Regulation of antigen-specific T cell infiltration and spatial architecture in multiple myeloma and premalignancy.

Entry of antigen-specific T cells into human tumors is critical for immunotherapy, but the underlying mechanisms are poorly understood. Here, we combined high-dimensional spatial analyses with in vitro and in vivo modeling to study the mechanisms underlying immune infiltration in human multiple myeloma (MM) and its precursor monoclonal gammopathy of undetermined significance (MGUS). Clustered tumor growth was a feature of MM but not MGUS biopsies, and this growth pattern was reproduced in humanized mouse models. MM biopsies exhibited intralesional as well as spatial heterogeneity, with coexistence of T cell-rich and T cell-sparse regions and the presence of areas of T cell exclusion. In vitro studies demonstrated that T cell entry into MM clusters was regulated by agonistic signals and CD2-CD58 interactions. Upon adoptive transfer, antigen-specific T cells localized to the tumor site but required in situ DC-mediated antigen presentation for tumor entry. C-type lectin domain family 9 member A-positive (CLEC9A+) DCs appeared to mark portals of entry for gradients of T cell infiltration in MM biopsies, and their proximity to T cell factor 1-positive (TCF1+) T cells correlated with disease state and risk status. These data illustrate a role for tumor-associated DCs and in situ activation in promoting the infiltration of antigen-specific T cells in MM and provide insights into spatial alterations in tumor/immune cells with malignant evolution.

Changes in Bone Marrow Tumor and Immune Cells Correlate with Durability of Remissions Following BCMA CAR T Therapy in Myeloma.

Chimeric antigen-receptor (CAR) T cells lead to high response rates in myeloma, but most patients experience recurrent disease. We combined several high-dimensional approaches to study tumor/immune cells in the tumor microenvironment (TME) of myeloma patients pre- and post-B-cell maturation antigen (BCMA)-specific CAR T therapy. Lower diversity of pretherapy T-cell receptor (TCR) repertoire, presence of hyperexpanded clones with exhaustion phenotype, and BAFF+PD-L1+ myeloid cells in the marrow correlated with shorter progression-free survival (PFS) following CAR T therapy. In contrast, longer PFS was associated with an increased proportion of CLEC9A+ dendritic cells (DC), CD27+TCF1+ T cells with diverse T-cell receptors, and emergence of T cells expressing marrow-residence genes. Residual tumor cells at initial response express stemlike genes, and tumor recurrence was associated with the emergence of new dominant clones. These data illustrate a dynamic interplay between endogenous T, CAR T, myeloid/DC, and tumor compartments that affects the durability of response following CAR T therapy in myeloma. SIGNIFICANCE: There is an unmet need to identify determinants of durable responses following BCMA CAR T therapy of myeloma. High-dimensional analysis of the TME was performed to identify features of immune and tumor cells that correlate with survival and suggest several strategies to improve outcomes following CAR T therapy. See related commentary by Graham and Maus, p. 478. This article is highlighted in the In This Issue feature, p. 476.

Co-evolution of Immune Response in Multiple Myeloma: Implications for Immune Prevention.

Multiple myeloma (MM), a malignant neoplasm of plasma cells that reside in the bone marrow (BM), is universally preceded by a precursor state termed monoclonal gammopathy of undetermined significance (MGUS). Many individuals with MGUS never progress to MM or progress over many years. Therefore, MGUS provides a unique opportunity to surveil changes in the BM tumor microenvironment throughout disease progression. It is increasingly appreciated that MGUS cells carry many of the genetic changes found in MM. Prior studies have also shown that MGUS cells can be recognized by the immune system, leading to early changes in the BM immune environment compared to that of healthy individuals, including alterations in both innate and adaptive immunity. Progression to clinical MM is associated with attrition of T cells with stem memory-like features and instead accumulation of T cells with more terminally differentiated features. Recent clinical studies have suggested that early application of immune-modulatory drugs, which are known to activate both innate and adaptive immunity, can delay the progression to clinical MM. Understanding the biology of how the immune response and tumors coevolve over time is needed to develop novel immune-based approaches to achieve durable and effective prevention of clinical malignancy.

Aberrant Extrafollicular B Cells, Immune Dysfunction, Myeloid Inflammation, and MyD88-Mutant Progenitors Precede Waldenstrom Macroglobulinemia.

Waldenstrom macroglobulinemia (WM) and its precursor IgM gammopathy are distinct disorders characterized by clonal mature IgM-expressing B-cell outgrowth in the bone marrow. Here, we show by high-dimensional single-cell immunogenomic profiling of patient samples that these disorders originate in the setting of global B-cell compartment alterations, characterized by expansion of genomically aberrant extrafollicular B cells of the nonmalignant clonotype. Alterations in the immune microenvironment preceding malignant clonal expansion include myeloid inflammation and naïve B- and T-cell depletion. Host response to these early lesions involves clone-specific T-cell immunity that may include MYD88 mutation-specific responses. Hematopoietic progenitors carry the oncogenic MYD88 mutations characteristic of the malignant WM clone. These data support a model for WM pathogenesis wherein oncogenic alterations and signaling in progenitors, myeloid inflammation, and global alterations in extrafollicular B cells create the milieu promoting extranodal pattern of growth in differentiated malignant cells. SIGNIFICANCE: These data provide evidence that growth of the malignant clone in WM is preceded by expansion of extrafollicular B cells, myeloid inflammation, and immune dysfunction in the preneoplastic phase. These changes may be related in part to MYD88 oncogenic signaling in pre-B progenitor cells and suggest a novel model for WM pathogenesis. This article is highlighted in the In This Issue feature, p. 549.

Risk-associated alterations in marrow T cells in pediatric leukemia.

Current management of childhood leukemia is tailored based on disease risk determined by clinical features at presentation. Whether properties of the host immune response impact disease risk and outcome is not known. Here, we combine mass cytometry, single cell genomics, and functional studies to characterize the BM immune environment in children with B cell acute lymphoblastic leukemia and acute myelogenous leukemia at presentation. T cells in leukemia marrow demonstrate evidence of chronic immune activation and exhaustion/dysfunction, with attrition of naive T cells and TCF1+ stem-like memory T cells and accumulation of terminally differentiated effector T cells. Marrow-infiltrating NK cells also exhibit evidence of dysfunction, particularly in myeloid leukemia. Properties of immune cells identified distinct immune phenotype-based clusters correlating with disease risk in acute lymphoblastic leukemia. High-risk immune signatures were associated with expression of stem-like genes on tumor cells. These data provide a comprehensive assessment of the immune landscape of childhood leukemias and identify targets potentially amenable to therapeutic intervention. These studies also suggest that properties of the host response with depletion of naive T cells and accumulation of terminal-effector T cells may contribute to the biologic basis of disease risk. Properties of immune microenvironment identified here may also impact optimal application of immune therapies, including T cell-redirection approaches in childhood leukemia.

Early alterations in stem-like/resident T cells, innate and myeloid cells in the bone marrow in preneoplastic gammopathy.

Preneoplastic lesions carry many of the antigenic targets found in cancer cells but often exhibit prolonged dormancy. Understanding how the host response to premalignancy is maintained and altered during malignant transformation is needed to prevent cancer. In order to understand the immune microenvironment in precursor monoclonal gammopathy of undetermined significance (MGUS) and myeloma, we analyzed bone marrow immune cells from 12 healthy donors and 26 MGUS/myeloma patients by mass cytometry and concurrently profiled transcriptomes of 42,606 single immune cells from these bone marrows. Compared to age-matched healthy donors, memory T cells from both MGUS and myeloma patients exhibit greater terminal-effector differentiation. However, memory T cells in MGUS show greater enrichment of stem-like TCF1/7hi cells. Clusters of T cells with stem-like and tissue-residence genes were also found to be enriched in MGUS by single-cell transcriptome analysis. Early changes in both NK and myeloid cells were also observed in MGUS. Enrichment of stem-like T cells correlated with a distinct genomic profile of myeloid cells and levels of Dickkopf-1 in bone-marrow plasma. These data describe the landscape of changes in both innate and adaptive immunity in premalignancy and suggest that attrition of the bone-marrow-resident T cell compartment due to loss of stem-like cells may underlie loss of immune surveillance in myeloma.

Metaboloptics: Visualization of the tumor functional landscape via metabolic and vascular imaging.

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO2 ("Warburg effect"), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO2, indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor's adaptive behavior.

Miniature spectral imaging device for wide-field quantitative functional imaging of the morphological landscape of breast tumor margins.

We have developed a portable, breast margin assessment probe leveraging diffuse optical spectroscopy to quantify the morphological landscape of breast tumor margins during breast conserving surgery. The approach presented here leverages a custom-made 16-channel annular photodiode imaging array (arranged in a 4 × 4 grid), a raster-scanning imaging platform with precision pressure control, and compressive sensing with an optimized set of eight wavelengths in the visible spectral range. A scalable Monte-Carlo-based inverse model is used to generate optical property [ ? s ? ( ? ) and ? a ( ? ) ] measures for each of the 16 simultaneously captured diffuse reflectance spectra. Subpixel sampling (0.75 mm) is achieved through incremental x , y raster scanning of the imaging probe, providing detailed optical parameter maps of breast margins over a 2 × 2 ?? cm 2 area in ? 9 ?? min . The morphological landscape of a tumor margin is characterized using optical surrogates for the fat to fibroglandular content ratio, which has demonstrated diagnostic utility in delineating tissue subtypes in the breast.

Delivery-corrected imaging of fluorescently-labeled glucose reveals distinct metabolic phenotypes in murine breast cancer.

When monitoring response to cancer therapy, it is important to differentiate changes in glucose tracer uptake caused by altered delivery versus a true metabolic shift. Here, we propose an optical imaging method to quantify glucose uptake and correct for in vivo delivery effects. Glucose uptake was measured using a fluorescent D-glucose derivative 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-deoxy-D-glucose (2-NBDG) in mice implanted with dorsal skin flap window chambers. Additionally, vascular oxygenation (SO2) was calculated using only endogenous hemoglobin contrast. Results showed that the delivery factor proposed for correction, "RD", reported on red blood cell velocity and injected 2-NBDG dose. Delivery-corrected 2-NBDG uptake (2-NBDG60/RD) inversely correlated with blood glucose in normal tissue, indicating sensitivity to glucose demand. We further applied our method in metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. The ratio 2-NBDG60/RD was increased in 4T1 tumors relative to 4T07 tumors yet average SO2 was comparable, suggesting a shift toward a "Warburgian" (aerobic glycolysis) metabolism in the metastatic 4T1 line. In heterogeneous regions of both 4T1 and 4T07, 2-NBDG60/RD increased slightly but significantly as vascular oxygenation decreased, indicative of the Pasteur effect in both tumors. These data demonstrate the utility of delivery-corrected 2-NBDG and vascular oxygenation imaging for differentiating metabolic phenotypes in vivo.