Collaboration between IL-7 and IL-15 enables adaptation of tissue-resident and circulating memory CD8+ T cells.

Interleukin-7 (IL-7) is considered a critical regulator of memory CD8+ T cell homeostasis, but this is primarily based on analysis of circulating and not tissue-resident memory (TRM) subsets. Furthermore, the cell-intrinsic requirement for IL-7 signaling during memory homeostasis has not been directly tested. Using inducible deletion, we found that Il7ra loss had only a modest effect on persistence of circulating memory and TRM subsets and that IL-7Rα was primarily required for normal basal proliferation. Loss of IL-15 signaling imposed heightened IL-7Rα dependence on memory CD8+ T cells, including TRM populations previously described as IL-15-independent. In the absence of IL-15 signaling, IL-7Rα was upregulated, and loss of IL-7Rα signaling reduced proliferation in response to IL-15, suggesting cross-regulation in memory CD8+ T cells. Thus, across subsets and tissues, IL-7 and IL-15 act in concert to support memory CD8+ T cells, conferring resilience to altered availability of either cytokine.

An In Vivo Model of Human Macrophages in Metastatic Melanoma.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome, and high-dimensional analyses of patient samples reveal the heterogeneity of the immune TME. Macrophages infiltrate TMEs and generally associate with tumor progression, but the underlying mechanisms are incompletely understood. Because experimental systems are needed to elucidate the functional properties of these cells, we developed a humanized mouse model reconstituted with human immune cells and human melanoma. We used two strains of recipient mice, supporting or not supporting the development of human myeloid cells. We found that human myeloid cells favored metastatic spread of the primary tumor, thereby recapitulating the cancer-supportive role of macrophages. We next analyzed the transcriptome of human immune cells infiltrating tumors versus other tissues. This analysis identified a cluster of myeloid cells present in the TME, but not in other tissues, which do not correspond to canonical M2 cells. The transcriptome of these cells is characterized by high expression of glycolytic enzymes and multiple chemokines and by low expression of gene sets associated with inflammation and adaptive immunity. Compared with humanized mouse results, we found transcriptionally similar myeloid cells in patient-derived samples of melanoma and other cancer types. The humanized mouse model described here thus complements patient sample analyses, enabling further elucidation of fundamental principles in melanoma biology beyond M1/M2 macrophage polarization. The model can also support the development and evaluation of candidate antitumor therapies.

Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics.

We developed a chemically inducible Cas9 (ciCas9) and a droplet digital PCR assay for double-strand breaks (DSB-ddPCR) to investigate the kinetics of Cas9-mediated generation and repair of DSBs in cells. ciCas9 is a rapidly activated, single-component Cas9 variant engineered by replacing the protein's REC2 domain with the BCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCL-xL-BH3 interaction within minutes. DSB-ddPCR demonstrates time-resolved, highly quantitative, and targeted measurement of DSBs. Combining these tools facilitated an unprecedented exploration of the kinetics of Cas9-mediated DNA cleavage and repair. We find that sgRNAs targeting different sites generally induce cleavage within minutes and repair within 1 or 2 h. However, we observe distinct kinetic profiles, even for proximal sites, and this suggests that target sequence and chromatin state modulate cleavage and repair kinetics.

Massively parallel digital transcriptional profiling of single cells.

Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.

Mitochondrial DNA exhibits resistance to induced point and deletion mutations.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations contributes to the pathogenesis of human disease. Currently, mitochondrial mutations are largely considered results of inaccurate processing of its heavily damaged genome. However, mainly from a lack of methods to monitor mtDNA mutations with sufficient sensitivity and accuracy, a link between mtDNA damage and mutation has not been established. To test the hypothesis that mtDNA-damaging agents induce mtDNA mutations, we exposed MutaTMMouse mice to benzo[a]pyrene (B[a]P) or N-ethyl-N-nitrosourea (ENU), daily for 28 consecutive days, and quantified mtDNA point and deletion mutations in bone marrow and liver using our newly developed Digital Random Mutation Capture (dRMC) and Digital Deletion Detection (3D) assays. Surprisingly, our results demonstrate mutagen treatment did not increase mitochondrial point or deletion mutation frequencies, despite evidence both compounds increase nuclear DNA mutations and demonstrated B[a]P adduct formation in mtDNA. These findings contradict models of mtDNA mutagenesis that assert the elevated rate of mtDNA mutation stems from damage sensitivity and abridged repair capacity. Rather, our results demonstrate induced mtDNA damage does not readily convert into mutation. These findings suggest robust mitochondrial damage responses repress induced mutations after mutagen exposure.

Alemtuzumab-related thyroid dysfunction in a phase 2 trial of patients with relapsing-remitting multiple sclerosis.

CONTEXT: Alemtuzumab, an anti-CD52 monoclonal antibody, increased the risk of thyroid dysfunction in CAMMS223, a phase 2 trial in relapsing-remitting multiple sclerosis. OBJECTIVE: The objective of the study was a detailed description of thyroid dysfunction in CAMMS223. DESIGN: Relapsing-remitting multiple sclerosis patients (n=334) were randomized 1:1:1 to 44 µg sc interferon-ß-1a (SC IFNB-1a, Rebif) or annual courses of 12 or 24 mg iv alemtuzumab. Thyroid function tests (TSH, free T3, free T4) and thyrotropin-binding inhibitory immunoglobulin (TBII) were assessed at screening, month 1, and quarterly thereafter; antithyroid peroxidase antibodies were assessed at screening and every 6 months. Thyroid dysfunction episodes were categorized post hoc by an endocrinologist. RESULTS: During a median follow-up of 57.3 months, 34% of alemtuzumab and 6.5% of SC IFNB-1a patients had thyroid dysfunction (P<.0001). Ten percent of alemtuzumab and 3% of SC IFNB-1a patients had more than one episode of thyroid dysfunction. With alemtuzumab, Graves' hyperthyroidism occurred in 22%, hypothyroidism in 7%, and subacute thyroiditis in 4%. Of patients with overt Graves' hyperthyroidism, 23% spontaneously became euthyroid and an additional 15% spontaneously developed hypothyroidism. Of patients with overt hypothyroidism, 74% were TBII positive. The annual incidence of a first episode of thyroid dysfunction increased each year through year 3 and then decreased each subsequent study year. CONCLUSIONS: Thyroid dysfunction was more common with alemtuzumab than with SC IFNB-1a. There were few serious episodes. Regular monitoring facilitated early detection. Unique features of this population included high prevalence of Graves' hyperthyroidism, multiple episodes of thyroid dysfunction in individual patients, spontaneous hypothyroidism after overt Graves' hyperthyroidism, and a high prevalence of TBII-positive overt hypothyroidism.