Tissue‐resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from ...tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T‐bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild‐type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin‐draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP‐NKT cells produce the majority of the IL‐4 in Peyer's patches and provide indirect help for B‐cell class switching to IgG1 in both transnuclear and wild‐type mice. Oral vaccination with α‐galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell‐sufficient mice. Transcriptional profiling reveals a unique signature of PP‐NKT cells, characterized by tissue residency. We thus define PP‐NKT as potentially important for surveillance for mucosal pathogens.
Synopsis
We used iNKT transnuclear mice to detect iNKT cells from tissues where they are rare, including adipose, lung, skin‐draining lymph nodes, and a previously undescribed population in Peyer's patches that produces IL‐4 and helps B‐cell class switching to IgG1.
iNKT transnuclear mice faithfully model tissue‐resident iNKT cell populations.
iNKT cells are found in Peyer's patches of both wild type and transnuclear mice and can be used as cellular adjuvants for oral vaccination.
PP‐NKT cells produce the majority of the IL‐4 in Peyer's patches, and provide indirect help for B‐cell class switching to IgG1.
A novel population of iNKT cells with a unique function in supporting IgG1 class switching in the gut mucosa may be important for surveillance for mucosal pathogens.
Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from ...tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.
We present a new foundational role for CXCR3
monocytes/macrophages in the process of tumor engraftment in the lung. CXCR3 is associated with monocytic and lymphocytic infiltration of inflamed or ...tumor-bearing lung. Although the requirement for tumor-expressed CXCR3 in metastatic engraftment has been demonstrated, the role of monocyte-expressed CXCR3 had not been appreciated. In a murine model of metastatic-like melanoma, engraftment was coordinate with CXCR3
monocyte/macrophage accumulation in the lungs and was sensitive to pharmacologic inhibition of CXCR3 signaling. Tumor engraftment to lung was impaired in CXCR3
mice, and transient reconstitution with circulating CXCR3-replete monocytes was sufficient to restore engraftment. These data illustrate the paradoxical pro-tumor role for CXCR3 in lung immunobiology wherein the CXCR3 axis drives both the anti-tumor effector cell chemoattraction and pro-tumor infiltration of the lungs and suggests a potential therapeutic target for lung-tropic metastasizing cancers.
Invariant NKT (iNKT) cell functional subsets are defined by key transcription factors and output of cytokines, such as IL-4, IFN-γ, IL-17, and IL-10. To examine how TCR specificity determines iNKT ...function, we used somatic cell nuclear transfer to generate three lines of mice cloned from iNKT nuclei. Each line uses the invariant Vα14Jα18 TCRα paired with unique Vβ7 or Vβ8.2 subunits. We examined tissue homing, expression of PLZF, T-bet, and RORγt, and cytokine profiles and found that, although monoclonal iNKT cells differentiated into all functional subsets, the NKT17 lineage was reduced or expanded depending on the TCR expressed. We examined iNKT thymic development in limited-dilution bone marrow chimeras and show that higher TCR avidity correlates with higher PLZF and reduced T-bet expression. iNKT functional subsets showed distinct tissue distribution patterns. Although each individual monoclonal TCR showed an inherent subset distribution preference that was evident across all tissues examined, the iNKT cytokine profile differed more by tissue of origin than by TCR specificity.
Inhibitor of apoptosis protein (IAP) antagonists are in clinical trials for a variety of cancers, and mouse models show synergism between IAP antagonists and anti-PD-1 immunotherapy. Although IAP ...antagonists affect the intrinsic signaling of tumor cells, their most pronounced effects are on immune cells and the generation of antitumor immunity. Here, we examined the effects of IAP antagonism on T-cell development using mouse fetal thymic organ culture and observed a selective loss of iNKT cells, an effector cell type of potential importance for cancer immunotherapy. Thymic iNKT-cell development probably failed due to increased strength of TCR signal leading to negative selection, given that mature iNKT cells treated with IAP antagonists were not depleted, but had enhanced cytokine production in both mouse and human
cultures. Consistent with this, mature mouse primary iNKT cells and iNKT hybridomas increased production of effector cytokines in the presence of IAP antagonists.
administration of IAP antagonists and α-GalCer resulted in increased IFNγ and IL-2 production from iNKT cells and decreased tumor burden in a mouse model of melanoma lung metastasis. Human iNKT cells also proliferated and increased IFNγ production dramatically in the presence of IAP antagonists, demonstrating the utility of these compounds in adoptive therapy of iNKT cells.
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Immune-mediated cancer regression requires tumor infiltration by antigen-specific effector T cells, but lymphocytes are commonly sparse in melanoma metastases. Activated T cells express CXCR3, whose ...cognate chemokines are CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. Little is known about expression of these chemokines in lymph node (LN) metastases of melanoma. We evaluated whether metastatic melanoma induces these CXCR3-cognate chemokines in human LN-derived tissues. In addition, as these chemokines can be induced by interferon (IFN), we evaluated whether type I or II IFNs (IFN-α or IFN-γ, respectively) can modulate chemokine expression in an in vitro model of the human tumor microenvironment. Production of CXCL9-11 by melanoma-infiltrated nodes (MIN) was no different than tumor-free nodes; both produced less chemokine than activated LN (sentinel immunized nodes, SIN). These data suggest that melanoma infiltration into LN neither induces nor reduces CXCL9-11. Stimulation with IFN-α or IFN-γ increased production of CXCL10-11 from MIN, but not tumor-free node or SIN. IFN-γ also increased production of CXCL9 in MIN. In IFN-treated SIN, CD14+ cells were the primary source of CXCL9-11, whereas melanoma cells were the source of chemokine in MIN. Melanoma cells in MIN express IFN receptors. Consistent with these observations, multiple human melanoma lines expressed IFN receptors and produced CXCL9-11 in response to IFN treatment. Thus, melanoma infiltration of LN is insufficient to induce the production of CXCL9-11, but melanoma may be a significant source of IFN-induced chemokines. Collectively, these data suggest that IFN-α or IFN-γ may act in the tumor microenvironment to increase the chemotactic gradient for CXCR3+ T cells.
CDK4/6 inhibitors are approved to treat breast cancer and are in trials for other malignancies. We examined CDK4/6 inhibition in mouse and human CD8
T cells during early stages of activation. Mice ...receiving tumor-specific CD8
T cells treated with CDK4/6 inhibitors displayed increased T-cell persistence and immunologic memory. CDK4/6 inhibition upregulated MXD4, a negative regulator of MYC, in both mouse and human CD8
T cells. Silencing of
in mouse CD8
T cells demonstrated the importance of this axis for memory formation. We used single-cell transcriptional profiling and T-cell receptor clonotype tracking to evaluate recently activated human CD8
T cells in patients with breast cancer before and during treatment with either palbociclib or abemaciclib. CDK4/6 inhibitor therapy in humans increases the frequency of CD8
memory precursors and downregulates their expression of MYC target genes, suggesting that CDK4/6 inhibitors in patients with cancer may augment long-term protective immunity. SIGNIFICANCE: CDK4/6 inhibition skews newly activated CD8
T cells toward a memory phenotype in mice and humans with breast cancer. CDK4/6 inhibitors may have broad utility outside breast cancer, particularly in the neoadjuvant setting to augment CD8
T-cell priming to tumor antigens prior to dosing with checkpoint blockade.
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Checkpoint blockade immunotherapy has failed in pancreatic cancer and other poorly responsive tumor types in part due to inadequate T cell priming. Naive T cells can receive costimulation not only ...via CD28 but also through TNF superfamily receptors that signal via NF-κB. Antagonists of the ubiquitin ligases cellular inhibitor of apoptosis protein (cIAP)1/2, also called second mitochondria-derived activator of caspases (SMAC) mimetics, induce degradation of cIAP1/2 proteins, allowing for the accumulation of NIK and constitutive, ligand-independent activation of alternate NF-κB signaling that mimics costimulation in T cells. In tumor cells, cIAP1/2 antagonists can increase TNF production and TNF-mediated apoptosis; however, pancreatic cancer cells are resistant to cytokine-mediated apoptosis, even in the presence of cIAP1/2 antagonism. Dendritic cell activation is enhanced by cIAP1/2 antagonism in vitro, and intratumoral dendritic cells show higher expression of MHC class II in tumors from cIAP1/2 antagonism-treated mice. In this study, we use in vivo mouse models of syngeneic pancreatic cancer that generate endogenous T cell responses ranging from moderate to poor. Across multiple models, cIAP1/2 antagonism has pleiotropic beneficial effects on antitumor immunity, including direct effects on tumor-specific T cells leading to overall increased activation, increased control of tumor growth in vivo, synergy with multiple immunotherapy modalities, and immunologic memory. In contrast to checkpoint blockade, cIAP1/2 antagonism does not increase intratumoral T cell frequencies. Furthermore, we confirm our previous findings that even poorly immunogenic tumors with a paucity of T cells can experience T cell-dependent antitumor immunity, and we provide transcriptional clues into how these rare T cells coordinate downstream immune responses.
Despite immunogenicity, melanoma-specific vaccines have demonstrated minimal clinical efficacy in patients with established disease but enhanced survival when administered in the adjuvant setting. ...Therefore, we hypothesized that organs bearing metastatic-like melanoma may differentially produce T-cell chemotactic proteins over the course of tumor development. Using an established model of metastatic-like melanoma in lungs, we assessed the production of specific cytokines and chemokines over a time course of tumor growth, and we correlated chemokine production with chemokine receptor-specific T-cell infiltration. We observed that the interferon (IFN)-inducible CXCR3-cognate chemokines (CXCL9 and CXCL10) were significantly increased in lungs bearing minimal metastatic lesions, but chemokine production was at or below basal levels in lungs with substantial disease. Chemokine production was correlated with infiltration of the organ compartment by adoptively transferred CD8(+) tumor antigen-specific T cells in a CXCR3- and host IFNγ-dependent manner. Adenosine signaling in the tumor microenvironment (TME) suppressed chemokine production and T-cell infiltration in the advanced metastatic lesions, and this suppression could be partially reversed by administration of the adenosine receptor antagonist aminophylline. Collectively, our data demonstrate that CXCR3-cognate ligand expression is required for efficient T-cell access of tumor-infiltrated lungs, and these ligands are expressed in a temporally restricted pattern that is governed, in part, by adenosine. Therefore, pharmacologic modulation of adenosine activity in the TME could impart therapeutic efficacy to immunogenic but clinically ineffective vaccine platforms.
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