The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1␥) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1␥ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1⌬DAD), indicating that deregulation of autoinhibition is effective in FMNL1␥. Expression of both FMNL1␥ and FMNL1⌬DAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.Formins represent a protein family indispensable for many fundamental actin-dependent processes, including migration, vesicle trafficking, morphogenesis, and cytokinesis (1). Because these polarized processes are also involved in inflammation, deregulated proliferation, and metastasis, formins have been suggested to represent attractive drug targets for inflammatory and malignant diseases. Formin-like 1 (FMNL1) 3 is expressed restrictedly in hematopoietic lineagederived cells and overexpressed in malignant cells of different origin. This restricted expression suggests FMNL1 to be an attractive target for novel immunotherapies in malignant and inflammatory diseases (2, 3). However, function and regulation of FMNL1 are less well characterized. Previous work has shown involvement of FMNL1 in the reorientation of the microtubuleorganizing center toward the immunological synapse and cytotoxicity of T cells (4). The murine homolog FRL, which has 85% homology to the human counterpart, has been additionally shown to be involved in cell adhesion and motility of macrophages as well as Fc␥ receptor-mediated phagocytosis (5, 6). To date, it is not clear how these different membrane-associated processes are regulated.Formins are defined by a unique and highly conserved C-terminal formin homology (FH) 2 domain that mediates the effects on actin (7-11). The FH2 domain is proceeded by a proline-rich FH1 domain that binds with low micromolar affinity to profilin (12, 13). In a conserved subfamily of formins known as diaphanous-related formins (DRFs), the FH1 and FH2 domains are flanked by an array of regulatory domains at the N terminus and by a single C-terminal diaphanous autoregulatory domain (DAD) (14). The large N-terminal regulatory region includes a binding domain for small G proteins like RhoGTPase followed by an adjacent diaphanous-inhibitory domain (DID) and a dimerization domain (13,(15)(16)(17). The DAD, which comprised only a small stretch of amino acid residues, binds to the DID. Interaction of DA...

The triple-negative breast cancer (TNBC), with a particularly poor prognosis, is increasingly recognized as heterogeneous in molecular signatures. MicroRNA expression profiles have been used for the classification and prognostication of breast cancer, numerous significantly upregulated microRNAs, i.e. miR-21, have been verified oncogenic in non-TNBCs. In present study, we determined the miR-21 levels in TNBC specimens, and TNBC cell levels in vitro, and then identified the role of miR-21 on tumor cell proliferation, apoptosis, and then identified PTEN as the possible target of the microRNA. It was shown that miR-21 expression is upregulated generally, and heterogeneous in TNBC specimens, posing a correlation with poor prognosis for TNBC patients. Further results demonstrated that the upregulated miR-21 promoted the tumor proliferation and inhibited cell apoptosis in vitro. And pro-apoptotic PTEN had been shown being targeted and downregulated. Therefore, our finding emphasized the oncogenic role of miR-21 in TNBC.

T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2369) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCRα-chain of this TCR specifically recognized HER2369 not only in combination with the original β-chain but also with four other β-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one β-chain derived from another HER2369-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells.

CMV is a prevalent human pathogen. The virus cannot be eliminated from the body, but is kept in check by CMV-specific T cells. Patients with an insufficient T cell response, such as transplant recipients, are at high risk of developing CMV disease. However, the CMV-specific T cell repertoire is complex, and it is not yet clear which T cells protect best against virus reactivation and disease. In this study, we present a highly resolved characterization of CMV-specific human CD8 + T cells based on enrichment by specific peptide stimulation and mRNA sequencing of their TCR b-chains (TCRb). Our analysis included recently identified T cell epitopes restricted through HLA-C, whose presentation is resistant to viral immunomodulation, and well-studied HLA-Brestricted epitopes. In eight healthy virus carriers, we identified a total of 1052 CMV-specific TCRb sequences. HLA-C-restricted, CMV-specific TCRb clonotypes dominated the ex vivo T cell response and contributed the highest-frequency clonotype of the entire repertoire in two of eight donors. We analyzed sharing and similarity of CMV-specific TCRb sequences and identified 63 public or related sequences belonging to 17 public TCRb families. In our cohort, and in an independent cohort of 352 donors, the cumulative frequency of these public TCRb family members was a highly discriminatory indicator of carrying both CMV infection and the relevant HLA type. Based on these findings, we propose CMV-specific TCRb signatures as a biomarker for an antiviral T cell response to identify patients in need of treatment and to guide future development of immunotherapy.

Colorectal cancer (CRC) is one of the most lethal diseases with high morbidity and mortality worldwide. Clinically, tumors located in colon and rectum have diverse prognosis and therapeutic outcome. Here, we performed data mining derived from 20 CRC patient samples to compare proteomic difference between colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ). We found that differential expressed proteins (DEPs) upregulated in COAD were mainly enriched in immune response, moreover, higher immune scores were found in COAD than READ, as calculated by The Cancer Genome Atlas (TCGA) data. To identify the core protein of DEPs with high prognostic value for COAD, we performed topological overlap matrix (TOM) to investigate the hub proteins using 77 immune-relevant DEPs, and identified complement component 3 (C3) as the core protein in the immune-relevant DEPs matrix between the COAD and READ. Moreover, we found that C3 was up-regulated in COAD, and its expression was negatively associated with overall survival of COAD patients but not READ. In conclusion, we identified C3-mediated immune response as key feature to distinguish COAD and READ, and highlighted C3 as potential biomarker with high prognostic value for clinical application, which provided new clue for precise treatment of COAD.

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T-cell responses to the immediate-early 1 (IE-1) protein of human cytomegalovirus (HCMV) are associated with protection from viral disease. Thus, IE-1 is a promising target for immunotherapy. CD8 T-cell responses to IE-1 are generally strong. In contrast, CD4 T-cell responses to IE-1 were described to be comparatively infrequent or undetectable in HCMV carriers, and information on their target epitopes and their function has been limited. To analyze the repertoire of IE-1-specific CD4 T cells, we expanded them from healthy donors with autologous IE-1-expressing mini-Epstein–Barr virus-transformed B-cell lines and established IE-1-specific CD4 T-cell clones. Clones from seven out of seven HCMV-positive donors recognized endogenously processed IE-1 epitopes restricted through HLA-DR, DQ, or DP. Three to seven IE-1 epitopes were recognized per donor. Cumulatively, about 27 different HLA/peptide class II complexes were recognized by 117 IE-1-specific clones. Our results suggest that a highly diversified repertoire of IE-1-specific CD4 T cells targeting multiple epitopes is usually present in healthy HCMV carriers. Therefore, multiepitope approaches to immunomonitoring and immunotherapy will make optimal use of this potentially important class of HCMV-specific effector cells.

SummaryAdoptive transfer of T cells genetically modified with tumour-specific T-cell receptors (TCR) is a promising novel approach in the treatment of cancer. We have previously isolated an allorestricted MHC class I-restricted TCR with specificity for Formin-like protein 1 (FMNL1) with potent activity against chronic lymphocytic leukaemia cells. CD4 + T cells have been described to be highly important for tumour elimination although TCR derived from CD4 + T cells with anti-tumour reactivity have been only rarely described. In this study we aimed to isolate MHC class-II-restricted CD4 + T cells and TCR with specificity for leukaemia antigens.We used professional antigen-presenting cells pulsed with the leukaemiaassociated and tumour-associated antigen FMNL1 for stimulation of autologous T cells in vitro. We isolated two CD4 + HLA-DR-restricted T-cell clones and T-cell-derived TCR with so far unknown specificity but high reactivity against lymphoma cells and native malignant cells derived from HLA-matched patients with diverse leukaemias. Moreover, characterization of the TCR after TCR gene transfer revealed that specific characteristics of isolated TCR as reactivity in response to Toll-like receptors were transferable on effector cells. Our results have a major impact on the development of novel immunotherapies. They demonstrate that TCR with potent HLA-DR-restricted anti-leukaemic reactivity against so far undefined self-restricted antigens can be isolated from the healthy autorestricted CD4 + T-cell repertoire and these TCR are highly interesting candidate tools for novel immunotherapies.