Lymphocyte networks are dynamic cellular communities in the immunoregulatory landscape of lung adenocarcinoma.

Lymphocytes are key for immune surveillance of tumors, but our understanding of the spatial organization and physical interactions that facilitate lymphocyte anti-cancer functions is limited. We used multiplexed imaging, quantitative spatial analysis, and machine learning to create high-definition maps of lung tumors from a Kras/Trp53-mutant mouse model and human resections. Networks of interacting lymphocytes ("lymphonets") emerged as a distinctive feature of the anti-cancer immune response. Lymphonets nucleated from small T cell clusters and incorporated B cells with increasing size. CXCR3-mediated trafficking modulated lymphonet size and number, but T cell antigen expression directed intratumoral localization. Lymphonets preferentially harbored TCF1+ PD-1+ progenitor CD8+ T cells involved in responses to immune checkpoint blockade (ICB) therapy. Upon treatment of mice with ICB or an antigen-targeted vaccine, lymphonets retained progenitor and gained cytotoxic CD8+ T cell populations, likely via progenitor differentiation. These data show that lymphonets create a spatial environment supportive of CD8+ T cell anti-tumor responses.

Visinity: Visual Spatial Neighborhood Analysis for Multiplexed Tissue Imaging Data.

New highly-multiplexed imaging technologies have enabled the study of tissues in unprecedented detail. These methods are increasingly being applied to understand how cancer cells and immune response change during tumor development, progression, and metastasis, as well as following treatment. Yet, existing analysis approaches focus on investigating small tissue samples on a per-cell basis, not taking into account the spatial proximity of cells, which indicates cell-cell interaction and specific biological processes in the larger cancer microenvironment. We present Visinity, a scalable visual analytics system to analyze cell interaction patterns across cohorts of whole-slide multiplexed tissue images. Our approach is based on a fast regional neighborhood computation, leveraging unsupervised learning to quantify, compare, and group cells by their surrounding cellular neighborhood. These neighborhoods can be visually analyzed in an exploratory and confirmatory workflow. Users can explore spatial patterns present across tissues through a scalable image viewer and coordinated views highlighting the neighborhood composition and spatial arrangements of cells. To verify or refine existing hypotheses, users can query for specific patterns to determine their presence and statistical significance. Findings can be interactively annotated, ranked, and compared in the form of small multiples. In two case studies with biomedical experts, we demonstrate that Visinity can identify common biological processes within a human tonsil and uncover novel white-blood cell networks and immune-tumor interactions.

Deep Margins Melanoma: How Deep Is Deep Enough?

BACKGROUND: Wide excision (WE) to muscular fascia for invasive melanoma is common practice but excision to subcutaneous tissue may be adequate. We evaluated practice patterns regarding depth of biopsy and excision as well as risks for recurrence. METHODS: Retrospective review of patients with pT1-4 melanoma (cN0) treated with WE at a single institution was performed. Patient factors were evaluated. Biopsy and excision techniques were compared to pathology and reviewed for recurrence. RESULTS: 385 patients from 2006 to 2020 were included. Lesions were on the extremity (n = 189), head/neck (n = 48), trunk (n = 148). Biopsy techniques included shave (n = 330), excisional (n = 36), punch (n = 10), incisional (n = 9). Deep biopsy margins were positive for IM/melanoma in situ in 139 patients. WE specimens were taken to muscular fascia (n = 218) or mid/deep fat (n = 144). 51 patients had recurrent disease or a new primary lesion: locoregional (n = 31), distant (3), or new lesions (n = 17). DISCUSSION: Patient characteristics associated with recurrence include older age and female gender. Tumor characteristics associated with recurrence include lesions located on the trunk, superficial spreading melanoma, ulceration, perineural invasion, and clinical T and P stage. Patients that recurred were more likely to have WE taken to or including muscular fascia. Biopsy type, deep margin on biopsy, and depth of dissection was not associated with recurrence.

Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice.

Existing preclinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates, we extrapolated half-life times in the circulation of between 40 and 260 s and intravasation rates between 60 and 107,000 CTCs/hour in mouse models of small-cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). Additionally, direct transfer of only 1-2% of daily-shed CTCs using our blood-exchange technique from late-stage, SCLC-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.

Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

Antigen dominance hierarchies shape TCF1+ progenitor CD8 T cell phenotypes in tumors.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1+ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1+ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6+ TCF1+ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6+ TCF1+ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

T cell-specific inhibition of multiple apoptotic pathways blocks negative selection and causes autoimmunity.

T cell self-tolerance is thought to involve peripheral tolerance and negative selection, involving apoptosis of autoreactive thymocytes. However, evidence supporting an essential role for negative selection is limited. Loss of Bim, a Bcl-2 BH3-only protein essential for thymocyte apoptosis, rarely results in autoimmunity on the C57BL/6 background. Mice with T cell-specific over-expression of Bcl-2, that blocks multiple BH3-only proteins, are also largely normal. The nuclear receptor Nur77, also implicated in negative selection, might function redundantly to promote apoptosis by associating with Bcl-2 and exposing its potentially pro-apoptotic BH3 domain. Here, we report that T cell-specific expression of a Bcl2 BH3 mutant transgene results in enhanced rescue of thymocytes from negative selection. Concomitantly, Treg development is increased. However, aged BH3 mutant mice progressively accumulate activated, autoreactive T cells, culminating in development of multi-organ autoimmunity and lethality. These data provide strong evidence that negative selection is crucial for establishing T cell tolerance.

Premalignant PTEN-deficient thymocytes activate microRNAs miR-146a and miR-146b as a cellular defense against malignant transformation.

Cancer develops by a multistep process during which cells acquire characteristics that allow them to evade apoptosis and proliferate unchecked. Sequential acquisition of genetic alterations drives this process but also causes cellular stress, frequently prompting cells to enter a premalignant period during which they mount a defense against transformation. T cell-specific deletion of the tumor suppressor PTEN in mice induces premalignancy in the thymus and development of CD4(+) T-cell lymphomas in the periphery. Here we sought to identify factors mediating the cellular defense against transformation during the premalignant period. We identified several microRNAs upregulated specifically in premalignant thymocytes, including miR-146a, miR-146b, and the miR-183/96/182 cluster. CD4-driven T cell-specific transgenic overexpression of mir-146a and mir-146b significantly delayed PTEN-deficient lymphomagenesis and delayed c-myc oncogene induction, a key driver of transformation in PTEN-deficient T-cell malignancies. We found that miR-146a and miR-146b targeting of Traf6 attenuates TCR signaling in the thymus and inhibits downstream NF-κB-dependent induction of c-myc. Additionally, c-myc repression in mature CD4 T cells by miR-146b impaired TCR-mediated proliferation. Hence, we have identified 2 miRNAs that are upregulated as part of the cellular response against transformation that, when overrepresented, can effectively inhibit progression to malignancy in the context of PTEN deficiency.

Vascular endothelial cadherin modulates renal interstitial fibrosis.

BACKGROUND/AIMS: Renal interstitial fibrosis is a final common pathway of all chronic, progressive kidney diseases. Peritubular capillary rarefaction is strongly correlated with fibrosis. The adherens junction protein vascular endothelial cadherin (VE-cadherin) is thought to play a critical role in vascular integrity. We hypothesized that VE-cadherin modulates the renal microcirculation during fibrogenesis and ultimately affects renal fibrosis. METHODS: Unilateral ureteral obstruction (UUO) was used as a renal fibrosis model in VE-cadherin heterozygote (VE+/-) and wild-type (WT) mice, and the kidneys were harvested at days 3, 7, and 14. Peritubular capillary changes and fibrogenesis were investigated. RESULTS: VE+/- mice had lower levels of VE-cadherin protein than WT mice at 3 and 7, but not 14 days after UUO. Vascular permeability was significantly greater in VE+/- mice 7 days after UUO, while peritubular capillary density was not significantly different in VE+/- and WT mice. Interstitial myofibroblast numbers and collagen I and III mRNA levels were significantly higher in VE+/- mice, consistent with a stronger early fibrogenic response. Expression of the pericyte marker neuron-glial antigen 2 was upregulated after UUO, but was not greater in VE+/- mice compared to the WT mice. CONCLUSION: Our data suggest that VE-cadherin controls vascular permeability and limits fibrogenesis after UUO.

Protein kinase C regulates mitochondrial targeting of Nur77 and its family member Nor-1 in thymocytes undergoing apoptosis.

Nur77 orphan steroid receptor and its family member Nor-1 are required for apoptosis of developing T cells. In thymocytes, signals from the TCR complex induce Nur77 and Nor-1 expression followed by translocation from the nucleus to mitochondria. Nur77 and Nor-1 associate with Bcl-2 in the mitochondria, resulting in a conformation change that exposes the Bcl-2 BH3 domain, a presumed pro-apoptotic molecule of Bcl-2. As Nur77 and Nor-1 are heavily phosphorylated, we examined the requirement of Nur77 and Nor-1 phosphorylation in mitochondria translocation and Bcl-2 BH3 exposure. We found that HK434, a PKC agonist, in combination with calcium ionophore, can induce Nur77 and Nor-1 phosphorylation, translocation, Bcl-2 BH3 exposure and thymocyte apoptosis. Inhibitors of both classical and novel forms of PKC were able to block this process. In contrast, only the general but not classical PKC-specific inhibitors were able to block the same process initiated by PMA, a commonly used PKC agonist. These data demonstrate a differential activation of PKC isoforms by PMA and HK434 in thymocytes, and show the importance of PKC in mitochondria translocation of Nur77/Nor-1 and Bcl-2 conformation change during TCR-induced thymocyte apoptosis.