Various TLR agonists are currently under investigation in clinical trials for their ability to orchestrate antitumor immunity. The antitumor responses are largely attributed to their aptitude to stimulate APCs such as DCs which in turn, activate tumor-specific T cell responses. However, there is a potential for TLR signaling to occur on cells other than professional APCs that could negate antitumor responses or even worse, promote tumor growth. The impetus for this review is twofold. First, there is accumulating data demonstrating that the engagement of TLRs on different T cell subsets and different cancer types could promote tumor growth or conversely, contribute to antitumor responses. Second, the efficacy of TLR agonists as monotherapies to treat cancer patients has been limited. In this review, we discuss how TLR signaling within different T cell subsets and cancer cells can potentially impact the generation of antitumor responses. Based on evidence from preclinical models and clinical trials, we draw attention to several criteria that we believe must be considered when selecting TLR agonists for developing effective immunotherapeutic strategies against cancer.

Purpose: To develop an adaptable gene-based vector that will confer immune cell specificity to various cancer types.Experimental Design: Human and mouse T cells were genetically engineered to express a chimeric antigen receptor (CAR) that binds a fluorescein isothiocyanate (FITC) molecule, termed anti-FITC CAR T cells. Various antibodies (Ab) currently in clinical use including cetuximab (Ctx), trastuzumab (Her2), and rituximab (Rtx) were conjugated with FITC and tested for their ability to bind tumor cells, activate T cells, and induce antitumor effects in vitro and in vivo.

Chronic inflammation has long been associated with the development of cancer. Among the various signaling pathways within cancer cells that can incite the expression of inflammatory molecules are those that activate IL-1 receptor-associated kinases (IRAK). The IRAK family is comprised of four family members, IRAK-1, IRAK-2, IRAK-3 (also known as IRAK-M), and IRAK-4, which play important roles in both positively and negatively regulating the expression of inflammatory molecules. The wide array of inflammatory molecules that are expressed in response to IRAK signaling within the tumor microenvironment regulate the production of factors which promote tumor growth, metastasis, immune suppression, and chemotherapy resistance. Based on published reports we propose that dysregulated activation of the IRAK signaling pathway in cancer cells contributes to disease progression by creating a highly inflammatory tumor environment. In this article, we present both theoretical arguments and reference experimental data in support of this hypothesis.

The development of therapeutic anti-cancer vaccines designed to elicit CTL responses with anti-tumor activity has become a reality thanks to the identification of several tumor-associated Ags and their corresponding peptide T cell epitopes. However, peptide-based vaccines, in general, fail to elicit sufficiently strong CTL responses capable of producing therapeutic anti-tumor effects (i.e., prolongation of survival, tumor reduction). Here we report that repeated administration of synthetic oligonucleotides containing foreign cytosine-phosphorothiolated guanine (CpG) motifs increased 10- to 100-fold the CTL response to immunization with various synthetic peptides corresponding to well-known T cell epitopes. Moreover, repeated CpG administration allowed the induction of CTL to soluble protein even in the absence of additional adjuvant. Our results indicate that the potentiating effect of CpG in CTL responses required the participation of Th lymphocytes. Repeated CpG administration resulted in overt splenomegaly and lymphadenopathy with a significant increase in the numbers of CTL precursors and dendritic cells. Protein vaccination in combination with repeated CpG therapy was effective in delaying tumor cell growth and extending survival in mice bearing melanoma tumors. These findings support the contention that repeated administration of CpG-oligonucleotides enhances the effect of peptide and protein vaccines leading to potent anti-tumor responses, presumably through the induction of Th1 and dendritic cells, which are essential for optimal CTL responses. The immunostimulatory properties of CpG motifs may be key in inducing a consistent long term immunity to tumor-associated Ags when using peptides or proteins as T cell-inducing vaccines.

Toll-like receptors (TLRs) are among the fundamental molecules that alert the immune system to the presence of an infection by recognizing pathogen-associated molecules. Much of our understanding regarding TLR function stems from the study of innate immune cells. Recent studies by several groups, including ours, have shown that TLRs can function as costimulatory receptors for antigen-specific T cells, resulting in enhanced T-cell survival and increased expression of effector molecules. We report that the ligation of the TLR1/2 heterodimer on OT-1 cytotoxic T-lymphocytes (CTL) but not TLR2(-/-)OT-1 T cells increased cytolytic activity in vitro and in vivo. On the basis of these data, we tested the hypothesis that TLR1/2 stimulation on CTLs would enhance antitumor activity in a therapeutic model of B16-Ova melanoma. Adoptive OT-1 T-cell transfer into wild-type and MyD88(-/-) mice, followed by injection with TLR1/2 ligand, resulted in a synergistic antitumor effect, which correlated with the induction of CD8 T cells specific to various tumor antigens. In contrast, mice receiving TLR2(-/-)OT-1 T cells and TLR1/2 ligand showed minimal therapeutic efficacy. These findings emphasize the physiological significance of TLR2 engagement on CTLs and could make possible new approaches for the development of effective immunotherapies by manipulating TLR signaling within CTLs.

Background-Experience with non-antigenic galactose α1,3 galactose (αGal) polymers and development of αGal deficient pigs has reduced or eliminated the significance of this antigen in xenograft rejection. Despite these advances, delayed xenograft rejection (DXR) continues to occur most likely due to antibody responses to non-Gal endothelial cell (EC) antigens.

Cancer metastasis and immune suppression are critical issues in cancer therapy. Here, we show that a β-galactoside-binding lectin [galectin-3 (gal3)] that recognizes the Thomsen-Friedenreich disaccharide (TFD, Galβ1,3GalNAc) present on the surface of most cancer cells is involved in promoting angiogenesis, tumor-endothelial cell adhesion, and metastasis of prostate cancer cells, as well as evading immune surveillance through killing of activated T cells. To block gal3-mediated interactions, we purified a glycopeptide from cod (designated TFD 100 ) that binds gal3 with picomolar affinity. TFD 100 blocks gal3-mediated angiogenesis, tumor-endothelial cell interactions, and metastasis of prostate cancer cells in mice at nanomolar levels. Moreover, apoptosis of activated T cells induced by either recombinant gal3 or prostate cancer patient serum-associated gal3 was inhibited at nanomolar concentration of TFD 100 . Because the gal3-TFD interaction is a key factor driving metastasis in most epithelial cancers, this high-affinity TFD 100 should be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma.antifreeze glycoprotein | PC3-luciferase cells | galectin-3 knockout PC3-luciferase cells | TF antigen | surface plasmon resonance

Emerging reports reveal that activating Toll-like receptor-2 (TLR2)-MyD88 signals in CD8 T lymphocytes enhances cytokine production and cytotoxicity; however, the signaling pathway remains undefined. In the present study, we examined the physiologic significance and molecular mechanisms involved in this process. We found that TLR2 engagement on T-cell receptor transgenic CD8 OT-1 T cells increased T-bet transcription factor levels consequently, augmenting effector transcript and protein levels both IntroductionThe molecular and cellular bases for the costimulatory effects of Toll-like receptor (TLR) agonists are being gradually unraveled, adding to our understanding of how TLR signals enhance T cell-mediated immune responses. Expressed primarily on cells of the innate immune system, such as dendritic cells (DCs), TLRs recognize microbial-derived molecules. Certain TLRs, such as TLR1 and TLR2, can form heterodimers to facilitate the detection of a broader array of microbes. 1 TLR engagement on professional antigen-presenting cells (APCs) induces their maturation, resulting in the increased expression of costimulatory molecules and cytokines necessary for optimal T-cell activation. 1 Thus, given their potent effects on APCs, TLR agonists are believed to influence T-cell responses principally by stimulating TLRs on cells of the innate immune system. 1,2 However, recent advances by several groups, including ours, indicate that the adjuvant effects of certain TLR agonists may also be attributed to the activation of TLRs and the TLR adapter molecule myeloid differentiation factor (MyD88) directly in T cells. Both CD4 [3][4][5][6][7][8][9][10][11]12,13 T cells express functional TLRs. In CD8 T cells, concomitant engagement of the T-cell receptor (TCR) and TLR3 enhanced interferon-␥ (IFN-␥) production, 10 whereas TLR2 engagement on CD8 T cells increased the expression levels of granzyme B in vitro. 13 We recently reported that TLR2 engagement on CD8 T cells also increased IFN-␥, granzyme B, and perforin production (referred to as effector molecules), and, consequently, enhanced T-cell cytotoxicity both in vivo and in vitro. 12 However, the molecular pathway by which TLR-MyD88 signals in T cells augment effector function is poorly defined. Only recently have studies implicated the PI3K signaling pathway in TLR2-mediated T-cell activation 14 ; however, the molecular mechanisms through which these signals influence activation and cytotoxicity remain largely undefined.Among the various signals involved in T-cell activation, 2 transcription factors, T-bet and eomesodermin (EOMES), profoundly influence CD8 T-cell differentiation into effector cytotoxic T lymphocytes (CTLs). 15 Both these transcription factors can regulate IFN-␥, perforin, and granzyme B transcription, and in their absence, CD8 T cells fail to effectively transition from a naive T cell into an effector or memory cell. 15-17 T-bet expression is known to be induced by signals mediated via the TCR and IFN-␥R; however, additional signals capable of influencing th...