Cloning and Expression of Human Membrane-Bound and Soluble Engineered T Cell Receptors for Immunotherapy

We report here the design and construction of several gene vectors for expression in mammalian cells of membrane-bound and soluble human T cell receptors (TR). We designed a vector (TR-ALPHA-IRES-TR-BETA pEF4) that encodes high-level expression of the full-length TR on the surface of T cells. Furthermore, we engineered TR that does not require the presence of endogenous CD3 molecules for surface expression and thus expression is not limited to T cells. We also constructed a vector encoding a single-chain TR (scTR) as a fusion protein of V-ALPHA-V-BETA-C-BETA with CD3Z. Since it is encoded and expressed as a single molecule, this scTR is well suited for gene therapy. Lastly, we successfully used a mammalian expression vector for generation of soluble human TR. The approaches we used here for manipulation of a human tumor-specific TR can be useful for other investigators interested in TR-based immunotherapy

Transcriptomic profiling disclosed the role of DNA methylation and histone modifications in tumor-infiltrating myeloid-derived suppressor cell subsets in colorectal cancer

Increased numbers of myeloid-derived suppressor cells (MDSCs) are positively correlated with poor prognosis and reduced survivals of cancer patients. They play central roles in tumor immune evasion and tumor metastasis. However, limited data are available on phenotypic/transcriptomic characteristics of the different MDSCs subsets in cancer. These cells include immature (I-MDSCs), monocytic (M-MDSCs), and polymorphonuclear/granulocytic (PMN-MDSCs). Phenotypic characterization of myeloid subsets from 27 colorectal cancer (CRC) patients was assessed by flow cytometric analyses. RNA-sequencing of sorted I-MDSCs, PMN-MDSCs, and antigen-presenting cells (APCs) was also performed. We found that the levels of I-MDSCs and PMN-MDSCs were increased in tumor tissues (TT), compared with normal tissues (NT) in colorectal cancer. Our functional annotation analyses showed that genes associated with histone deacetylase (HDAC) activation- and DNA methylation-mediated transcriptional silencing were upregulated, and histone acetyl transferase (HAT)-related genes were downregulated in tumor-infiltrating I-MDSCs. Moreover, pathways implicated in cell trafficking and immune suppression, including Wnt, interleukin-6 (IL-6), and mitogen-activated protein kinase (MAPK) signaling, were upregulated in I-MDSCs. Notably, PMN-MDSCs showed downregulation in genes related to DNA methylation and HDAC binding. Using an ex vivo model, we found that inhibition of HDAC activation or neutralization of IL-6 in CRC tumor tissues downregulates the expression of genes associated with immunosuppression and myeloid cell chemotaxis, confirming the importance of HDAC activation and IL-6 signaling pathway in MDSC function and chemotaxis. This study provides novel insights into the epigenetic regulations and other molecular pathways in different myeloid cell subsets within the CRC tumor microenvironment (TME), giving opportunities to potential targets for therapeutic benefits

MHC-UNRESTRICTED MUC1-SPECIFIC T CELL RECEPTOR FOR CANCER IMMUNOTHERAPY/GENE THERAPY

MUC1 glycoprotein is overexpressed on the surface of a variety of epithelial tumors and has been under investigation as a target for immunotherapy. A number of cytotoxic lymphocyte clones were generated in our laboratory from breast and pancreatic cancer patients that recognized MUC1 on the surface of tumor cells in a TCR-mediated MHC-unrestricted manner. The purpose of this study was to test the feasibility, efficacy and safety of using MHC-unrestricted MUC1-specific T cell receptor (TCR) gene transfer as a tool for cancer immunotherapy. The TCR £ and £] chains were cloned from one MHC-unrestricted MUC1-specific CTL clone (MA). Various configurations of chimeric TCRs were constructed and were expressed on the surface of a variety of cell lines in vitro. The TCR-deficient T cell line, Jurkat JRT3.5, transfected with the TCR £ and £] chains from MA CTL clone fluxed calcium in response to stimulation by a MUC1+ pancreatic human tumor, HPAF. BWZ murine thymoma cells transfected with a single-chain TCR (scTCR) consisting of the TCR extracellular domain and the CD3 ƒê signaling domain) were triggered to secrete IL-2 in response to stimulation with different MUC1+ tumor cells. The tumor recognition and rejection functions of this scTCR were tested in vivo when SCID mice were reconstituted with bone marrow (BM) cells transduced with scTCR-MFG retroviral supernatant and challenged with HPAF tumor cells. Tumor growth in mice reconstituted with scTCR-transduced BM cells was significantly slower (P<0.05) than that seen in the control group. Tumor sections from TCR-reconstituted mice were infiltrated by neutrophils and macrophages, and to lesser extent, by NK cells. FACS analyses showed that BM cells transduced with scTCR-MFG could differentiate in vivo into multiple immune lineages including T cells, B cells, granulocytes, monocytes and NK cells that express the scTCR. The scTCR was expressed on higher percentages of cells of the innate immune system when compared to T and B cells. Human MUC1 transgenic (Tg.) mice reconstituted with BM cells transduced with this MUC1-specific TCR did not show any signs of autoimmunity, abnormal cellular infiltration or destruction of MUC1-expressing tissues. Transduction of BM with tumor-specific TCR represents a potentially efficacious gene therapy/immunotherapy approach. MUC1-specific MHC-unrestricted TCR will make this treatment applicable to all cancer patients with MUC1+ tumors, regardless of their HLA type

Transcriptomic analyses of myeloid-derived suppressor cell subsets in the circulation of colorectal cancer patients

Myeloid-derived suppressor cells (MDSCs) promote tumor immune evasion and favor tumorigenesis by activating various tumor-promoting downstream signals. MDSC expansion is evident in the circulation and tumor microenvironment of many solid tumors including colorectal cancer (CRC). We have recently reported the transcriptomic profiles of tumor-infiltrating MDSCs in CRC patients and uncovered pathways, which could potentially assist tumor progression. In this study, we sorted different subsets of circulating MDSCs in CRC patients and investigated their transcriptomic profiles in order to disclose pathways, which could potentially contribute to disease progression. The sorted subsets included polymorphonuclear/granulocytic MDSCs (PMN-MDSCs), immature MDSCs (I-MDSCs), and monocytic MDSCs (M-MDSCs). Our functional annotation analyses revealed that multiple pathways including DNA damage-, chemotaxis-, apoptosis-, mitogen-activated protein kinase-, transforming growth factor β-, and myeloid differentiation–related transcripts were higher in PMN-MDSCs, compared with monocytic antigen-presenting cells (APCs) or I-MDSCs. Furthermore, genes related to Janus kinase (JAK)–signal transducer and activator of transcription (STAT) were also elevated in PMN-MDSCs. These data suggest that upregulation of JAK-STAT pathway could trigger multiple downstream targets in PMN-MDSCs, which favor tumor progression. Additionally, we found that pathways including phosphatidyl inositol 3-kinase (PI3K), interleukin 6, and TGF-β in M-MDSCs and cell cycle–related pathways in I-MDSCs were upregulated, compared with monocytic APCs. Moreover, acetylation-related genes were upregulated in both PMN-MDSCs and M-MDSCs. This latter finding implicates that epigenetic modifications could also play a role in the regulation of multiple tumor-promoting genes in PMN-MDSCs and M-MDSCs. Taken together, this study reveals various signaling pathways, which regulate the function of MDSC subsets in the circulation of CRC patients. However, functional studies are warranted to support these findings

Transcriptomic profiling of tumor-infiltrating CD4 + TIM-3 + T Cells reveals their suppressive, exhausted, and metastatic characteristics in colorectal cancer patients

T cell immunoglobulin mucin-3 (TIM-3) is an immune checkpoint identified as one of the key players in regulating T-cell responses. Studies have shown that TIM-3 is upregulated in the tumor microenvironment (TME). However, the precise role of TIM-3 in colorectal cancer (CRC) TME is yet to be elucidated. We performed phenotypic and molecular characterization of TIM-3+ T cells in the TME and circulation of CRC patients by analyzing tumor tissues (TT, TILs), normal tissues (NT, NILs), and peripheral blood mononuclear cells (PBMC). TIM-3 was upregulated on both CD4+ and CD3+CD4− (CD8+) TILs. CD4+TIM-3+ TILs expressed higher levels of T regulatory cell (Tregs)-signature genes, including FoxP3 and Helios, compared with their TIM-3− counterparts. Transcriptomic and ingenuity pathway analyses showed that TIM-3 potentially activates inflammatory and tumor metastatic pathways. Moreover, NF-κB-mediated transcription factors were upregulated in CD4+TIM-3+ TILs, which could favor proliferation/invasion and induce inflammatory and T-cell exhaustion pathways. In addition, we found that CD4+TIM-3+ TILs potentially support tumor invasion and metastasis, compared with conventional CD4+CD25+ Tregs in the CRC TME. However, functional studies are warranted to support these findings. In conclusion, this study discloses some of the functional pathways of TIM-3+ TILs, which could improve their targeting in more specific therapeutic approaches in CRC patients

CDH1 and IL1-beta expression dictates FAK and MAPKK-dependent cross-talk between cancer cells and human mesenchymal stem cells

Is Figure S1 showing transfer of cellular components between hMSCs and cancer cells. (DOCX 214 kb

Bone morphogenetic protein 2 (BMP2) induces growth suppression and enhances chemosensitivity of human colon cancer cells

Additional file 5: Figure S4. BMP2 expression in CRC cell lines from the Cancer Cell Line Encyclopedia. Data are presented as mRNA expression (Log2)

In vitro differentiation of human skin-derived multipotent stromal cells into putative endothelial-like cells

<p>Abstract</p> <p>Background</p> <p>Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs) possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs) and adult dermal skin (hADSSCs) using explants cultures and were compared with bone marrow (hMSC-TERT) and adipose tissue-derived mesenchymal stem cells (hADMSCs) for their potential differentiation into osteoblasts, adipocytes, and endothelial cells.</p> <p>Results</p> <p>Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC), with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA) was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues.</p> <p>Conclusions</p> <p>Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs represents a novel source of stem/stromal cells for tissue regeneration and the vascularization of engineered tissues. Moreover, the CD146 investigations suggested that the microenvironmental niche might contribute to direct stromal cells multipotency toward certain lineages, which warrants further investigation.</p