ABSTRACT During chronic infection, the persistence of antigen and inflammation leads to the differentiation of CD8 + T cells into an exhausted state characterised by expression of inhibitory receptors (IRs) and the progressive loss of T cell functions. Among the different subsets of exhausted CD8 + T (Tex) cells, Tex progenitors expressing SLAMF6 and the transcription factor TCF-1 (TCF-1+) give rise to Tex effector-like cells expressing CX3CR1 and Tex terminal cells expressing CD101. PD-1/PD-L1 blockade acts on TCF-1+ Tex progenitor cells and promotes their differentiation into Tex effector-like cells. The molecular events controlling CD8 + Tex cell differentiation are still poorly defined. As Notch signaling may be sustained during chronic infection by persistent TCR stimulation and inflammation, we tested whether Notch signaling influences CD8 + T cell exhaustion. Using mice lacking (N1N2 Δ/Δ ) or not (N1N2 fl/fl ) Notch1/2 expression only in mature CD8 + T cells, we showed that the absence of Notch signal causes severe CD8+ T cell exhaustion during chronic LCMV infection. N1N2 Δ/Δ Tex cells express higher levels of IRs and are less functional when compared to their wild-typee counterpart. In the absence of N1N2 receptors, Tex progenitor and Tex terminal cells accumulate and Tex cells cannot be reinvigorated by PD-1/PD-L1 blockade. We further demonstrated that Notch signaling is essential to promote the differentiation of Tex progenitors into Tex effector-like cells. Moreover, Notch signals, provided by stromal cells expressing the ligands Delta-like 1 and 4, are necessary during all stages of the infection to prevent severe exhaustion. Single-nucleus RNA and ATAC multiome profiling identifies Notch signaling as a critical role on effector transcriptional programming in exhausted CD8 T cells. Loss of Notch signaling impairs transcriptional program associated with migration and perception of CD4 + T cell help. Together, these alterations drive the differentiation of Tex progenitor cells toward a terminally exhausted Tex fate.

Adaptive immune responses occur lymph nodes (LNs) in a microenvironment established by resident stromal cells. LNs are also a site of proliferation of chronic lymphocytic leukemia (CLL), a B cell cancer that alters LN structure in a stereotypic manner. To deeply characterize reactive and CLL LNs, we developed a single-cell RNA sequencing pipeline. We find that proliferation of CLL cells in proliferation centers (PCs), a CLL-specific niche, begins with transient upregulation of MYC, subsequent downregulation of which may limit CLL growth. PCs contain a distinct fibroblast population expressing CCL19 while CLL cells express the CCL19 receptor CCR7, providing a recruitment mechanism for CLL cells to PCs. Using informatic, spatial, and in situ analyses to identify ligand-receptor pairs involving PC CLL cells and nearby immune and stromal cells, we observe that PCs are enriched for macrophages expressing BAFF, the integrin αXβ2 heterodimer, and Galectin9, factors implicated in cell growth, adhesion, and immunosuppression. The most common predicted interactions in PCs involve CD74 and ligands such as MIF, and we find that CD74 blockade consistently inhibits CLL cell growth in culture. Our work highlights key features of the CLL proliferative niche and provides a roadmap for identifying vulnerabilities and new therapeutic strategies.

A better understanding of the mechanisms regulating CD8+ T cell differentiation is essential to develop new strategies to fight infections and cancer. Using genetic mouse models and blocking antibodies, we uncovered cellular and molecular mechanisms by which Notch signaling favors the efficient generation of effector CD8+ T cells. Fibroblastic reticular cells from secondary lymphoid organs, but not dendritic cells, were the dominant source of Notch signals in T cells via Delta-like1/4 ligands within the first 3 days of immune responses to vaccination or infection. Using transcriptional and epigenetic studies, we identified a unique Notch-driven T cell-specific signature. Early Notch signals were associated with chromatin opening in regions occupied by bZIP transcription factors, specifically BATF, known to be important for CD8+ T cell differentiation. Overall, we show that fibroblastic reticular cell niches control the ultimate molecular and functional fate of CD8+ T cells after vaccination or infection through the delivery of early Notch signals.

Innate-like splenic marginal zone (MZ) B (MZB) cells play unique roles in immunity due to their rapid responsiveness to blood-borne microbes. How MZB cells integrate cell-extrinsic and -intrinsic processes to achieve accelerated responsiveness is unclear. We found that Delta-like1 (Dll1) Notch ligands in splenic fibroblasts regulated MZB cell pool size, migration, and function. Dll1 could not be replaced by the alternative Notch ligand Dll4. Dll1-Notch2 signaling regulated a Myc-dependent gene expression program fostering cell growth and a Myc-independent program controlling cell-movement regulators such as sphingosine-1 phosphate receptor 1 (S1PR1). S1pr1-deficient B cells experienced Notch signaling within B cell follicles without entering the MZ and were retained in the spleen upon Notch deprivation. Key elements of the mouse B cell Notch regulome were preserved in subsets of human memory B cells and B cell lymphomas. Thus, specialized niches program the poised state and patrolling behavior of MZB cells via conserved Myc-dependent and Myc-independent Notch2-regulated mechanisms.

Lymph nodes (LNs) are secondary lymphoid organs where lymphocytes interact with antigen presenting cells to initiate adaptive immune responses within microenvironments established by resident stromal cells. LNs are also the major site of growth of follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL), B cell neoplasms that alter the stromal architecture of LNs in highly stereotypic ways. To characterize FL and CLL cells within the LN microenvironment, we developed a pipeline for single-cell RNA sequencing of all resident LN cells. We observed that proliferation of FL and CLL cells within specialized niches commences with transient upregulation of MYC, subsequent downregulation of which may act to limit the growth potential of these indolent neoplasms. Proliferating FL cells within neoplastic follicles co-localized with follicular dendritic cells, whereas proliferating CLL cells were spatially associated with a distinct set of fibroblasts expressing CCL19 that localized to proliferation centers. We used informatic analyses and microscopy to identify and validate interacting sets of ligand-receptor pairs between proliferating neoplastic B cells, immune cells and stromal fibroblasts, including interactions involving CD74-MIF, TNFRSF13C (BAFF receptor), immunomodulatory factors such as CD55 and Galectin-9 (Gal9), and adhesion molecules. Our analyses highlight common features of these two microenvironment-dependent neoplasms and provide a roadmap for identifying vulnerabilities and new therapeutic strategies. ### Competing Interest Statement I.M. has received research funding from Genentech and Regeneron and is a member of Garuda Therapeutics scientific advisory board (all unrelated to this work). S.C.B. is on the board of directors for the non-profit Institute for Protein Innovation, is on the scientific advisory board and receives funding from Erasca, Inc., for an unrelated project; is an advisor to MPM Capital; and is a consultant for IFM, Scorpion Therapeutics, Odyssey Therapeutics, and Ayala Pharmaceuticals for unrelated projects. J.C.A. is a consultant for Ayala Pharmaceuticals and Remix Therapeutics and is on the scientific advisory board of Cellestia, Inc., all for work unrelated to that described herein.

Supplementary Table S10. p value of NOTCH2 target genes and members of its transcriptional complex that are inversely correlated to BCL6 in primary GC B-cells.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullinreally interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemo-resistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degra-dation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective gamma-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.

Supplementary Table S3. Excel file containing the statistical analysis of the 184 FL targets with significant inverse correlation with BCL6.

Supplementary Table S7. BCL6 target genes in GCB-DLBCL. Spearman and rho. (See Supplementary Figure S1F).

Supplementary Table S1. BCL6 target genes on FL patient samples ChIp-onChIP.

Marginal zone (MZ) B cells are naïve innate-like B cells that function as a first line of defense in the spleen within the blood-rich marginal zone. Previous research revealed a critical role for Notch2 receptors in B cells and Delta-like1 (Dll1) ligands in Ccl19-Cre + fibroblastic stromal cells to support the development and maintenance of MZ B cells in mice. Yet, little is known about how MZ B cells integrate extracellular signals and Notch-regulated transcriptional programs to support their positioning and function. To understand the basis of Dll1/Notch2's specificity, we used monoclonal antibodies to acutely block Notch2 receptors vs. Dll1 or Dll4 Notch ligands in vivo. Dll1 and Notch2 blockade, but not Dll4 inhibition, rapidly decreased MZ B cell numbers and Notch2-regulated transcription in B cells. Analysis of fluorescent reporter alleles revealed co-expression of Ccl19-Cre;ROSA26 YFP and Dll1-mCherry or Dll4-mCherry within white pulp follicles, showing that both Delta-like ligands are available in fibroblastic stromal cells, although with differential abundance and spatial distribution. To determine if the reliance of MZ B cells on Dll1 was due to ligand availability in the appropriate stromal niche, we inactivated endogenous Dll1 and Dll4 via Ccl19-Cre-mediated recombination, and restored Dll1 or Dll4 expression selectively via separate Cre-inducible expression alleles in the Hprt locus. Dll4 could not sustain Notch2-dependent MZ B cells even when expressed in the correct stromal niche, suggesting that Dll1/Notch2 interactions have unique biochemical and functional properties. As MZ B cells are known to shuttle between the follicle and the MZ across the marginal sinus, we assessed where Dll1/Notch2-mediated signals are delivered to MZ B cells. High abundance of intracellular Notch2 and expression of the Notch target gene, Hes1 (as revealed via a Hes1-GFP reporter) was apparent in CD1d high MZ B cells across the marginal sinus, including prominent signaling within B cell follicles (and not only in the MZ) (Figure 1A). We next evaluated the transcriptional programs controlled by Notch in MZ B cells. Notch2/Dll1-regulated genes included both Myc-dependent and Myc-independent cohorts, with the latter highly enriched for integrin and chemotactic receptor genes including S1pr1, shown previously to guide B cells towards the MZ across the marginal sinus. Myc itself was dispensable for MZ B cell positioning and retention in the spleen. Without S1pr1, B cells experienced Notch signaling within B cell follicles even without entering the MZ. Unlike wild-type B cells, mislocalized S1pr1-deficient MZ-like B cells were retained in the spleen upon Notch deprivation (Figure 1B). These findings identify splenic B cell follicles and not the MZ as a central hub for stroma-driven Dll1/Notch2 signaling, with Notch empowering subsequent B cell migration and positioning to the MZ through a Myc-independent transcriptional program. As many Notch-regulated transcriptional targets are conserved in human Notch-driven B cell lymphomas, we speculate that principles of stroma-driven Notch signaling and its downstream effects in B cells have been conserved during evolution from mouse to human B cells - with the Notch signature tagging the B cell subsets that rely on conserved Notch programs and the B cell malignancies that hijack them.

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4β7 in conventional T cells while preserving α4β7 in regulatory T cells, with findings suggesting increased β1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4β7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Supplementary Table S8. DLBCL p-values and rho values on GCB-DLBCL probesets for Notch2 pathway genes. (See Supplementary Figure S1G).

Supplementary Table S4. p value of NOTCH2 target genes and members of its transcriptional complex that are inversely correlated to BCL6 in primary FL specimens.

Supplementary Table S6. DLBCL p-values on DLBCL signatures. (See Supplementary Figure S1E).

Supplementary Figure S1. BCL6 displays a specific genomic localization pattern in FL. Supplementary Figure S2. Inverse correlation between BCL6 and NOTCH2 complex genes in primary GC B-cells. Supplementary Figure S3. GC reaction is impaired in Notch2 knock-in mice. Supplementary Figure S4. GC reaction is impaired in Notch2 knock-in mice. Supplementary Figure S5. BCL6 represses NOTCH2 complex genes and Notch activity. Supplementary Figure S6. FL cells are dependent on BCL6 in a NOTCH2-dependent manner. Supplementary Figure S7. Evaluation of the in vivo potency and specificity of anti-Notch2 blockade. Supplementary Figure S8. RI-BPI suppresses FL tumors in vivo and ex vivo. Supplementary Table S5. Primers used for RT-PCR.

Supplementary Table S2. Gene sets that are enriched among BCL6 target genes in FL samples.

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesinR171W). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesinR171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesinR171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesinR171W. Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesinR171W, and will provide new insights into how the actin cortex regulates lymphocyte function.

Notch signaling promotes T-cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like ligand DLL4. To assess if Notch’s effects are evolutionarily conserved and identify key mechanisms, we studied antibody-mediated DLL4 blockade in a non-human primate model similar to human allo-HCT. Short-term DLL4 blockade improved post-transplant survival with striking, durable protection from gastrointestinal GVHD, out of proportion to other disease sites. Unlike prior immunosuppressive strategies, anti-DLL4 interfered with a T-cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin a4b7 in conventional T-cells via b1 competition for a4 binding, while preserving a4b7 in regulatory T-cells. Thereby, DLL4/Notch blockade decreased effector T-cell infiltration into the gut, with increased regulatory to conventional T-cell ratios early after allo-HCT. Our results identify a conserved, biologically unique and targetable role of DLL4/Notch signaling in GVHD. One Sentence Summary Notch signaling promotes pathogenic effector T cell infiltration of the intestine during acute graft-versus-host disease.

Early T cell development is supported by signals mediated by the Notch1 receptor in T lineage progenitors and Delta-like4 (Dll4) ligand in thymic epithelial cells. Although T cell development is normally restricted to the thymus, extrathymic T cell development has been reported in athymic Foxn1nu/nu nude mice as well as during times of thymopoietic stress such as post-bone marrow transplantation (BMT). A population of CD4+CD8a double positive T cell progenitors can be found in the mesenteric lymph nodes (MLN) in both athymic mice and early post-BMT, suggesting the presence of an extrathymic niche conducive to T cell development. However, whether Notch signaling is required, as well as the cellular source(s) of Notch ligand throughout the process in the MLN, remains unknown. We hypothesize that MLNs harbor a unique environment in which Notch ligands are available to circulating progenitors and critical to sustain extrathymic T cell development in these contexts. To test this, we utilized systemic neutralizing antibodiesas well as loss-of-function genetic models to assess whether the Notch ligands Dll4 or Dll1 are important for extrathymic T cell development. We found that, like homeostatic thymocyte development, Dll4 appears to play an essential role in generation of early T cell progenitors found in the MLN. Furthermore, the source of Dll4 appears to reside within subsets of non-hematopoietic fibroblastic stromal cells lineage traced by a Ccl19-Cre transgene. In sum, these findings shed new light on the cellular and molecular cues regulating T cell development outside of the thymus.