Do structures exist within the embryonic central nervous system that guide axons across the midline during development of the great cerebral commissures (corpus callosum, anterior commissure)? With the use of serial section and reconstructive computer graphic techniques we have found that during normal ontogeny of the mouse forebrain and before the arrival of the pioneer fibers of the corpus callosum at the midline, a population of primitive glial cells migrates medially (through the fused walls of the dorsal septum) from the ependymal zones of each hemisphere. At the midline, and well rostral to the lamina terminalis, these cells unite to form a bridgelike structure or "sling" suspended below the longitudinal cerebral fissure. The first callosal axons grow along the surface of this cellular bridge as they travel toward the contralateral side of the brain. The "sling" disappears neonatally. The fibers of the anterior commissure grow within the lamina terminalis along a different type of preformed glial structure. Movement of these axons occurs through an aligned system of glial processes separated by wide extracellular spaces. Do these transient glial tissues actually provide guidance cues to the commissural axons? Analyses of three situations in which the glial "sling" is genetically or surgically impaired or nonexistent indicate that this structure does, indeed, play an essential role in the development of the corpus callosum. We have analyzed (1) the embryonic stages of a congenitally acallosal mouse mutant (strain BALB/cCF), (2) several pouch stages of a primitive acallosal marsupial, Didelphys virginiana (opossum), and (3) animals in which the "sling" had been lesioned surgically through the uterine wall in the normal embryo (strain C57BL/6J). In the acallosal mouse mutant fusion of the septal midline is delayed by about 72 hours and the "sling" does not form. Although the would-be callosal axons approach the midline on schedule, they do not cross. Instead, the callosal fibers whirl into a pair of large neuromas adjacent to the longitudinal fissure. Similarly, in the opossum, fusion of the medial septal walls and formation of the glial "sling" are also lacking. However, in this species, instead of traveling dorsally, the "callosal" axons turn ventrally and pass contralaterally by way of the anterior commissure pathway. Surgical disunion of the glial "sling" also resulted in acallosal individuals. The callosal pathology in these affected animals mimicked exactly that of the genetically lesioned mutant. Our observations suggest that many different types of oriented glial tissues exist within the embryonic neural anlage. We propose that such tissues have the ability to influence the directionality of axonal movements and, thereby, play a crucial role in establishing orderly fiber projections within the developing central nervous system.

Introduction: Regulatory T cells (Tregs), a highly immunosuppressive subset of CD4 T cells, are enriched in B-cell non-Hodgkin lymphoma (NHL) and constitute a barrier to potent antitumor immune responses. Despite extensive studies, the significance of tumor-infiltrating Tregs on disease outcome is unclear and while Tregs may express co-inhibitory and co-stimulatory receptors, the role of intratumoral Tregs in the context of immune checkpoint therapy remains elusive. Emerging evidence suggests heterogeneity among Tregs and their suppressive capacities in cancer, emphasizing the need for additional markers to identify highly suppressive Tregs. Therefore, an in-depth characterization of Treg heterogeneity in NHL could provide important insight into the disease pathogenesis and have implications for rational drug design. Methods: Expression of checkpoint receptors in Tregs was characterized by fluorescence flow cytometry and mass cytometry analysis of single-cell suspensions from diffuse large B-cell lymphoma (DLBCL; n = 16), follicular lymphoma (FL; n = 8), mantle cell lymphoma (MCL; n = 10), marginal zone lymphoma (MZL; n = 2), chronic lymphocytic lymphoma (CLL; n = 7), as well as tonsils (n = 8) and peripheral blood (n = 4) from healthy donors. Functional characterization of intratumoral Tregs was performed by a proliferation assay using FACS-sorted Tregs as suppressor cells and autologous CellTrace Violet-labelled T effector cells as responder cells. Single-cell RNA sequencing (scRNA-seq) was performed on FACS-sorted CD4 T cells from 3 DLBCL, 3 FL and 3 healthy donor tonsils using the 10X Genomics single cell 5' based library construction and VDJ libraries for TCR-sequencing. Additionally, for simultaneous profiling of phenotypic features with the mRNA expression in single cells, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq) was applied. The Treg compartment was characterized by clustering into distinct transcriptional Treg states and differential expression of marker genes. Results: TIGIT and CTLA-4 were identified as common markers of intratumoral Tregs, in addition to FOXP3 and CD25. Unsupervised computational analysis revealed two distinct Treg subsets, based on contrasting expression of PD-1, OX40, CD226 and ICOS (Figure 1A). One subset displayed a checkpoint receptorlow phenotype that corresponded to peripheral blood Tregs. The second subset had a checkpoint receptorhigh phenotype with elevated levels of PD-1, OX40, ICOS, TIGIT, CTLA-4 and increased levels of activation markers CD28, CD69 and CD95/Fas. The frequency of checkpoint receptorhigh Tregs was significantly increased in NHL tumors, compared to PBMCs and tonsils from healthy donors. FL tumors had the highest frequency of Tregs with receptorhigh phenotype among the NHL entities (median frequency of 86%, range 71-92%) and DLBCL had the highest donor-to-donor variation (median frequency of 77%, range 35-98%) (Figure 1B). This phenotypic heterogeneity of the Treg compartment reflected different suppressive capacities of the two subsets. Checkpoint receptorhigh Tregs were more potent mediators of immunosuppression in terms of suppressing the proliferation of autologous effector CD4 and CD8 T cells (Figure 1C). Furthermore, transcriptomic analysis of CD4 T cells by scRNA-seq and CITEseq revealed distinct transcriptomic signatures of the checkpoint receptorhigh and -receptorlow subsets. In addition, a third subset of Tregs, characterized by increased expression of LAG3 and immunosuppression-associated genes (CTLA-4, IL10, CD38, KLRB1) but lack of FOXP3, was identified (Figure 1D-E). Analysis of scTCR-sequences to compare TCR repertoires and to identify developmental trajectories will further add to our knowledge of intratumoral Tregs. Conclusions: These results reveal heterogeneity within the Treg compartment in NHL based on expression of checkpoint receptors, transcriptional profiles and suppressive capacities. As intratumoral Treg phenotypes differ from peripheral blood Tregs, this presents new therapeutic opportunities. Specific targeting of intratumoral Tregs would lead to stronger antitumor effects while limiting immune-related adverse events. A deeper understanding of Treg heterogeneity within the tumor microenvironment could therefore open new paths for rational design of immune checkpoint therapy. Disclosures Kolstad: Merck: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees, Research Funding. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding.

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Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Both transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: We performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients. An average sequencing coverage of 700X was achieved for both tumor and normal samples, which ensured high data quality and ability to detect SNVs and InDels using our bench-marked bioinformatics pipeline. SNP6.0 data was available for 93 sequenced tumors and used to infer allele-specific copy number alterations. Several computational tools were applied to identify potential cancer driver genes (MutSig2CV, 2020plus), mutational signatures (MutationalPatterns), and to study clonal evolution (PyClone, ClonEvol). Results: Twenty-two of the 44 FL patients experienced relapses without transformation (referred to as the nFL group), and 22 patients experienced transformation (the tFL group). Nineteen patients (including both groups) experienced POD24. Both transformation and POD24 were associated with inferior overall survival. The median non-synonymous mutational burden was 96 per biopsy (range 10-326). Pre-treatment FL biopsies from the tFL group had significantly higher mutational burden compared to the nFL group (p<0.02). The application of two different approaches for driver gene discovery resulted in a total of 55 as putative drivers. Sixteen of these genes were identified by both tools, including the known FL driver genes CREBBP, KMT2D, TNFRSF14 and BCL2, all being mutated in more than 40 % of cases, and the novel cancer driver genes CTSS and VPS39. By applying MutSig2CV to different categories of biopsies, we identified RRAGC, ATP6V1B2, and HNRNPU as being significantly mutated in pre-treatment tumors of the nFL group, whereas TNFRSF14 and EZH2 were significantly mutated in pre-treatment tumors of the tFL group. When comparing pretreatment and relapsed FL biopsies, we identified CTSS, ZNF493 and HLA-A as significantly mutated in relapsed FL, thus potentially linking these genes to FL relapse. Analysis of mutational signatures demonstrated the presence of the same signatures for longitudinal samples, and no contrasting difference was found between nFL and tFL biopsies. Finally, we constructed clonal phylogenetic trees for 31 patients with serial biopsies and observed that both non-transformed and transformed tumors can evolve directly from CPCs. Contrary to previous findings, we did not find a preference for divergent evolution from FL to tFL, as compared to FL to relapsed FL. There was no significant correlation between evolution pattern and time intervals in the nFL and tFL groups. Based on the mutational history, we confirmed that CREBBP and KMT2D were the most frequently mutated genes seen in CPCs and PCLO, ATP6V1B2 and LRRN3 were also identified as early mutated genes. TNFRSF14, TBL1XR1 and GNAI2 were often mutated later, but before clonal expansion. Conclusions: We show that at diagnosis, the mutational burden in the tFL group is significantly higher than in the nFL group. By systematically applying driver discovery tools we have confirmed known driver genes and discovered novel genes that may be of importance for FL progression and transformation. Divergent evolution is a common feature both during relapse of FL and transformation to DLBCL, regardless of the time interval between the longitudinal biopsies. A better characterization of the CPCs may provide additional therapeutic opportunities towards a cure for FL patients. Disclosures Holte: Novartis: Honoraria, Other: Advisory board.

Neutrophils are innate cells that have been suggested to play a critical role in terminal differentiation of NK cells. Whether this is a direct effect or a consequence of global immune changes with effects on NK cell homeostasis remains unknown. Here, we used high-resolution flow and mass cytometry to examine NK cell repertoires in 64 neutropenic patients and 27 healthy age- and gender-matched controls. A subgroup of neutropenic patients had lower frequencies and absolute numbers of NK cells, yet increased frequencies of CD56bright among NK cells (Figure 1A-C). Moreover, their CD56dim compartment was characterized by a block in differentiation, with a relative lack of NKG2A-CD57+KIR+ NK cells. In line with the differentiation arrest, no expansion of adaptive NK cells could be detected in CMV-seropositive patients from this subgroup. Furthermore, CD56dim NK cells showed increased frequencies of Ki-67+, Tim-3+ and TIGIT+ cells suggestive of activation and exhaustion (Figure 1D). The systemic imprint in the NK cell repertoire was associated with a blunted tumor target cell response with inefficient killing and a lower proportion of degranulating CD56dim cells (Figure 1E). RNA sequencing of the NK cell compartment further revealed that the differentiation arrest was linked to increased expression of transcription factors and genes involved in proliferation and cytokine signaling (Figure 1F). Serum protein profiling of 264 proteins showed upregulation of pathways related to apoptosis and cell turnover, as well as immune regulation and inflammation including higher levels of IL-10, IL-18 and IL-27 in these patients (Figure 1G-H). Notably, the majority of patients with perturbed NK cell compartment exhibited high-grade neutropenia, overall suggesting that the profoundly altered NK cell homeostasis was tightly connected to the severity of their underlying etiology (Figure 1I). Disclosures Meinke: XNK Therapeutics AB: Consultancy. Palmblad:Roche Sweden Inc: Speakers Bureau; Chieti Canada Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Malmberg:Vycellix: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Patents & Royalties.