Antitumoral immunity requires organized, spatially nuanced interactions between components of the immune tumor microenvironment (iTME). Understanding this coordinated behavior in effective versus ineffective tumor control will advance immunotherapies. We re-engineered co-detection by indexing (CODEX) for paraffin-embedded tissue microarrays, enabling simultaneous profiling of 140 tissue regions from 35 advanced-stage colorectal cancer (CRC) patients with 56 protein markers. We identified nine conserved, distinct cellular neighborhoods (CNs)—a collection of components characteristic of the CRC iTME. Enrichment of PD-1⁺CD4⁺ T cells only within a granulocyte CN positively correlated with survival in a high-risk patient subset. Coupling of tumor and immune CNs, fragmentation of T cell and macrophage CNs, and disruption of inter-CN communication was associated with inferior outcomes. This study provides a framework for interrogating how complex biological processes, such as antitumoral immunity, occur through concerted actions of cells and spatial domains.

The intestine is a complex organ that promotes digestion, extracts nutrients, participates in immune surveillance, maintains critical symbiotic relationships with microbiota and affects overall health¹. The intesting has a length of over nine metres, along which there are differences in structure and function². The localization of individual cell types, cell type development trajectories and detailed cell transcriptional programs probably drive these differences in function. Here, to better understand these differences, we evaluated the organization of single cells using multiplexed imaging and single-nucleus RNA and open chromatin assays across eight different intestinal sites from nine donors. Through systematic analyses, we find cell compositions that differ substantially across regions of the intestine and demonstrate the complexity of epithelial subtypes, and find that the same cell types are organized into distinct neighbourhoods and communities, highlighting distinct immunological niches that are present in the intestine. We also map gene regulatory differences in these cells that are suggestive of a regulatory differentiation cascade, and associate intestinal disease heritability with specific cell types. These results describe the complexity of the cell composition, regulation and organization for this organ, and serve as an important reference map for understanding human biology and disease.

Neutropenia is probably the strongest known predisposition to infection with otherwise harmless environmental or microbiota-derived species. Because initial swarming of neutrophils at the site of infection occurs within minutes, rather than the hours required to induce “emergency granulopoiesis,” the relevance of having high numbers of these cells available at any one time is obvious. We observed that germ-free (GF) animals show delayed clearance of an apathogenic bacterium after systemic challenge. In this article, we show that the size of the bone marrow myeloid cell pool correlates strongly with the complexity of the intestinal microbiota. The effect of colonization can be recapitulated by transferring sterile heat-treated serum from colonized mice into GF wild-type mice. TLR signaling was essential for microbiota-driven myelopoiesis, as microbiota colonization or transferring serum from colonized animals had no effect in GF MyD88−/−TICAM1−/− mice. Amplification of myelopoiesis occurred in the absence of microbiota-specific IgG production. Thus, very low concentrations of microbial Ags and TLR ligands, well below the threshold required for induction of adaptive immunity, sets the bone marrow myeloid cell pool size. Coevolution of mammals with their microbiota has probably led to a reliance on microbiota-derived signals to provide tonic stimulation to the systemic innate immune system and to maintain vigilance to infection. This suggests that microbiota changes observed in dysbiosis, obesity, or antibiotic therapy may affect the cross talk between hematopoiesis and the microbiota, potentially exacerbating inflammatory or infectious states in the host.

Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease with a characteristic chronic phase (cp) of several years before progression to blast crisis (bc). The immune system may contribute to disease control in CML. We analyzed leukemia-specific immune responses in cpCML and bcCML in a retroviral-induced murine CML model. In the presence of cpCML and bcCML expressing the glycoprotein of lymphocytic choriomeningitis virus as a model leukemia antigen, leukemia-specific cytotoxic T lymphocytes (CTLs) became exhausted. They maintained only limited cytotoxic activity, and did not produce interferon-γ or tumor necrosis factor-α or expand after restimulation. CML-specific CTLs were characterized by high expression of programmed death 1 (PD-1), whereas CML cells expressed PD-ligand 1 (PD-L1). Blocking the PD-1/PD-L1 interaction by generating bcCML in PD-1–deficient mice or by repetitive administration of αPD-L1 antibody prolonged survival. In addition, we found that PD-1 is up-regulated on CD8⁺ T cells from CML patients. Taken together, our results suggest that blocking the PD-1/PD-L1 interaction may restore the function of CML-specific CTLs and may represent a novel therapeutic approach for CML.

Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1⁺ CD4⁺ T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies.