Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice.

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

Enzyme-mediated depletion of methylthioadenosine restores T cell function in MTAP-deficient tumors and reverses immunotherapy resistance.

Chromosomal region 9p21 containing tumor suppressors CDKN2A/B and methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic deletions in cancer. 9p21 loss is correlated with reduced tumor-infiltrating lymphocytes (TILs) and resistance to immune checkpoint inhibitor (ICI) therapy. Previously thought to be caused by CDKN2A/B loss, we now show that it is loss of MTAP that leads to poor outcomes on ICI therapy and reduced TIL density. MTAP loss causes accumulation of methylthioadenosine (MTA) both intracellularly and extracellularly and profoundly impairs T cell function via the inhibition of protein arginine methyltransferase 5 (PRMT5) and by adenosine receptor agonism. Administration of MTA-depleting enzymes reverses this immunosuppressive effect, increasing TILs and drastically impairing tumor growth and importantly, synergizes well with ICI therapy. As several studies have shown ICI resistance in 9p21/MTAP null/low patients, we propose that MTA degrading therapeutics may have substantial therapeutic benefit in these patients by enhancing ICI effectiveness.

A Molecular Analysis of Memory B Cell and Antibody Responses Against Plasmodium falciparum Merozoite Surface Protein 1 in Children and Adults From Uganda.

Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum (Pf) merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against Pf develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, full-length Pf merozoite surface protein 1 (PfMSP1FL), in individuals from a region in Uganda with high Pf transmission. Our results showed that PfMSP1FL-specific B cells in adults with immunological protection against malaria were predominantly IgG+ classical MBCs, while children with incomplete protection mainly harbored IgM+ PfMSP1FL-specific classical MBCs. In contrast, anti-PfMSP1FL plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against PfMSP1FL, with broadening of the response against non-3D7 strains in adults. The B cell receptors encoded by PfMSP1FL-specific IgG+ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable PfMSP1 protein. Proteomics analysis of PfMSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to B cell receptors of PfMSP1FL-specific MBCs, anti-PfMSP119 IgGs had high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the PfMSP1-specific humoral immune response with cumulative Pf exposure, with a shift from IgM+ to IgG+ B cell memory, diversification of B cells from germline, and stronger recognition of PfMSP1 variants by the plasma IgG repertoire.

Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity.

Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.

Reversal of indoleamine 2,3-dioxygenase-mediated cancer immune suppression by systemic kynurenine depletion with a therapeutic enzyme.

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8+ lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

The vitamin E analogue α-TEA stimulates tumor autophagy and enhances antigen cross-presentation.

The semisynthetic vitamin E derivative alpha-tocopheryloxyacetic acid (α-TEA) induces tumor cell apoptosis and may offer a simple adjuvant supplement for cancer therapy if its mechanisms can be better understood. Here we report that α-TEA also triggers tumor cell autophagy and that it improves cross-presentation of tumor antigens to the immune system. α-TEA stimulated both apoptosis and autophagy in murine mammary and lung cancer cells and inhibition of caspase-dependent apoptosis enhanced α-TEA-induced autophagy. Cell exposure to α-TEA generated double-membrane-bound vesicles indicative of autophagosomes, which efficiently cross-primed antigen-specific CD8(+) T cells. Notably, vaccination with dendritic cells pulsed with α-TEA-generated autophagosomes reduced lung metastases and increased the survival of tumor-bearing mice. Taken together, our findings suggest that both autophagy and apoptosis signaling programs are activated during α-TEA-induced tumor cell killing. We suggest that the ability of α-TEA to stimulate autophagy and enhance cross-priming of CD8(+) T cells might be exploited as an adjuvant strategy to improve stimulation of antitumor immune responses.

The small molecule TGF-ß signaling inhibitor SM16 synergizes with agonistic OX40 antibody to suppress established mammary tumors and reduce spontaneous metastasis.

Effective tumor immunotherapy may require not only activation of anti-tumor effector cells, but also abrogation of tumor-mediated immunosuppression. The cytokine TGF-ß, is frequently elevated in the tumor microenvironment and is a potent immunosuppressive agent and promoter of tumor metastasis. OX40 (CD134) is a member of the TNF-α receptor superfamily and ligation by agonistic antibody (anti-OX40) enhances effector function, expansion, and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of a small molecule TGF-ß signaling inhibitor, SM16, plus anti-OX40 in the poorly immunogenic, highly metastatic, TGF-ß-secreting 4T1 mammary tumor model. Our data show that SM16 and anti-OX40 mutually enhanced each other to elicit a potent anti-tumor effect against established primary tumors, with a 79% reduction in tumor size, a 95% reduction in the number of metastatic lung nodules, and a cure rate of 38%. This positive treatment outcome was associated with a 3.2-fold increase of tumor-infiltrating, activated CD8+ T cells, an overall accumulation of CD4+ and CD8+ T cells, and an increased tumor-specific effector T cell response. Complete abrogation of the therapeutic effect in vivo following depletion of CD4+ and CD8+ T cells suggests that the anti-tumor efficacy of SM16+ anti-OX40 therapy is T cell dependent. Mice that were cured of their tumors were able to reject tumor re-challenge and manifested a significant tumor-specific peripheral memory IFN-γ response. Taken together, these data suggest that combining a TGF-ß signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.

α-Tocopheryloxyacetic acid: a novel chemotherapeutic that stimulates the antitumor immune response.

INTRODUCTION: α-Tocopheryloxyacetic acid (α-TEA) is a novel ether derivative of α-tocopherol that has generated interest as a chemotherapeutic agent because of its selective toxicity toward tumor cells and its ability to suppress tumor growth in various rodent and human xenograft models. We previously reported that oral α-TEA inhibited the growth of both a transplanted (4T1) and a spontaneous MMTV-PyMT mouse model of breast cancer. METHODS: Because little is known about the possible immunological mechanisms underlying the in vivo α-TEA effects, we evaluated the impact of α-TEA therapy on the immune response by characterizing immune cell populations infiltrating the tumor site. RESULTS: α-TEA treatment resulted in higher frequencies of activated T cells in the tumor microenvironment and twofold and sixfold higher ratios of CD4⁺ and CD8⁺T cells to regulatory T cells, respectively. This finding was correlated with an increased ability of tumor-draining lymph node cells and splenocytes from α-TEA-treated mice to secrete interferon (IFN)-γ in response to CD3 or to mediate a cytolytic response in a tumor-specific fashion, respectively. That the α-TEA-mediated antitumor effect had a T cell-dependent component was demonstrated by the partial abrogation of tumor suppression when CD4⁺ and CD8⁺ T cells were depleted. We also determined the intratumoral cytokine and chemokine profile and found that α-TEA treatment increased intratumoral IFN-γ levels but decreased interleukin (IL)-4 levels, suggesting a shift toward a TH1 response. In addition, α-TEA induced higher levels of the inflammatory cytokine IL-6 and the chemokine CCL5. CONCLUSIONS: Taken together, these data suggest that α-TEA treatment, in addition to its direct cytotoxic effects, enhanced the anti-tumor immune response. This study provides a better understanding of the mechanisms of action of α-TEA and its effect on the immune system and may prove useful in designing immune-stimulating strategies to boost the antitumor effects of α-TEA in breast cancer patients.

Differential gene expression of p27Kip1 and Rb knockout pituitary tumors associated with altered growth and angiogenesis.

Mice lacking the p27Kip1 Cdk inhibitor, like mice lacking Rb, develop pituitary tumors involving pars intermedia melanotrophs, yet p27(Kip1) tumors are genetically distinct from Rb derived tumors as they exhibit haploid insufficiency. We compared tumors from mice with p27( Kip1) constitutive and tissue specific null mutations to tumors arising in tissue specific Rb knockout mice with the aim of determining whether they are distinguished by quantitative or qualitative differences. The rate of p27Kip1 knockout tumor development was strongly influenced by strain background due to polygenic strain modifiers in the C57BL/6J versus 129S4 strains but, unlike a prior report of Rb mutants, this impacted tumor incidence but not the tumor spectrum. p27Kip1 tumors were oligoclonal or polyclonal based on studies of X-chromosomal inactivation of Dock11. In contrast, Rb null tissue developed monoclonal neoplasms even in the absence of a requirement for Rb mutant clonal selection. Rb null tumors exhibited a higher proliferation rate and developed ischemic necrosis associated with an aberrant vasculature. p27Kip1 null tumors maintained normal vascular density, through a tumor cell dependent mechanism, but were more often hemorrhagic. Gene expression profiles distinguished p27Kip1 from Rb null tumors including significant differences in expression of Rb and E2F signature genes. Rb null tumors expressed higher levels of VEGF which, in other systems, is associated with dilated vessels, ineffective perfusion and tissue hypoxia. Mouse models lacking p27Kip1 and Rb may help us better understand the pathophysiology of MEN syndromes, retinoblastoma and other cancers that disrupt these important cell cycle inhibitors.

Genetic mosaics reveal both cell-autonomous and cell-nonautonomous function of murine p27Kip1.

Loss of the cyclin-dependent kinase inhibitor p27(Kip1) leads to an overall increase in animal growth, pituitary tumors, and hyperplasia of hematopoietic organs, yet it is unknown whether all cells function autonomously in response to p27(Kip1) activity or whether certain cells take cues from their neighbors. In addition, there is currently no genetic evidence that tumor suppression by p27(Kip1) is cell-autonomous because biallelic gene inactivation is absent from tumors arising in p27(Kip1) hemizygous mice. We have addressed these questions with tissue-specific targeted mouse mutants and radiation chimeras. Our results indicate that the suppression of pars intermedia pituitary tumors by p27(Kip1) is cell-autonomous and does not contribute to overgrowth or infertility phenotypes. In contrast, suppression of spleen growth and hematopoietic progenitor expansion is a consequence of p27(Kip1) function external to the hematopoietic compartment. Likewise, p27(Kip1) suppresses thymocyte hyperplasia through a cell-nonautonomous mechanism. The interaction of p27(Kip1) loss with epithelial cell-specific cyclin-dependent kinase 4 overexpression identifies the thymic epithelium as a relevant site of p27(Kip1) activity for the regulation of thymus growth.