Efficient CD4Cre-Mediated Conditional KRas Expression in Alveolar Macrophages and Alveolar Epithelial Cells Causes Fatal Hyperproliferative Pneumonitis.

The CD4Cre transgenic model has been widely used for T cell-specific gene manipulation. We report unexpected highly efficient Cre-mediated recombination in alveolar macrophages (AMFs), bronchial epithelial cells (BECs), and alveolar epithelial cells (AECs) in this strain of mice. Different from CD4 T cells, AMFs, AECs, and BECs do not express detectable Cre protein, suggesting that Cre protein is either very transiently expressed in these cells or only expressed in their precursors. Mice carrying a conditional constitutively active KRas (caKRas) allele and the CD4Cre transgene contain not only hyperactivated T cells but also develop severe AMF accumulation, AEC and BEC hyperplasia, and adenomas in the lung, leading to early lethality correlated with caKRas expression in these cells. We propose that caKRas-CD4Cre mice represent, to our knowledge, a novel model of proliferative pneumonitis involving macrophages and epithelial cells and that the CD4Cre model may offer unique usefulness for studying gene functions simultaneously in multilineages in the lung. Our observations, additionally, suggest that caution in data interpretation is warranted when using the CD4Cre transgenic model for T cell-specific gene manipulation, particularly when lung pathophysiological status is being examined.

STAT3 in CD8+ T Cells Inhibits Their Tumor Accumulation by Downregulating CXCR3/CXCL10 Axis.

One of the obstacles for cancer immunotherapy is the inefficiency of CD8(+) T-cell recruitment to tumors. STAT3 has been shown to suppress CD8(+) T-cell antitumor functions in various cancer models, in part by restricting accumulation of CD8(+) T cells. However, the underlying molecular mechanism by which STAT3 in CD8(+) T cells inhibits their accumulation in tumors remains to be defined. Here, we show that STAT3 signaling in CD8(+) T cells inhibits chemokine CXCL10 production by tumor-associated myeloid cells by reducing IFNγ expression by T cells. We further demonstrate that ablating STAT3 in T cells allows expression of CXCR3, the receptor of CXCL10, on CD8(+) T cells, resulting in efficient accumulation of CD8(+) T cells at tumor sites. Blocking IFNγ or CXCR3 impairs the accumulation of STAT3-deficient CD8(+) T cells in tumor and their antitumor effects. Together, our study reveals a negative regulation by STAT3 signaling in T cells on cross-talk between myeloid cells and T cells through IFNγ/CXCR3/CXCL10, which is important for CD8(+) T cells homing to tumors. Our results thus provide new insights applicable to cancer immunotherapy and adoptive T-cell strategies.

CD8+ T-cell immunosurveillance constrains lymphoid premetastatic myeloid cell accumulation.

Increasing evidence suggests that premetastatic niches, consisting mainly of myeloid cells, provide microenvironment critical for cancer cell recruitment and survival to facilitate metastasis. While CD8(+) T cells exert immunosurveillance in primary human tumors, whether they can exert similar effects on myeloid cells in the premetastatic environment is unknown. Here, we show that CD8(+) T cells are capable of constraining premetastatic myeloid cell accumulation by inducing myeloid cell apoptosis in C57BL/6 mice. Ag-specific CD8(+) T-cell cytotoxicity against myeloid cells in premetastatic lymph nodes is compromised by Stat3. We demonstrate here that Stat3 ablation in myeloid cells leads to CD8(+) T-cell activation and increased levels of IFN-γ and granzyme B in the premetastatic environment. Furthermore, Stat3 negatively regulates soluble Ag cross-presentation by myeloid cells to CD8(+) T cells in the premetastatic niche. Importantly, in tumor-free lymph nodes of melanoma patients, infiltration of activated CD8(+) T cells inversely correlates with STAT3 activity, which is associated with a decrease in number of myeloid cells. Our study suggested a novel role for CD8(+) T cells in constraining myeloid cell activity through direct killing in the premetastatic environment, and the therapeutic potential by targeting Stat3 in myeloid cells to improve CD8(+) T-cell immunosurveillance against metastasis.

S1PR1 is crucial for accumulation of regulatory T cells in tumors via STAT3.

S1PR1 signaling has been shown to restrain the number and function of regulatory T (Treg) cells in the periphery under physiological conditions and in colitis models, but its role in regulating tumor-associated T cells is unknown. Here, we show that S1PR1 signaling in T cells drives Treg accumulation in tumors, limits CD8(+) T cell recruitment and activation, and promotes tumor growth. T-cell-intrinsic S1PR1 affects Treg cells, but not CD8(+) T cells, as demonstrated by adoptive transfer models and transient pharmacological S1PR1 modulation. An increase in S1PR1 in CD4(+) T cells promotes STAT3 activation and JAK/STAT3-dependent Treg tumor migration, whereas STAT3 ablation in T cells diminishes tumor-associated Treg accumulation and tumor growth. Our study demonstrates a stark contrast between the consequences of S1PR1 signaling in Treg cells in the periphery versus tumors.

Diacylglycerol kinase zeta positively controls the development of iNKT-17 cells.

Invariant natural killer T (iNKT) cells play important roles in bridging innate and adaptive immunity via rapidly producing a variety of cytokines. A small subset of iNKT cells produces IL-17 and is generated in the thymus during iNKT-cell ontogeny. The mechanisms that control the development of these IL-17-producing iNKT-17 cells (iNKT-17) are still not well defined. Diacylglycerol kinase ζ (DGKζ) belongs to a family of enzymes that catalyze the phosphorylation and conversion of diacylglycerol to phosphatidic acid, two important second messengers involved in signaling from numerous receptors. We report here that DGKζ plays an important role in iNKT-17 development. A deficiency of DGKζ in mice causes a significant reduction of iNKT-17 cells, which is correlated with decreased RORγt and IL-23 receptor expression. Interestingly, iNKT-17 defects caused by DGKζ deficiency can be corrected in chimeric mice reconstituted with mixed wild-type and DGKζ-deficient bone marrow cells. Taken together, our data identify DGKζ as an important regulator of iNKT-17 development through iNKT-cell extrinsic mechanisms.

Regulation of adipose tissue T cell subsets by Stat3 is crucial for diet-induced obesity and insulin resistance.

Dysregulated inflammation in adipose tissue, marked by increased proinflammatory T-cell accumulation and reduced regulatory T cells (Tregs), contributes to obesity-associated insulin resistance. The molecular mechanisms underlying T-cell-mediated inflammation in adipose tissue remain largely unknown, however. Here we show a crucial role for signal transducer and activator of transcription 3 (Stat3) in T cells in skewing adaptive immunity in visceral adipose tissue (VAT), thereby contributing to diet-induced obesity (DIO) and insulin resistance. Stat3 activity is elevated in obese VAT and in VAT-resident T cells. Functional ablation of Stat3 in T cells reduces DIO, improves insulin sensitivity and glucose tolerance, and suppresses VAT inflammation. Importantly, Stat3 ablation reverses the high Th1/Treg ratio in VAT of DIO mice that is likely secondary to elevated IL-6 production, leading in turn to suppression of Tregs. In addition, Stat3 in T cells in DIO mice affects adipose tissue macrophage accumulation and M2 phenotype. Our study identifies Stat3 in VAT-resident T cells as an important mediator and direct target for regulating adipose tissue inflammation, DIO, and its associated metabolic dysfunctions.

G-protein-coupled receptor agonist BV8/prokineticin-2 and STAT3 protein form a feed-forward loop in both normal and malignant myeloid cells.

BACKGROUND: Signaling pathways underlying BV8-mediated oncogenesis remain unknown. RESULTS: BV8-STAT3 forms a feed-forward loop in both normal and malignant myeloid cells and promotes tumor growth. CONCLUSION: JAK2/STAT3 signaling plays critical roles in BV8-mediated myeloid cell-dependent oncogenesis. SIGNIFICANCE: This study identifies a novel role of BV8-STAT3 signaling in mediating cross-talk between tumor microenvironment and tumor cells. An important role of BV8 in mobilization of myeloid cells and myeloid cell-dependent angiogenesis has been established. Recently, it has also been shown that granulocyte colony-stimulating factor (G-CSF)-induced BV8 expression is STAT3 dependent in CD11b(+)Gr1(+) myeloid cells. However, the BV8 downstream signaling pathway(s) intrinsic to myeloid cells crucial for angiogenesis, and potentially also for development of cancers of myeloid origin, remains largely unknown. Here we show that BV8 activates STAT3, which is critical for regulating genes important for both tumor cell proliferation/survival and tumor angiogenesis, in both normal and malignant myeloid cells. Further, BV8-induced STAT3 activation requires Janus-activated kinase 2 (JAK2) activity as shown by both genetic and pharmacologic inhibition. Knocking down BV8 in human myeloid leukemia cells inhibits STAT3 activity and expression of STAT3 downstream angiogenic and pro-proliferation/survival genes, leading to a decrease in tumor cell viability. BV8 shRNA expressing leukemia cells exhibit reduced STAT3 activity and tumor growth in vivo. Taken together, we have delineated a signaling pathway downstream of BV8 that plays critical roles in both the tumor microenvironment and malignant myeloid cells for angiogenesis and tumor cell proliferation/survival.

S1PR1-STAT3 signaling is crucial for myeloid cell colonization at future metastatic sites.

Recent studies underscore the importance of myeloid cells in rendering distant organs hospitable for disseminating tumor cells to colonize. However, what enables myeloid cells to have an apparently superior capacity to colonize distant organs is unclear. Here, we show that S1PR1-STAT3 upregulation in tumor cells induces factors that activate S1PR1-STAT3 in various cells in premetastatic sites, leading to premetastatic niche formation. Targeting either S1PR1 or STAT3 in myeloid cells disrupts existing premetastatic niches. S1PR1-STAT3 pathway enables myeloid cells to intravasate, prime the distant organ microenvironment and mediate sustained proliferation and survival of their own and other stromal cells at future metastatic sites. Analyzing tumor-free lymph nodes from cancer patients shows elevated myeloid infiltrates, STAT3 activity, and increased survival signal.

Murine regulatory T cells contain hyperproliferative and death-prone subsets with differential ICOS expression.

Regulatory T cells (Treg) are crucial for self-tolerance. It has been an enigma that Treg exhibit an anergic phenotype reflected by hypoproliferation in vitro after TCR stimulation but undergo vigorous proliferation in vivo. We report in this study that murine Treg are prone to death but hyperproliferative in vitro and in vivo, which is different from conventional CD4(+)Foxp3(-) T cells (Tcon). During in vitro culture, most Treg die with or without TCR stimulation, correlated with constitutive activation of the intrinsic death pathway. However, a small portion of the Treg population is more sensitive to TCR stimulation, particularly weak stimulation, proliferates more vigorously than CD4(+) Tcon, and is resistant to activation-induced cell death. Treg proliferation is enhanced by IL-2 but is less dependent on CD28-mediated costimulation than that of Tcon. We demonstrate further that the surviving and proliferative Treg are ICOS(+) whereas the death-prone Treg are ICOS(-). Moreover, ICOS(+) Treg contain much stronger suppressive activity than that of ICOS(-) Treg. Our data indicate that massive death contributes to the anergic phenotype of Treg in vitro and suggest modulation of Treg survival as a therapeutic strategy for treatment of autoimmune diseases and cancer.

Critical roles of RasGRP1 for invariant NKT cell development.

The invariant NKT (iNKT) cell lineage contains CD4(+) and CD4(-) subsets. The mechanisms that control such subset differentiation and iNKT cell maturation in general have not been fully understood. RasGRP1, a guanine nucleotide exchange factor for TCR-induced activation of the Ras-ERK1/2 pathway, is critical for conventional αß T cell development but dispensable for generating regulatory T cells. Its role in iNKT cells has been unknown. In this study, we report severe decreases of iNKT cells in RasGRP1(-/-) mice through cell intrinsic mechanisms. In the remaining iNKT cells in RasGRP1(-/-) mice, there is a selective absence of the CD4(+) subset. Furthermore, RasGRP1(-/-) iNKT cells are defective in TCR-induced proliferation in vitro. These observations establish that RasGRP1 is not only important for early iNKT cell development but also for the generation/maintenance of the CD4(+) iNKT cells. Our data provide genetic evidence that the CD4(+) and CD4(-) iNKT cells are distinct sublineages with differential signaling requirements for their development.

Tight regulation of diacylglycerol-mediated signaling is critical for proper invariant NKT cell development.

Type I NKT cells, or invariant NKT (iNKT) cells, express a semi-invariant TCR characterized by its unique Vα14-Jα18 usage (iVα14TCR). Upon interaction with glycolipid/CD1d complexes, the iVα14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the protein kinase C-IκB kinase (IKK)α/ß-NF-κB pathway, known to be crucial for iNKT development, as well as the RasGRP1-Ras-Erk1/2 pathway in T cells. DAG kinases play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. In this article, we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NF-κB and Ras-Erk1/2 activation. Moreover, constitutive IKKß and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development.

The importance of LAT in the activation, homeostasis, and regulatory function of T cells.

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T(reg) cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.

The role of the LAT-PLC-gamma1 interaction in T regulatory cell function.

The interaction between the linker for activation of T cells (LAT) with PLC-gamma1 is important for TCR-mediated Ca(2+) signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells, implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect, we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wild type LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling, proliferation, and IL-2 production. Furthermore, the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3(+) natural Treg cells were present in these mice after deletion, they were unable to suppress the proliferation of conventional T cells. Our data indicate that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4(+)CD25(+) regulatory T cells.

The essential role of LAT in thymocyte development during transition from the double-positive to single-positive stage.

The linker for activation of T cells (LAT) is an adaptor protein that couples TCR engagement to downstream signaling cascades. LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the double-negative (DN) 3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by the Cre recombinase. Analysis of these mice showed that deletion of LAT after the DN3 stage allowed thymocytes to develop past the DN3 to DN4 checkpoint and to generate double-positive thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to differentiate into single-positive thymocytes efficiently. Consequently, few LAT-deficient mature T cells could be found in the periphery. These T cells had undergone extensive homeostatic proliferation and expressed low levels of the TCR on their surface. Collectively, our data indicate that in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during transition from the double-positive to single-positive stage.

The importance of Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons sterile-alpha motif domain in thymic selection and T-cell activation.

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76) is a cytosolic adaptor protein essential for thymocyte development and T-cell activation. It contains a sterile-alpha motif (SAM) domain, 3 phosphotyrosine motifs, a proline-rich region, and a Src homology 2 domain. Whereas the other domains have been extensively studied, the role of the SAM domain in SLP-76 function is not known. To understand the function of this domain, we generated SLP-76 knockin mice with the SAM domain deleted. Analysis of these mice showed that thymocyte development was partially blocked at the double-positive to single-positive transition. Positive and negative thymic selection was also impaired. In addition, we analyzed T-cell receptor (TCR)-mediated signaling in T cells from these mutant mice. TCR-mediated inositol 1,4,5-triphosphate production, calcium flux, and extracellular signal-regulated kinase activation were decreased, leading to defective interleukin-2 production and proliferation. Moreover, despite normal association between Gads and SLP-76, TCR-mediated formation of SLP-76 microclusters was impaired by the deletion of the SAM domain. Altogether, our data demonstrated that the SAM domain is indispensable for optimal SLP-76 signaling.

Negative regulation of T cell activation and autoimmunity by the transmembrane adaptor protein LAB.

LAB (linker for activation of B cells), also known as NTAL (non-T cell activation linker), is a LAT (linker for activation of T cells)-like adaptor protein that is expressed in B, NK, and mast cells. Its role in lymphocytes has not been clearly demonstrated. Here, we showed that aged LAB-deficient (Lat2(-/-)) mice developed an autoimmune syndrome. Lat2(-/-) T cells were hyperactivated and produced more cytokines than Lat2(+/+) T cells. Even though LAB was absent in naive T cells, LAB could be detected in activated Lat2(+/+) T cells. LAT-mediated signaling events were enhanced in Lat2(-/-) T cells; however, they were suppressed in T cells that overexpressed LAB. Mice with the Lat2 gene conditionally deleted from T cells also developed the autoimmune syndrome like Lat2(-/-) mice. Together, these data demonstrated an important role of LAB in limiting autoimmune response and exposed a mechanism regulating T cell activation.

LAT-mediated signaling in CD4+CD25+ regulatory T cell development.

Engagement of the T cell receptor for antigen (TCR) induces formation of signaling complexes mediated through the transmembrane adaptor protein, the linker for activation of T cells (LAT). LAT plays an important role in T cell development, activation, and homeostasis. A knock-in mutation at Tyr136, which is the phospholipase C (PLC)-gamma1-binding site in LAT, leads to a severe autoimmune disease in mice. In this study, we show that CD4+CD25+ T reg cells that expressed Foxp3 transcription factor were nearly absent in both thymus and peripheral lymphoid organs of LAT(Y136F) mice. This defect was not a result of the autoimmune environment as LAT(Y136F) T reg cells also failed to develop in healthy LAT-/- mice that received mixed wild-type and LAT(Y136F) bone marrow cells. Moreover, adoptive transfer of normal CD4+CD25+ T reg cells protected neonatal LAT(Y136F) mice from developing this disease. These T reg cells effectively controlled expansion of CD4+ T cells in LAT(Y136F) mice likely via granzymes and/or TGF-beta-mediated suppression. Furthermore, ectopic expression of Foxp3 conferred a suppressive function in LAT(Y136F) T cells. Our data indicate that the LAT-PLC-gamma1 interaction plays a critical role in Foxp3 expression and the development of CD4+CD25+ T reg cells.

Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development.

It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.