T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture ...system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and
transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.
TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies ...indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.
Tumor blood vessels are frequently inefficient in their design and function, leading to high interstitial fluid pressure, hypoxia, and acidosis in the tumor microenvironment (TME), rendering tumors ...refractory to the delivery of chemotherapeutic agents and immune effector cells. Here we identified the NOTCH antagonist delta-like 1 homologue (DLK1) as a vascular pericyte-associated antigen expressed in renal cell carcinomas (RCC), but not in normal kidney tissues in mice and humans. Vaccination of mice bearing established RCC against DLK1 led to immune-mediated elimination of DLK1+ pericytes and to blood vessel normalization (i.e., decreased vascular permeability and intratumoral hypoxia) in the TME, in association with tumor growth suppression. After therapeutic vaccination, tumors displayed increased prevalence of activated VCAM1+CD31+ vascular endothelial cells (VECs) and CXCL10, a type-1 T cell recruiting chemokine, in concert with increased levels of type-1 CD8+ tumor-infiltrating lymphocytes (TIL). Vaccination against DLK1 also yielded (i) dramatic reductions in Jarid1B+, CD133+, and CD44+ (hypoxia-responsive) stromal cell populations, (ii) enhanced tumor cell apoptosis, and (iii) increased NOTCH signaling in the TME. Coadministration of a γ-secretase inhibitor (N-N-(3,5-Difluorophenacetyl-l-alanyl)-(S)-phenylglycine t-butyl ester (DAPT)) that interferes with canonical NOTCH signaling resulted in the partial loss of therapeutic benefits associated with lentivirus encoding full-length murine (lvDLK1)-based vaccination.
PD-1, TIM-3, and LAG-3 are molecules shown to have immune modulatory properties, and although initially classified as indicators of T cell hyporesponsiveness, it has become clear that they are also ...associated with the normal course of T cell activation. Functional studies have focused mainly on CD8(+) T cells during chronic inflammation due to interest in co-opting the cellular immune response to eliminate viral or cancerous threats; however, there remains a relative lack of data regarding the expression of these molecules on CD4(+) T cells. Here, we report that expression of the immune checkpoint (IC) molecules PD-1, LAG-3, and TIM-3 are differentially expressed on CD4(+) and CD8(+) T cells in the allogeneic response resulting from a mixed lymphocyte reaction. In these studies, PD-1 expression is higher on CD4(+) T cells compared to CD8(+) T cells. In contrast, TIM-3 is expressed at higher levels on CD8(+) T cells compared to CD4(+) T cells with an apparent reciprocity in that PD-1(+) CD4(+) T cells are frequently TIM-3(lo/-), while TIM-3-expressing CD8(+) T cells are largely PD-1(lo/-). In addition, there is a decrease in the frequency of TIM-3(+) CD4(+) cells producing IFN-γ and IL-5 compared to TIM-3(+) CD8(+) cells. Lastly, the memory T cell phenotype within each IC-expressing subset differs between CD4(+) and CD8(+) T cells. These findings highlight key differences in IC expression patterns between CD4(+) and CD8(+) T cells and may allow for more effective therapeutic targeting of these molecules in the future.
Remote inflammation monitoring with digital health technologies (DHTs) would provide valuable information for both clinical research and care. Controlled perturbations of the immune system may reveal ...physiological signatures which could be used to develop a digital biomarker of inflammatory state. In this study, molecular and physiological profiling was performed following an in vivo lipopolysaccharide (LPS) challenge to develop a digital biomarker of inflammation. Ten healthy volunteers received an intravenous LPS challenge and were monitored for 24 h using the VitalConnect VitalPatch (VitalPatch). VitalPatch measurements included heart rate (HR), heart rate variability (HRV), respiratory rate (RR), and skin temperature (TEMP). Conventional episodic inpatient vital signs and serum proteins were measured pre‐ and post‐LPS challenge. The VitalPatch provided vital signs that were comparable to conventional methods for assessing HR, RR, and TEMP. A pronounced increase was observed in HR, RR, and TEMP as well as a decrease in HRV 1–4 h post‐LPS challenge. The ordering of participants by magnitude of inflammatory cytokine response 2 h post‐LPS challenge was consistent with ordering of participants by change from baseline in vital signs when measured by VitalPatch (r = 0.73) but not when measured by conventional methods (r = −0.04). A machine learning model trained on VitalPatch data predicted change from baseline in inflammatory protein response (R2 = 0.67). DHTs, such as VitalPatch, can improve upon existing episodic measurements of vital signs by enabling continuous sensing and have the potential for future use as tools to remotely monitor inflammation.
When compared with vascular cells in normal tissues, pericytes and vascular endothelial cells (VEC) in tumor blood vessels exhibit altered morphology and epigenetic programming that leads to the ...expression of unique antigens that allow for differential recognition by CD8
+
T cells. We have previously shown that the Notch antagonist delta-like homolog 1 (DLK1) is a tumor pericyte-associated antigen expressed in setting of melanoma and a range of carcinomas. In this report, we show that therapeutic vaccination against DLK1 in murine models results in slowed tumor growth, but also to the compensatory expression of the DLK1 homolog, DLK2, by tumor-associated pericytes. Vaccines targeting both DLK1 and DLK2 resulted in superior antitumor benefits in association with improved activation and recruitment of antigen-specific Type 1 CD8
+
T cells, reduced presence of myeloid-derived suppressive cells, T regulatory cell and tumor vascular normalization. The antitumor efficacy of vaccines coordinately targeting DLK1 and DLK2 was further improved by inclusion of PD-L1 blockade, thus defining a combination immunotherapy theoretically suitable for the treatment of a broad range of solid (vascularized) cancers.
Abstract
The tumor microenvironment (TME) promotes unique epigenetic programming of stromal cell populations, including vascular endothelial cells (VEC) and pericytes, leading to differential ...expression of antigens that may be recognized by therapeutic T cells. These antigenic differences may be targeted immunologically via specific vaccination. We show that pericytes in human and murine renal cell carcinoma (RCC) and melanoma differentially overexpress the antigen Delta-like homolog 1 (DLK-1). DLK-1 is an EGF-like protein that functions as a Notch pathway antagonist. We demonstrate that both peptide and genetic vaccination of RENCA-bearing BALB/c mice against DLK-1 results in tumor growth inhibition and removal of DLK-1 within the TME. However, DLK-1 removal also led to a compensatory increase in DLK-2 expression (a DLK-1 homolog and NOTCH antagonist) in the murine RENCA TME. Using recombinant lentiviruses encoding murine DLK-1 and DLK-2, we show that coordinate genetic vaccination against these two antigens leads to superior therapeutic inhibition of RENCA tumor growth compared to vaccination against either DLK-1 or DLK-2 alone. The coordinate vaccination also resulted in the activation of DLK-1- and DLK-2-specific CD8+ T cells, recruitment of T cells into the TME and normalization of tumor blood vessels. We conclude that vaccination against DLK-1 and/or DLK-2 may represent an effective strategy for vascularized, solid forms of cancer, including RCC and melanoma, among others.
Abstract
Previous studies suggest that by improving Notch signaling in the tumor microenvironment (TME), one may rescue tumor infiltrating T lymphocyte function and inhibit tumor growth in vivo. ...Delta-like homolog 1 (DLK-1) and Delta-like homolog 2 (DLK-2) are homologous transmembrane proteins whose ectodomains may be shed from the cell surface by the action of the tumor necrosis factor alpha converting enzyme. Both the membrane-associated and soluble forms of DLK-1/-2 serve as antagonists of Notch receptor-mediated signaling. We report that DLK-1 is differentially expressed by vascular pericytes of murine renal cell carcinomas (RENCA) and have shown that vaccination of RENCA-bearing BALB/c mice with DLK-1-derived peptides or cDNA results in the activation of specific CD8+ T cells, the recruitment of these T cells into the TME and the normalization of tumor vasculature in vivo. However, DLK-1 and DLK-2 have been reported to counter-modulate each other’s expression. Here we show that DLK-1-based vaccination leads to the T cell-dependent loss of DLK-1 expression in RENCA tumors, but to an apparent compensatory increase in TME expression of DLK-2. We have recently defined H-2d class I-presented peptide epitopes recognized by CD8+ T cells and are currently evaluating the therapeutic benefits of vaccines targeting both DLK-1 and DLK-2 in our RENCA model. We believe that such vaccines will have significant translational value for the treatment of many solid, vascularized forms of cancer.
Ineffective recognition of tumor cells by CD8+ T cells is a limitation of cancer immunotherapy. Therefore, treatment regimens that coordinately promote enhanced antitumor CD8+ T-cell activation, ...delivery, and target cell recognition should yield greater clinical benefit. Using an MCA205 sarcoma model, we show that in vitro treatment of tumor cells with the HSP90 inhibitor 17-DMAG results in the transient (proteasome-dependent) degradation of the HSP90 client protein EphA2 and the subsequent increased recognition of tumor cells by Type-1 anti-EphA2 CD8+ T cells. In vivo administration of 17-DMAG to tumor-bearing mice led to slowed tumor growth, enhanced/prolonged recognition of tumor cells by anti-EphA2 CD8+ T cells, reduced levels of myeloid-derived suppressor cells and regulatory T cells in the tumor microenvironment, and activation of tumor-associated vascular endothelial cells in association with elevated levels of Type-1 tumor-infiltrating lymphocytes. When combined with EphA2-specific active vaccination or the adoptive transfer of EphA2-specific CD8+ T cells, 17-DMAG cotreatment yielded a superior tumor therapeutic regimen that was capable of rendering animals free of disease. Taken together, our findings indicate that 17-DMAG functions as an immune adjuvant in the context of vaccines targeting EphA2.
Abstract
Background and Aims
Tesnatilimab, a monoclonal antibody targeting NKG2D, was evaluated in Crohn’s disease CD patients who had failed or were intolerant to biologic or conventional therapy.
...Methods
TRIDENT was a phase 2b, two-part, randomised, double-blind, placebo-controlled, parallel-arm, multicenter study. In Part 1 proof of concept, 145 patients who were biologic intolerant or refractory Bio-IR or had not failed biologic therapy Bio-NF were randomised in a 1:1 ratio to placebo subcutaneously SC or tesnatilimab 400 mg SC. In Part 2 dose ranging, 243 Bio-IR and Bio-NF patients were randomised in a 1:1:1:1:1 ratio to placebo, tesnatilimab 50 mg, 150 mg, 400 mg, or intravenous infusion of ustekinumab ~6 mg/kg at Week 0 and 90 mg SC at Weeks 8 and 16. The primary endpoint was mean change from baseline in Crohn’s Disease Activity Index CDAI at Week 8 Part 1 and Week 12 Part 2. Clinical and endoscopic remission/response were evaluated. Efficacy analyses were also assessed by NKG2D and MICB single nucleotide polymorphism SNP status SNP-positive means positive in at least one of two SNPs. Safety events were summarised.
Results
In Part 1, mean change from baseline in CDAI score was significantly greater with tesnatilimab vs placebo at Week 8 -103.6 vs -60.0; p < 0.01. In Part 2, no dose-response signal was detected. Mean changes from baseline in CDAI at Week 12 were -93.2, -72.2, and -84.3 for low, middle, and high doses of tesnatilimab, respectively, vs -59.2 for placebo and -148.8 for ustekinumab. Similar reductions from baseline in CDAI score were observed in patients receiving tesnatilimab, regardless of SNP status. Clinical remission rates were greater with tesnatilimab than placebo in Parts 1 and 2, whereas endoscopic response rates were greater with tesnatilimab only in Part 1. No unexpected safety events occurred.
Conclusions
Tesnatilimab was well tolerated. The efficacy of tesnatilimab in patients with CD was significant for the primary endpoint in Part 1; however, no dose-response signal was detected for the primary endpoint in Part 2. Based on these inconsistent findings, tesnatilimab was not considered an effective treatment for patients with CD and no further development is planned.
ClinicalTrials.gov Identifier
NCT02877134
Graphical Abstract
Graphical Abstract
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