Abstract T cell receptor (TCR)-based immunotherapies leverage the ability of a TCR to bind with high specificity and selectivity to peptides derived from intracellular targets, including those overexpressed on tumors. Consequently, TCRs identified from tumor-infiltrating lymphocytes (TILs) that respond to antigens in the tumor microenvironment represent attractive substrates for TCR immunotherapy development. However, de-orphaning TCRs is exceedingly challenging given the diversity of TCR and target sequence space. Although multiplexed peptide-MHC (pMHC) staining assays have improved throughput of direct, ex vivo measurement of TCR-pMHC binding, they are limited by TIL sample quality, T cell infiltration, TCR repertoire diversity, and limited pMHC library size. Machine-learning based techniques may overcome these limitations by training models to predict TCR-pMHC interactions and then inferring TCR-pMHC binding on large databases collected in other contexts. Here, we applied a contrastive model of TCR-pMHC binding prediction (1) to 382 tumor samples from 9 indications containing 490,000 T cells. We found that 5,244/490,000 (∼1.1%) of TCRs from this dataset were predicted to recognize one of the 4,438 pMHCs in our model training set. Of these, 1,949 were predicted to bind common viral peptides, an observation indicative of bystander recruitment and infiltration. An additional 2,549 TCRs were predicted to bind to Class I onco-antigens, including QLLALLPSL [PRAME], YLEPGPVTA [GP100] and GLYDGMEHLI [MAGEA10]. The model was able to capture TIL TCRs with diverse complementarity-determining regions (CDRs) as well as highly homologous sequences. TCRs predicted to bind self-antigens were phenotyped using Repertoire’s DECODE™ platform (2), showing that cancer-specific TCRs tended to have a more cytotoxic and effector like phenotype compared to a memory-like signature on viral specific TCRs (e.g. TCRs specific to cytomegalovirus, Epstein-Barr virus, and influenza). These results demonstrate a way to computationally de-orphan TCR specificities and phenotypically characterize T cells in a high-throughput manner to further our understanding of the TCR landscape in cancer patients. Importantly, this strategy could enable the discovery of TCR sequences suitable for therapeutic development using existing datasets. Future work should focus on increasing the diversity of validated TCR-pMHC specificities and in-vitro validation of predicted TCR-pMHC interactions (as well as reported model performance metrics).1. Abel, John, et al. "REPTRA: Mapping Immune T Cell Receptor Activity from Full Sequences with a Debiased Contrastive Loss." bioRxiv (2025): 2025-10.2. Francis, Joshua M., et al. "Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2." Science immunology 7.67 (2021): eabk3070. Citation Format: Brinda Vijaykumar, Qiaomu Tian, Jack Prazich, Preet Joshi, Neel Patel, John Abel, Anthony Coyle, Daniel Pregibon, . Predicting self-antigen recognition of TCRs derived from tumor infiltrating CD8+ T cells via contrastive learning and T cell phenotyping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4196.

Effective anti-tumor immunity is driven by cytotoxic CD8+ T cells with specificity for tumor antigens. However, the factors that control successful tumor rejection are not well understood. Here we identify a subpopulation of CD8+ T cells that are tumor-antigen-specific and can be identified by KIR expression but paradoxically impair anti-tumor immunity in patients with melanoma. These tumor-antigen-specific KIR+CD8+ regulatory T cells target other tumor-antigen-specific CD8+ T cells, can be detected in both the tumor and the blood, have a conserved transcriptional program and are associated with a poor overall survival. These findings broaden our understanding of the transcriptional and functional heterogeneity of human CD8+ T cells and implicate KIR+CD8+ regulatory T cells as a cellular mediator of immune evasion in human cancer. Tumor-antigen-specific CD8+ T cells are generally thought to help fight against cancer, but here the authors identify a subpopulation of CD8+ T cells that are associated with a poor clinical outcome in melanoma. Although these cells can recognize tumor antigens, they suppress cancer immunity.

Supplementary Figures S1-S6. Supplementary Figure S1. Liquid chromatography electrospray ionization tandem mass spectrometry analysis; Supplementary Figure S2. TM4SF1 expression in human breast and colon cancers; Supplementary Figure S3A. Clearance of anti-TM4SF1 antibody from HUVEC surface; Supplementary Figure S3B. Uptake of anti-TM4SF1 antibody in HUVEC; Supplementary Figure S4A. NSCLC killing by anti-TM4SF1 ADC; Supplementary Figure S4B. Tumor cell killing by anti-TM4SF1 ADC; Supplementary Figure S5. Expression level of TM4SF1 and other endothelial cell markers in tumor xenografts; Supplementary Figure S6. Clearance of anti-TM4SF1 ADCs; tolerance in mice.

Background High-risk human papillomavirus (HPV) is a primary cause of an increasing number of oropharyngeal squamous cell carcinomas (OPSCCs). The viral etiology of these cancers provides the opportunity for antigen-directed therapies that are restricted in scope compared with cancers without viral components. However, specific virally-encoded epitopes and their corresponding immune responses are not fully defined.Methods To understand the OPSCC immune landscape, we conducted a comprehensive single-cell analysis of HPV16+ and HPV33+ primary tumors and metastatic lymph nodes. We used single-cell analysis with encoded peptide-human leukocyte antigen (HLA) tetramers to analyze HPV16+ and HPV33+ OPSCC tumors, characterizing the ex vivo cellular responses to HPV-derived antigens presented in major Class I and Class II HLA alleles.Results We identified robust cytotoxic T-cell responses to HPV16 proteins E1 and E2 that were shared across multiple patients, particularly in HLA-A*01:01 and HLA-B*08:01. Responses to E2 were associated with loss of E2 expression in at least one tumor, indicating the functional capacity of these E2-recognizing T cells and many of these interactions validated in a functional assay. Conversely, cellular responses to E6 and E7 were limited in quantity and cytotoxic capacity, and tumor E6 and E7 expression persisted.Conclusions These data highlight antigenicity beyond HPV16 E6 and E7 and nominate candidates for antigen-directed therapies.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/β sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.

Background Human papillomavirus (HPV) infection is a risk factor for oropharyngeal squamous cell carcinoma (OPSCC). HPV+ OPSCC is characterized by distinct biology, a heterogenous immune landscape, and accounts for approximately 14,400 new diagnoses per year in the U.S. For patients with locoregional recurrence or distant metastasis, systemic treatment options beyond immune checkpoint inhibitors are limited. Though a decline in HPV+ HNSCC is expected due to HPV vaccination campaigns, incidence is currently increasing, prompting a wave of therapeutic development. While most therapeutic efforts have been focused on eliciting a cellular response to HPV early antigens E6 and E7, we sought to identify additional highly prevalent, immunogenic targets in both early stage as well as in relapsed/refractory HPV+ HNSCC. Methods Treatment-naïve HPV+ OPSCC patients (n=19) were prospectively enrolled under an institutional review board (IRB)-approved tissue collection protocol (DF/HCC#09–472) for collection of surgical specimens. Tumor tissue from a biopsy or definitive oncologic transoral robotic-assisted resection specimen was collected prospectively for analysis. Multiplexed, barcoded peptide-MHC-I tetramer libraries containing epitopes derived from the HPV16 genome were used to probe dissociated tumors followed by single cell RNA sequencing using the 10x Genomics platform. SingleR and published gene marker sets were used to phenotype and perform broad lineage assignment. Results We found broad T-cell reactivity to several HPV proteins across multiple HLA alleles and epitopes. E1 and E2-reactive T-cells were cytotoxic and highly expanded, whereas E5, E6 and E7-reactive T-cells were rare and displayed exhausted or naïve phenotypes. Cytotoxic E2 T-cell responses were associated with loss of E2 expression in at least one tumor suggesting functional capacity of these T-cells. We also identified evidence of pseudo-public TCR sequences, as TCR alpha, beta or paired CDR3 sequences associated with E2 reactivity were shared across multiple patients with common HLAs. TCR specificity was confirmed for several TCR-epitope interactions using recombinant TCRs and cognate peptide. Conclusions We employed high-throughput, single-cell immune synapse profiling to characterize the cellular response to HPV+ HNSCC across common HLA haplotypes and identified new candidates for immunotherapeutic intervention. We found that HPV16 proteins E1 and E2 induce robust, effective cytotoxic responses in HPV16-driven OPSCC. These findings indicate HPV16 E1 and E2-directed immunotherapy may be effective among patients with OPSCC expressing these antigens. Ethics Approval Patients were consented and enrolled under an institutional review board (IRB)-approved tissue collection protocol (DF/HCC#09–472).

Background The evolving dynamics of human papillomavirus (HPV) genotypes has the potential to impact the prognosis of HPV-associated malignancies. HPV33+ oropharyngeal squamous cell carcinoma (OPSCC) patients in particular may have inferior survival rates compared to the other high-risk HPV subtypes. Despite heterogeneity in disease biology and varying clinical outcomes, ambiguity exists for the optimal clinical management of non-HPV16 high-risk subtypes. Here, we interrogated tumor from HPV33+ HNSCC patients to gain an in-depth understanding of cellular heterogeneity, identify high-risk MHC alleles, and explore disease-relevant targets that may inform strain-specific disease management. Methods Treatment-naïve HPV+ OPSCC patients (n=19) were prospectively enrolled under an institutional review board (IRB)-approved tissue collection protocol (DF/HCC#09-472) for collection of surgical specimens. Subtype was assessed using TTMV-HPV DNA. Multiplexed, barcoded peptide-MHC-I tetramer libraries containing epitopes derived from the HPV33 genome were used to probe dissociated tumors followed by single cell RNA sequencing using the 10x Genomics platform. SingleR and published gene marker sets were used to phenotype and perform broad lineage assignment. Bulk RNA-sequencing data from additional HNSCC dataset was interrogated to validate select findings. Results Our cohort (Total patients n=237; HPV33+ n=17, 7.2%) showed a ~2.4 fold higher rate of HPV33-driven OPSCC compared to published reports (3%). Single cell RNA-seq of tumor cells revealed that HPV33+ tumors exhibited a distinct HPV gene expression profile compared to HPV16+ tumors. HPV33+ tumors showed evidence of less infiltration of cytotoxic T-cells, exhausted T-cells and overall CD8+ T-cells. Interrogation of an independent HPV+ OPSCC cohort confirmed an overrepresentation of A*02:01 allele in HPV33+ OPSCC patients compared to HPV16+ OPSCC. Comparative epitope prediction analysis revealed that HPV33 lacks a key epitope found in HPV16 that is known to play a key role in immune recognition in A02:01+ patient samples. Conclusions Targeted therapeutic trials have focused on HPV16+, rather than other high-risk HPV subtypes such as HPV33. Our data highlight reduced CD8+ T cell infiltrates as a potential correlate to poor outcomes in these patients. In addition, the observed A*02:01 ratios demonstrates potential immunoediting in HPV16+ patients. Increasing prevalence underscores an unmet need and presents a unique opportunity to design novel precision-based immunotherapeutic approaches for HPV33+ malignancies and potentially improve clinical outcome in this growing patient subpopulation.

The introduction and development of T cell-based therapies to treat a variety of both hematological and solid tumors have led to substantial clinical benefits in some patients. In this brief review we introduce the different T cell modalities used and some of their key features and limitations. Emerging avenues to overcome current bottlenecks are put into perspective, especially the issues of limited persistence and efficacy that make it challenging to achieve durable clinical responses in solid tumor indications.

Background High dose IL-2 treatment in metastatic renal cell carcinoma and metastatic melanoma patients induced complete remission in 5%–10% of patients without recurrence for over 25 years and potentially cured 70% of these patients.1 Although FDA approved for treatment of metastatic melanoma and renal cell carcinoma, the clinical utility of high-dose IL-2 is limited by significant multisystem toxicity, treatment-related mortalities in up to 4% of patients, and a lack of response in some patients. As a pleiotropic cytokine, IL-2 not only boosts the desired proliferation and effector function of T and NK cells, but also enhances detrimental immune suppression by expanding high affinity IL-2R(αβγ) expressing Treg cells. Contributing to its toxicity, IL-2 also activates innate lymphoid cells (ILC) and IL-2Rα+ endothelial cells to cause vascular leak syndrome. A key challenge in developing IL-2 as a safe and efficacious cancer therapeutic is uncoupling its efficacy from its toxicity. Here, we present data to support that this could be achieved by linking wildtype IL-2 to an anti-CD8 antibody and selectively delivering IL2 to CD8 T cells. Methods We separately linked wild type human IL-2 to anti-murine and anti-human CD8 or untargeted RSV antibodies for studies in murine and human systems, respectively. In vitro pharmacology was studied with mouse splenocytes and human PBMC. In vivo pharmacodynamics, efficacy, and toxicity were assessed in naive mice, B16F10 and MC38 syngeneic tumor models. Results Incubating respective CD8-IL2s with mouse splenocytes and human PBMC selectively loaded IL2 on CD8 T cells. This resulted in potent pSTAT5 activation downstream of the IL-2R and CD8 T cell expansion compared to incubating with untargeted IL-2 antibody fusions. CD8-IL2 localization is driven by CD8 antibody affinity where as untargeted IL2 localization is driven by IL-2 affinity for its receptors. In naïve mice, CD8-IL2 preferentially expanded CD8 T cells over Treg and NK cells. In contrast, untargeted IL-2 primarily expanded high-affinity IL-2R positive Treg and NK cells. In B16F10 syngeneic tumor bearing mice, untargeted IL-2 induced a dose dependent increase in inflammatory cytokines responsible for high toxicity and body weight loss. In contrast, treatment with CD8-IL2 significantly reduced toxicities but potently inhibited B16F10 and MC38 syngeneic tumor growth. Conclusions Our data supports that selective targeting of IL-2 to CD8+ T cells minimizes exposure of other cell types to IL2 and reduce IL2 mediated toxicity. CD8-IL2 is expected to be a safe and effective cancer immunotherapy. Reference Rosenberg SA. IL-2: the first effective immunotherapy for human cancer. J Immunol. 2014;192(12):5451–58 Ethics Approval All experimental animal procedures were approved by the Institutional Ethics Review Board.

Effective presentation of antigens by HLA class I molecules to CD8 + T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multi-omic technology to generate the first unified ex vivo characterization of the CD8 + T cell response to SARS-CoV-2 across 4 major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCR α/β sequence diversity, and the utilization of pre-existing SARS-CoV-2 reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations influence pre-existing immunity to SARS-CoV-2 and shape the immune repertoire upon subsequent viral exposure. One-Sentence Summary We perform a unified, multi-omic characterization of the CD8 + T cell response to SARS-CoV-2, revealing pre-existing immunity conditioned by HLA genotype.

Abstract PRIME IL-15 (RPTR-147) is a novel non-genetically modified, autologous, multi-clonal T cell product loaded with an IL-15Fc nanogel. The product is derived from peripheral T cells that are primed and expanded against five tumor associated antigens (TAAs) known to be overexpressed in multiple different tumor types: PRAME, NY-ESO, SSX-2, WT-1 and Survivin. 17 patients with advanced or metastatic solid-tumor cancers were dosed in a Phase I dose escalation study. Where samples were available, we characterized the reactivity and specificity of the cellular product, and tracked its persistence and localization post administration in patients. We report cellular composition of the product, along with phenotype and TAA-specificity of the T cells. Antigen specificity of patient-derived product samples was determined by Repertoire Immune Medicines' proprietary DNA-barcoded pMHC tetramer platform (CIPHERTM), as well as by functional immunological methods including IFNg ELISpot and flow cytometry assessment of activation induced markers (AIM). The CIPHER platform was used to identify TAA-specific CD8 T cell clonotypes, allowing decoding of T cell receptor (TCR) sequences and their cognate epitope/MHC. Distinct peptides binding from all five TAAs were observed across patients, and were presented across multiple HLA alleles. The phenotype of antigen-specific T cells identified via CIPHER was also assessed via single cell gene expression. Consistent with our previous studies on healthy donors, reactivity via IFNg ELISpot and AIM was also observed to all targeted TAAs. TCR CDR3 sequences were used as molecular signatures of antigen-specific cells to track the product post administration. Bulk sequencing of TCRVβ was performed on tumors pre- and post-treatment. Several product-derived, TAA-specific CD8+ T cell clonotypes were observed in the post-, but not pre-treatment biopsies. We identified additional CD4+ and CD8+ clones that were enriched in the post-treatment biopsies, and some of these clonotypes were also enriched in the product. To de-orphan these tumor infiltrating lymphocytes (TIL) of interest, we cloned selected TIL TCRs to assess their reactivity using Repertoire Immune Medicines' proprietary MCR epitope screening platform. In summary, characterization of antigen-specific T cells in the PRIME IL-15 product was performed by a variety of functional and molecular immune-based methods. Antigen specific T cell reactivities and clonotypes were identified in the peripheral repertoire. We confirmed that T cells were successfully expanded against TAAs and that these T cells do infiltrate the tumors as evidenced by bulk TCR sequencing of post-treatment tumor biopsies. Citation Format: John David Pajerowski, William Gordon, Phil Bardwell, Christine McInnis, Ji Young Hwang, Tabasum Huseni, Parul Agnihotri, Josh Francis, Christina Tarr, Florian Renoux, Sebastian Heer, Nastassja Cerreghetti-Terraneo, Marisa Loi, Erika Hamilton, Sarah Nikiforow, George Blumenschein, Mihaela Christea, Keren Osman, Anthony Shields, Harriet Kluger, Franz-Josef Obermair, Daniel Pregibon, Jan Kisielow, Anthony Coyle, Mojca Skoberne. Tracking and decoding the antigen specificity of peripherally derived T cells that infiltrate into solid tumors in patients treated with an autologous T cell therapy, PRIME IL-15 (RPTR-147) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB027.

Drug development is hampered by poor target selection. Phenotypic screens using neurons differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we design a multi-step screening funnel using patient-derived motor neurons. High-content live cell imaging is used to evaluate neuronal excitability, and from a screen against a chemogenomic library of 2,899 target-annotated compounds, 67 reduce the hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identifies 13 targets that modulate motor neuron excitability, including two known ALS excitability modulators, AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identify D2 dopamine receptors as modulators of ALS motor neuron excitability. This screen demonstrates the power of human disease cell-based phenotypic screens for identifying clinically relevant targets for neurological disorders.

Several human autoimmune diseases are characterized by increased expression of type 1 IFN-stimulated genes in both the peripheral blood and tissue. The contributions of different type I IFNs to this gene signature are uncertain as the type I IFN family consists of 13 alphas and one each of β, ε, κ, and ω subtypes. We sought to investigate the contribution of various IFNs to IFN signaling in primary human cell types. We stimulated primary skin, muscle, kidney, and PBMCs from normal healthy human donors with various TLR ligands and measured the expression of type I IFN subtypes and activation of downstream signaling by quantitative PCR. We show that IFNB1 is the dominant type I IFN expressed upon TLR3 and TLR4 stimulation, and its expression profile is associated with subsequent MX1 transcription. Furthermore, using an IFN-β-specific neutralizing Ab, we show that MX1 expression is inhibited in a dose-dependent manner, suggesting that IFN-β is the primary driver of IFN-stimulated genes following TLR3 and TLR4 engagement. Stimulation with TLR7/8 and TLR9 ligands induced IFNB1 and IFNA subtypes and MX1 expression only in PBMCs and not in tissue resident cell types. Concordantly, IFN-β neutralization had no effect on MX1 expression in PBMCs potentially because of the combination of IFNB1 and IFNA expression. Combined, these data highlight the potential role for IFN-β in driving local inflammatory responses in clinically relevant human tissue types and opportunities to treat local inflammation by targeting IFN-β.

Slit guidance ligand 2 (SLIT2) is a large, secreted protein which binds roundabout (ROBO) receptors on multiple cell types including neurons and kidney podocytes. SLIT2–ROBO2 mediated signaling regulates neuronal migration and ureteric bud (UB) outgrowth during kidney development as well as glomerular filtration in adult kidneys. Additionally, SLIT2 binds Gremlin, an antagonist of bone morphogenetic proteins (BMPs), and BMP–Gremlin signaling also regulates UB formation. However, direct crosstalk between the ROBO2–SLIT2 and BMP–Gremlin signaling pathways has not been established. Here, we report the discovery of negative feedback between the SLIT2 and BMP–Gremlin signaling pathways. We found that the SLIT2–Gremlin interaction inhibited both SLIT2–ROBO2 signaling in neurons and Gremlin antagonism of BMP activity in myoblasts and fibroblasts. Furthermore, BMP2 downregulated SLIT2 expression and promoter activity through canonical BMP signaling. Gremlin treatment, BMP receptor inhibition, and SMAD family member 4 (SMAD4) knockdown rescued BMP-mediated repression of SLIT2. BMP2 treatment of nephron progenitor cells derived from human embryonic stem cells decreased SLIT2 expression, further suggesting an interaction between the BMP2–Gremlin and SLIT2 pathways in human kidney cells. In conclusion, our study has revealed direct negative crosstalk between two pathways, previously thought to be unassociated, that may regulate both kidney development and adult tissue maintenance. INTRODUCTION Slit guidance ligands (SLITs) are ~200 kDa proteins that contain four N-terminal leucinerich repeat domains (LRR), seven to nine epidermal growth factor (EGF)-like domains, a laminin G domain, and a C-terminal cysteine-rich domain (1-3). A cleavage site within the EGF domain of SLIT2 results in an N-terminal fragment (SLIT2N) which binds roundabout (ROBO) receptors, via an active site within the second LRR domain (D2), and a C-terminal fragment (SLIT2C) which can bind PlexinA1 and Glypican-1 (3-9). SLIT–ROBO signaling was originally identified as a repulsive axon guidance cue during nervous system development (6,10,11), and the interaction of SLIT2 with ROBO receptors has also been shown to regulate forebrain development (12) and metanephric kidney development (13,14). Disruption of SLIT2– ROBO2 signaling during kidney development can lead to congenital anomalies of the kidney and urinary tract (CAKUT) (14-18). In adult tissue, the SLIT–ROBO interaction can inhibit nephrininduced actin polymerization and the attachment of glomerular podocytes which are essential for glomerular filtration (19,20), mediate fibroblast activation and fibrotic disease progression (21-24), http://www.jbc.org/cgi/doi/10.1074/jbc.M117.804021 The latest version is at JBC Papers in Press. Published on January 9, 2018 as Manuscript M117.804021 Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 2, 2018 hp://w w w .jb.org/ D ow nladed from Direct negative crosstalk between SLIT2 and BMP-Gremlin signaling 2 regulate tumor growth (25-27), and promote angiogenesis (28,29). In addition to binding to ROBO receptors, SLIT2 was discovered to bind to the secreted protein Gremlin in a yeast two-hybrid screen (30). Gremlin, as well as its family member Noggin, is a secreted cysteine knot protein that antagonizes bone morphogenetic protein (BMP) signaling by preventing BMP ligands from binding to their receptors (31-35). BMP–Gremlin signaling has many known roles in the embryonic development of the kidney and urinary tract (36,37), osteoblast differentiation (38), renal function (39), and fibrotic disease progression (33,40,41). Metanephric kidney development is driven by epithelial and mesenchymal cell interactions between nephric ducts (ND), also referred to as Wolffian ducts (WD), and the metanephric mesenchyme (MM), which both originate from the intermediate mesoderm (IM) (42). Signals from the MM promote the outgrowth of the ureteric bud (UB) from the ND (42). The UB then invades the MM and begins to branch repeatedly to give rise to the fully formed nephron and metanephric kidney (42-44). UB outgrowth and branching is tightly regulated by the spatial and temporal expression of a number of signaling pathways and transcription factors (42,43). A key mechanism of regulation is the formation of a complex between glial cell derived neurotrophic factor (GDNF), which is expressed in the MM adjacent to the ND (45,46), with its receptor RET, a receptor tyrosine kinase, and co-receptor GDNF Family Receptor Alpha 1 (GFRα1) which are expressed by the ND (44,46-51). UB outgrowth is also regulated by SLIT2 which is expressed in the tips of the UB and ROBO2 which is expressed by the MM in the area surrounding the UB (13). ROBO2 knockout mice and SLIT2 knockout mice form supernumerary ureteric buds (14). SLIT2–ROBO2 signaling restricts the nephrogenic field and limits cell-cell interactions between the MM and ND, possibly by limiting GDNF–RET signaling by regulating ND– MM separation (14,52). Interestingly, BMP– Gremlin signaling is also an important regulator of UB outgrowth and branching. Gremlin-mediated inhibition of BMP is essential to induce UB outgrowth and establish RET–GDNF feedback signaling by mediating epithelial-mesenchymal signaling (37). In Gremlin-deficient mouse embryos, the UB fails to invade the MM because of uncontrolled BMP signaling resulting in renal agenesis (36,37). Conversely, recombinant Gremlin protein is able to induce ectopic UB outgrowth from the ND by upregulating GDNF expression (36). Given the roles of both SLIT2-ROBO2 signaling and BMP–Gremlin signaling in fibrotic disease progression and UB outgrowth and branching, as well as the opposite effects of SLIT2 and Gremlin genetic loss in mouse kidney development, we hypothesized that the interaction between SLIT2 and Gremlin serves to regulate their signaling. Here we report the identification of direct negative crosstalk between SLIT2 and BMP–Gremlin signaling. We found that SLIT2 bound to Gremlin via its D2 domain and that this interaction inhibited SLIT2–ROBO2 activity as well as Gremlin-mediated antagonism of BMP2 signaling. Furthermore, BMP2 downregulated SLIT2 expression by promoting transcriptional repression via SMAD signaling in mouse and human fibroblasts. Similar changes in SLIT2 expression were observed when nephron progenitor cells derived from human embryonic stem cells were treated with BMP2. These studies unveil a regulatory feedback loop between two previously unassociated pathways that could have implications for both kidney development and adult tissue maintenance in human disease. RESULTS Gremlin binds to the D2 domain of SLIT2—It had been previously reported that rat Gremlin interacts directly with the repulsive guidance cue SLIT2 (30). To determine if human Gremlin also interacts with the N-terminal domain of SLIT2, we performed an ELISA with recombinant, biotinylated human SLIT2N and human Gremlin on a streptavidin-coated plate. We found that Gremlin bound to SLIT2N, beginning at 62.5 nM, and binding increased with higher doses (Figure 1A). These results were confirmed by conducting surface plasmon resonance (SPR) experiments with human SLIT2N and human Gremlin (Figure 1B). SLIT2N was the ligand and was covalently immobilized to a C1 chip surface. It was then exposed to a dose range of Gremlin–the analyte in solution–to measure association and dissociation kinetics. A sensorgram (Figure 1B) and residuals graph by gest on Jauary 2, 2018 hp://w w w .jb.org/ D ow nladed from Direct negative crosstalk between SLIT2 and BMP-Gremlin signaling 3 (Figure S1A) show that the maximal residual difference of the observed data from a 1:1 Langmuir binding model is less than 10% of the global maximum capacity (Rmax). The average global ka of human Gremlin for human SLIT2N was 3.99 x 10 6 ± 2.87 x 10 5 M -1 s -1 , and the average global kd was 8.25 x 10 -2 ± 1.15 x 10 -2 s -1 for 3 independent biosensor surfaces (Table 1). The KD value of Gremlin for SLIT2N was mathematically derived from the ka and kd data and the average kinetic KD across all independent sensor surfaces was 20.6 ± 2.3 nM (Table 2 and Table S2). Steady state analysis of Gremlin binding to SLIT2N (Figure S1B) revealed an average KD of 15.1 ± 2.7 nM (Table 2 and Table S3) and confirmed our kinetic analysis. These data suggest that human SLIT2N interacts with human Gremlin with relatively low affinity. The LRR D2 domain of SLIT2 (SLIT2D2) binds to ROBO family members and mediates ROBO signaling (4). To determine if the SLIT2– Gremlin interaction also occurs at the same D2 domain, we performed SPR experiments by exposing immobilized human SLIT2D2 (ligand) to a range of Gremlin doses in solution (analyte) to measure association and dissociation kinetics. Interestingly, the D2 fragment of SLIT2 bound to immobilized Gremlin on 3 independent biosensor surfaces. A sensorgram (Figure 1C) and residuals graph (Figure S1C) show that the maximal residual difference of the observed data from a 1:1 Langmuir binding model is less than 10% of the Rmax. The average global ka of Gremlin for SLIT2D2 was 5.76 x 10 6 ± 7.17 x 10 5 M -1 s -1 , and the average global kd was 1.69 x 10 -1 ± 5.48 x 10 -2 s -1 (Table 1). The mathematically derived average KD of Gremlin for SLIT2D2 was 27.9 ± 5.5 nM (Table 2 and Table S2). The average steady state KD (Figure S1D) was 17.5 ± 2.5 nM (Table 2 and Table S3) and agreed with kinetic analysis. We also reversed the orientation and exposed immobilized Gremlin (ligand) to a range of SLIT2D2 concentrations in solution (analyte) to monitor association and dissociation kinetics. A sensorgram (Figure 1D) and residuals graph (Figure S1E) show that the observed data deviate minimally from a 1:1 Langmuir binding model– less than 10% of the Rmax. The average global ka of SLIT2D2 for Gremlin was 1.85 x 10 6 ± 2.03 x 10 5 M -1 s -1 , and the average global kd was 2.37 x 10 -2

Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Treg). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Treg cells. We report the generation of the first fully human anti-IL-2 antibody, F5111.2 that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalitis, and protected mice against xenogeneic Graft-versus-Host-Disease (GvHD). These results suggest that IL-2-F5111.2 may provide a new immunotherapy to treat autoimmune diseases and GvHD.

Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Tregs). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Tregs. We report the generation of a fully human anti-IL-2 antibody, F5111.2, that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalomyelitis and protected mice against xenogeneic graft-versus-host disease. These results suggest that IL-2-F5111.2 may provide an immunotherapy to treat autoimmune diseases and graft-versus-host disease.

Slit guidance ligand 2 (SLIT2) is a large, secreted protein that binds roundabout (ROBO) receptors on multiple cell types, including neurons and kidney podocytes. S LIT2-ROBO mediated signaling regulates neuronal migration and ureteric bud (UB) outgrowth during kidney development as well as glomerular filtration in adult kidneys. Additionally, SLIT2 binds Gremlin, an antagonist of bone morphogenetic proteins (BMPs), and BMP Gremlin signaling also regulates UB formation. However, direct cross-talk between the ROBO2-SLIT2 and BMP Gremlin signaling pathways has not been established. Here, we report the discovery of negative feedback between the SLIT2 and BMP Gremlin signaling pathways. We found that the SLIT2 Gremlin interaction inhibited both SLIT2-ROBO2 signaling in neurons and Gremlin antagonism of BMP activity in myoblasts and fibroblasts. Furthermore, BMP2 down-regulated SLIT2 expression and promoter activity through canonical BMP signaling. Gremlin treatment, BMP receptor inhibition, and SMAD family member 4 (SMAD4) knockdown rescued BMPmediated repression of SLIT2. BMP2 treatment of nephron progenitor cells derived from human embryonic stem cells decreased SLIT2 expression, further suggesting an interaction between the BMP2 Gremlin and SLIT2 pathways in human kidney cells. In conclusion, our study has revealed direct negative cross-talk between two pathways, previously thought to be unassociated, that may regulate both kidney development and adult tissue maintenance.