by supporting efficient translation of MCL1.

Loss of NF1 in Schwann cells leads to activation of the RAS-MAPK pathway, followed by immune cell recruitment and development of benign nerve tumors (PNFs). MEK inhibitors, which shrink most PNFs, also reduce tumor-associated myeloid cells. We tested whether SHP2 inhibition, predicted to block RAS-MAPK signaling and exert immunomodulatory effects, alters tumor volume or the immune microenvironment in PNFs, using flow cytometry and single-cell RNA sequencing. We found that both cobimetinib and daytime RMC-4550 similarly reduced tumor volume. The abundance of CD163-negative PNF-associated macrophages, derived from circulating monocytes, correlated with tumor size. Combining SHP2 inhibition with anti-PD1 altered tumor monocyte phenotype and reversed SHP2-mediated tumor shrinkage. Diurnal patterns of monocyte trafficking were disrupted in tumor-bearing mice, and SHP2 inhibition reduced tumor volume only when administered during the day, when myeloid infiltration was low. These findings suggest that SHP2 inhibitor-driven tumor shrinkage requires targeting monocyte-derived macrophages and is influenced by the timing of drug administration.

BACKGROUND & AIMS:Regulatory T cells (Tregs), a subset of CD4 lymphocytes, protect against inflammatory tissue injury. However, it is currently unknown how retention of bile acids (BA) in fibrosing cholangiopathies like biliary atresia or PSC shape hepatic Treg responses. METHODS:To induce sclerosing cholangitis (SC), mice were fed a diet containing 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine for 14 days, followed by 28 days on regular chow to assess tissue repair. Serial hepatic cell and nuclear preparations were subjected to single-cell RNA sequencing and ATAC-seq to define gene regulatory networks controlling Tregs under cholestatic conditions. Candidate molecules mediating the effects of tauro- and chenodeoxycholic acid (T/CDCA) on Tregs were validated in vitro, across three murine models of SC, and in liver tissue samples from 130 infants with biliary atresia. RESULTS:Single-cell analyses revealed that Tregs acquired a Th17-like transcriptional program during cholestasis and upregulated amphiregulin (Areg) during the repair phase. S1P receptors were identified as mediators of T/CDCA effects on Tregs, and this was validated both in vitro and in the Abcb4-/- model of SC. Deletion of Stat3 in CD4+ cells enhanced hepatic Treg responses following bile duct ligation. Pharmacologic reduction of hepatic BA concentrations using an IBAT (ileal bile acid transporter) inhibitor increased hepatic Treg numbers and attenuated liver injury and fibrosis in Abcb4-/- mice. These protective effects were lost upon Treg depletion or AREG neutralization. Finally, in infants with biliary atresia, a liver transcriptional profile consistent with Treg activation and AREG upregulation at diagnosis was associated with improved 2-year native liver survival. CONCLUSION:Bile acids suppress Treg regulatory function by promoting a Th17-like phenotype, thereby limiting their capacity to mitigate immune-mediated cholangiocyte injury. Restoring Treg function and amphiregulin expression may represent a novel therapeutic strategy in fibrosing cholangiopathies. IMPACT AND IMPLICATIONS:In this study, we examined the role of CD4 lymphocytes in controlling bile duct epithelial injury in fibrosing cholangiopathies, with potential implications for developing targeted therapies for BA and PSC. Using single-cell genomics, functional assays, and complementary mouse models of sclerosing cholangitis, we investigated the mechanisms by which chenodeoxycholic acid-derived bile acids determine polarization and suppressor functions of CD4 lymphocytes. Interventions such as reducing hepatic bile acid concentrations with an IBAT (ileal bile acid transporter) inhibitor, antagonizing STAT3 in CD4+ cells, or blocking S1P receptors enhanced hepatic regulatory T cell responses and protected against cholestatic liver injury in experimental models. These preclinical findings provide a foundation for future clinical trials in patients with fibrosing cholangiopathies.