Spatial mapping of human hematopoiesis at single-cell resolution reveals aging-associated topographic remodeling.

ABSTRACT: The spatial anatomy of hematopoiesis in the bone marrow (BM) has been extensively studied in mice and other preclinical models, but technical challenges have precluded a commensurate exploration in humans. Institutional pathology archives contain thousands of paraffinized BM core biopsy tissue specimens, providing a rich resource for studying the intact human BM topography in a variety of physiologic states. Thus, we developed an end-to-end pipeline involving multiparameter whole tissue staining, in situ imaging at single-cell resolution, and artificial intelligence-based digital whole slide image analysis and then applied it to a cohort of disease-free samples to survey alterations in the hematopoietic topography associated with aging. Our data indicate heterogeneity in marrow adipose tissue (MAT) content within each age group and an inverse correlation between MAT content and proportions of early myeloid and erythroid precursors, irrespective of age. We identify consistent endosteal and perivascular positioning of hematopoietic stem and progenitor cells (HSPCs) with medullary localization of more differentiated elements and, importantly, uncover new evidence of aging-associated changes in cellular and vascular morphologies, microarchitectural alterations suggestive of foci with increased lymphocytes, and diminution of a potentially active megakaryocytic niche. Overall, our findings suggest that there is topographic remodeling of human hematopoiesis associated with aging. More generally, we demonstrate the potential to deeply unravel the spatial biology of normal and pathologic human BM states using intact archival tissue specimens.

Multi-omic profiling of follicular lymphoma reveals changes in tissue architecture and enhanced stromal remodeling in high-risk patients.

Follicular lymphoma (FL) is a generally incurable malignancy that evolves from developmentally blocked germinal center (GC) B cells. To promote survival and immune escape, tumor B cells undergo significant genetic changes and extensively remodel the lymphoid microenvironment. Dynamic interactions between tumor B cells and the tumor microenvironment (TME) are hypothesized to contribute to the broad spectrum of clinical behaviors observed among FL patients. Despite the urgent need, existing clinical tools do not reliably predict disease behavior. Using a multi-modal strategy, we examined cell-intrinsic and -extrinsic factors governing progression and therapeutic outcomes in FL patients enrolled onto a prospective clinical trial. By leveraging the strengths of each platform, we identify several tumor-specific features and microenvironmental patterns enriched in individuals who experience early relapse, the most high-risk FL patients. These features include stromal desmoplasia and changes to the follicular growth pattern present 20 months before first progression and first relapse.

TIGIT is a key inhibitory checkpoint receptor in lymphoma.

BACKGROUND: PD-1 checkpoint blockade therapy (CBT) has greatly benefited patients with select solid tumors and lymphomas but has limited efficacy against diffuse large B-cell lymphoma (DLBCL). Because numerous inhibitory checkpoint receptors have been implicated in driving tumor-specific T cell dysfunction, we hypothesized that combinatorial CBT would enhance the activity of anti-PD-1-based therapy in DLBCL. T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is a coinhibitory receptor expressed on dysfunctional tumor-infiltrating T cells, and TIGIT blockade has demonstrated encouraging activity in combination with PD-1 blockade in murine tumor models and in clinical studies. However, the degree to which TIGIT mediates T cell dysfunction in DLBCL has not been fully explored. RESULTS: Here, we demonstrate that TIGIT is broadly expressed on lymphoma-infiltrating T cells (LITs) across a variety of human lymphomas and is frequently coexpressed with PD-1. TIGIT expression is particularly common on LITs in DLBCL, where TIGIT+ LITs often form distinct cellular communities and exhibit significant contact with malignant B cells. TIGIT+/PD-1+ LITs from human DLBCL and murine lymphomas exhibit hypofunctional cytokine production on ex vivo restimulation. In mice with established, syngeneic A20 B-cell lymphomas, TIGIT or PD-1 mono-blockade leads to modest delays in tumor outgrowth, whereas PD-1 and TIGIT co-blockade results in complete rejection of A20 lymphomas in most mice and significantly prolongs survival compared with mice treated with monoblockade therapy. CONCLUSIONS: These results provide rationale for clinical investigation of TIGIT and PD-1 blockade in lymphomas, including DLBCL.

Lack of intrafollicular memory CD4 + T cells is predictive of early clinical failure in newly diagnosed follicular lymphoma.

Despite a characteristic indolent course, a substantial subset of follicular lymphoma (FL) patients has an early relapse with a poor outcome. Cells in the microenvironment may be a key contributor to treatment failure. We used a discovery and validation study design to identify microenvironmental determinants of early failure and then integrated these results into the FLIPI. In total, 496 newly diagnosed FL grade 1-3 A patients who were prospectively enrolled into the MER cohort from 2002 to 2012 were evaluated. Tissue microarrays were stained for CD4, CD8, FOXP3, CD32b, CD14, CD68, CD70, SIRP-α, TIM3, PD-1, and PD-L1. Early failure was defined as failing to achieve event-free survival at 24 months (EFS24) in immunochemotherapy-treated patients and EFS12 in all others. CyTOF and CODEX analysis were performed to characterize intratumoral immunophenotypes. Lack of intrafollicular CD4 expression was the only predictor of early failure that replicated with a pooled OR 2.37 (95%CI 1.48-3.79). We next developed a bio-clinical risk model (BioFLIPI), where lack of CD4 intrafollicular expression moved patients up one FLIPI risk group, adding a new fourth high-risk group. Compared with BioFLIPI score of 1, patients with a score of 2 (OR 2.17; 95% CI 1.08-4.69), 3 (OR 3.53; 95% CI 1.78-7.54), and 4 (OR 8.92; 95% CI 4.00-21.1) had increasing risk of early failure. The favorable intrafollicular CD4 T cells were identified as activated central memory T cells, whose prognostic value was independent from genetic features. In conclusion, lack of intrafollicular CD4 expression predicts early failure in FL and combined with FLIPI improves identification of high-risk patients; however, independent validation is warranted.