TCR-dependent and -independent activation underlie liver-specific regulation of NKT cells.

The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.

Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediated mitigation of an immunosuppressive tumor microenvironment.

Treatment of mice bearing orthotopic, metastatic tumors with anti-CD40 antibody resulted in only partial, transient anti-tumor effects whereas combined treatment with IL-2/anti-CD40, induced tumor regression. The mechanisms for these divergent anti-tumor responses were examined by profiling tumor-infiltrating leukocyte subsets and chemokine expression within the tumor microenvironment after immunotherapy. IL-2/anti-CD40, but not anti-CD40 alone, induced significant infiltration of established tumors by NK and CD8(+) T cells. To further define the role of chemokines in leukocyte recruitment into tumors, we evaluated anti-tumor responses in mice lacking the chemokine receptor, CCR2. The anti-tumor effects and leukocyte recruitment mediated by anti-CD40 alone, were completely abolished in CCR2(-/-) mice. In contrast, IL-2/anti-CD40-mediated leukocyte recruitment and reductions in primary tumors and metastases were maintained in CCR2(-/-) mice. Treatment of mice with IL-2/anti-CD40, but not anti-CD40 alone, also caused an IFN-gamma-dependent increase in the expression of multiple Th1 chemokines within the tumor microenvironment. Interestingly, although IL-2/anti-CD40 treatment increased Tregs in the spleen, it also caused a coincident IFN-gamma-dependent reduction in CD4(+)/FoxP3(+) Tregs, myeloid-derived suppressor cells and Th2 chemokine expression specifically within the tumor microenvironment that was not observed after treatment with anti-CD40 alone. Similar effects were observed using IL-15 in combination with anti-CD40. Taken together, our data demonstrate that IL-2/anti-CD40, but not anti-CD40 alone, can preferentially reduce the overall immunosuppressive milieu within the tumor microenvironment. These results suggest that the use of anti-CD40 in combination with IL-2 or IL-15 may hold substantially more promise for clinical cancer treatment than anti-CD40 alone.

Crumbling the Castle: Targeting DNABII Proteins for Collapsing Bacterial Biofilms as a Therapeutic Approach to Treat Disease and Combat Antimicrobial Resistance.

Antimicrobial resistance (AMR) is a concerning global threat that, if not addressed, could lead to increases in morbidity and mortality, coupled with societal and financial burdens. The emergence of AMR bacteria can be attributed, in part, to the decreased development of new antibiotics, increased misuse and overuse of existing antibiotics, and inadequate treatment options for biofilms formed during bacterial infections. Biofilms are complex microbiomes enshrouded in a self-produced extracellular polymeric substance (EPS) that is a primary defense mechanism of the resident microorganisms against antimicrobial agents and the host immune system. In addition to the physical protective EPS barrier, biofilm-resident bacteria exhibit tolerance mechanisms enabling persistence and the establishment of recurrent infections. As current antibiotics and therapeutics are becoming less effective in combating AMR, new innovative technologies are needed to address the growing AMR threat. This perspective article highlights such a product, CMTX-101, a humanized monoclonal antibody that targets a universal component of bacterial biofilms, leading to pathogen-agnostic rapid biofilm collapse and engaging three modes of action-the sensitization of bacteria to antibiotics, host immune enablement, and the suppression of site-specific tissue inflammation. CMTX-101 is a new tool used to enhance the effectiveness of existing, relatively inexpensive first-line antibiotics to fight infections while promoting antimicrobial stewardship.

Development of regulatory T cells requires IL-7Ralpha stimulation by IL-7 or TSLP.

Interleukin-7 (IL-7), a cytokine produced by stromal cells, is required for thymic development and peripheral homeostasis of most major subsets of T cells. We examined whether regulatory T (Treg) cells also required the IL-7 pathway by analyzing IL-7Ralpha(-/-) mice. We observed a striking reduction in cells with the Treg surface phenotype (CD4, CD25, GITR (glucocorticoid-induced tumor necrosis factor [TNF]-like receptor), CD45RB, CD62L, CD103) or intracellular markers (cytotoxic T-lymphocyte-associated antigen-4, CTLA-4, and forkhead box transcription factor 3, Foxp3). Foxp3 transcripts were virtually absent in IL-7Ralpha(-/-) lymphoid tissues, and no Treg cell suppressive activity could be detected. There are 2 known ligands for IL-7Ralpha: IL-7 itself and thymic stromal lymphopoietin (TSLP). Surprisingly, mice deficient in IL-7 or the other chain of the TSLP receptor (TSLPR) developed relatively normal numbers of Treg cells. Combined deletion of IL-7 and TSLP receptor greatly reduced Treg cell development in the thymus but was not required for survival of mature peripheral Treg cells. We conclude that Treg cells, like other T cells, require signals from the IL-7 receptor, but unlike other T cells, do not require IL-7 itself because of at least partially overlapping actions of IL-7 and TSLP for development of Treg cells.

Enhanced antitumor response by divergent modulation of natural killer and natural killer T cells in the liver.

The use of interleukin-18 (IL-18) together with IL-12 induced high levels of IFN-gamma in tumor-bearing mice and regression of liver tumors that was abolished in IFN-gamma((-/-)) mice. Natural killer (NK) and NKT cells were the major producers of IFN-gamma in the livers of mice treated with IL-18 and/or IL-12. Liver NK cells were significantly increased by treatment with IL-18/IL-12, whereas the degree of liver NKT cell TCR detection was diminished by this treatment. Reduction of NK cells with anti-asGM1 decreased the antitumor activity of IL-18/IL-12 therapy and revealed NK cells to be an important component for tumor regression in the liver. In contrast, the antitumor effects of both IL-18 and IL-12 were further increased in CD1d((-/-)) mice, which lack NKT cells. Our data, therefore, show that the antitumor activity induced in mice by IL-18/IL-12 is NK and IFN-gamma dependent and is able to overcome an endogenous immunosuppressive effect of NKT cells in the liver microenvironment. These results suggest that immunotherapeutic approaches that enhance NK cell function while eliminating or altering NKT cells could be effective in the treatment of cancer in the liver.

Resistance to IFN-alpha-induced apoptosis is linked to a loss of STAT2.

Type I IFNs (IFN-alpha/beta) are pleitropic cytokines widely used in the treatment of certain malignancies, hepatitis B and C, and multiple sclerosis. IFN resistance is a challenging clinical problem to overcome. Hence, understanding the molecular mechanism by which IFN immunotherapy ceases to be effective is of translational importance. In this study, we report that continuous IFN-alpha stimulation of the human Jurkat variant H123 led to resistance to type I IFN-induced apoptosis due to a loss of signal transducers and activators of transcription 2 (STAT2) expression. The apoptotic effects of IFN-alpha were hampered as STAT2-deficient cells were defective in activating the mitochondrial-dependent death pathway and ISGF3-mediated gene activation. Reconstitution of STAT2 restored the apoptotic effects of IFN-alpha as measured by the loss of mitochondrial membrane potential, cytochrome c release from mitochondria, caspase activation, and ultimately cell death. Nuclear localization of STAT2 was a critical event as retention of tyrosine-phosphorylated STAT2 in the cytosol was not sufficient to activate apoptosis. Furthermore, silencing STAT2 gene expression in Saos2 and A375S.2 tumor cell lines significantly reduced the apoptotic capacity of IFN-alpha. Altogether, we show that STAT2 is a critical mediator in the activation of type I IFN-induced apoptosis. More importantly, defects in the expression or nuclear localization of STAT2 could lessen the efficacy of type I IFN immunotherapy.