10 research outputs found
Regulation of Immunity by Dendritic Cells in the Setting of Liver Transplantation
The immune system is the defense mechanism of the human body for protection against
infections and other unwanted biological invasions. There are two types of immune responses:
the innate immune response and the adaptive immune response.
Innate immune recognition relies on a limited number of germline-encoded innate immune
receptors that recognize conserved products of microbial metabolism produced
by pathogens, but not by the host. Recognition of these molecules allows the immune
system to distinguish infectious nonself from noninfectious self (1). Antigen presenting
cells (APC) form the bridge between the innate and the adaptive immune system,
which has more variability and specificity. B en T cells are the mediators of the adaptive
immune system. B-cells are the precursors of antibody secreting cells and can directly
recognize antigens via their B-cell receptors, however T cells need the antigen to be
processed and presented to them by an APC. The T-cell receptor (TCR) recognizes fragments
of antigen in combination with molecules of the major histocompatibility complex
(MHC). Two types of MHC molecules exist: MHC class I that is expressed by all nucleated
cells in the human body and MHC class II that is expressed exclusively by APC,
which present antigenic peptides to cytotoxic T cells and Thelper cells, respectively
Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ T-cell proliferations
Clonality assessment through Southern blot (SB) analysis of TCRB genes or
polymerase chain reaction (PCR) analysis of TCRG genes is important for
diagnosing suspect mature T-cell proliferations. Clonality assessment
through reverse transcription (RT)-PCR analysis of Vbeta-Cbeta transcripts
and flow cytometry with a Vbeta antibody panel covering more than 65% of
Vbeta domains was validated using 28 SB-defined clonal T-cell receptor
(TCR)alphabeta(+) T-ALL samples and T-cell lines. Next, the diagnostic
applicability of the V(beta) RT-PCR and flow cytometric clonality assays
was studied in 47 mature T-cell proliferations. Clonal Vbeta-Cbeta RT-PCR
products were detected in all 47 samples, whereas single Vbeta domain
usage was found in 31 (66%) of 47 patients. The suspect leukemic cell
populations in the other 16 patients showed a complete lack of Vbeta
monoclonal antibody reactivity that was confirmed by molecular data
showing the usage of Vbeta gene segments not covered by the applied Vbeta
monoclonal antibodies. Nevertheless, this could be considered indirect
evidence for the "clonal" character of these cells. Remarkably, RT-PCR
revealed an oligoclonal pattern in addition to dominant Vbeta-Cbeta
products and single Vbeta domain expression in many T-LGL proliferations,
providing further evidence for the hypothesis raised earlier that T-LGL
derive from polyclonal and oligoclonal proliferations of antigen-activated
cytotoxic T cells. It is concluded that molecular Vbeta analysis serves to
assess clonality in suspect T-cell proliferations. However, the faster and
cheaper Vbeta antibody studies can be used as a powerful screening method
for the detection of single Vbeta domain expression, followed by molecular
studies in patients with more than 20% single Vbeta domain expression or
large suspect T-cell populations (more than 50%-60%) without Vbeta
reactivity
Characterization of Antigen-Presenting Cell Subsets in Human Liver-Draining Lymph Nodes
T-cell immunity in the liver is tightly regulated to prevent chronic liver inflammation in
response to antigens and toxins derived from food and intestinal bacterial flora. Since
the main sites of T cell activation in response to foreign components entering solid
tissues are the draining lymph nodes (LN), we aimed to study whether Antigen-Presenting
Cell (APC) subsets in human liver lymph-draining LN show features that may contribute
to the immunologically tolerant liver environment. Healthy liver LN, iliac LN, spleen
and liver perfusates were obtained from multi-organ donors, while diseased liver
LN were collected from explanted patient livers. Inguinal LN were obtained from
kidney transplant recipients. Mononuclear cells were isolated from fresh tissues, and
immunophenotypic and functional characteristics of APC subsets were studied using
flowcytometry and in ex vivo cultures. Healthy liver-draining LN contained significantly
lower relative numbers of CD1c+ conventional dendritic cells (cDC2), plasmacytoid DC
(PDC), and CD14+CD163+DC-SIGN+ macrophages (MF) compared to inguinal LN.
Compared to spleen, both types of LN contained low relative numbers of CD141hi
cDC1. Both cDC subsets in liver LN showed a more activated/mature immunophenotype
than those in inguinal LN, iliacal LN, spleen and liver tissue. Despite their more
mature status, cDC2 isolated from hepatic LN displayed similar cytokine production
capacity (IL-10, IL-12, and IL-6) and allogeneic T cell stimulatory capacity as their
counterparts from spleen. Liver LN from patients with inflammatory liver diseases showe
PD-L1, Galectin-9 and CD8+ tumor-infiltrating lymphocytes are associated with survival in hepatocellular carcinoma
Novel systemic treatments for hepatocellular carcinoma (HCC) are strongly needed. Immunotherapy is a promising strategy that can induce specific antitumor immune responses. Understanding the mechanisms of immune resistance by HCC is crucial for development of suitable immunotherapeutics. We used immunohistochemistry on tissue-microarrays to examine the co-expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM and IDO, as well as tumor CD8+ lymphocyte infiltration in HCC, in two independent cohorts of patients. We found that at least some expression in tumor cells was seen in 97% of cases for HVEM, 83% for PD-L1, 79% for Gal-9 and 66% for IDO. In the discovery cohort (n = 94), we found that lack of, or low, tumor expression of PD-L1 (p < 0.001), Galectin-9 (p < 0.001) and HVEM (p < 0.001), and low CD8+TIL count (p = 0.016), were associated with poor HCC-specific survival. PD-L1, Galectin-9 and CD8+TIL count were predictive of HCC-specific survival independent of baseline clinicopathologic characteristics and the combination of these markers was a powerful predictor of HCC-specific survival (HR 0.29; p <0.001). These results were confirmed in the validation cohort (n = 60). We show that low expression levels of PD-L1 and Gal-9 in combination with low CD8+TIL count predict extremely poor HCC-specific survival and it requires a change in two of these parameters to significantly improve prognosis. In conclusion, intra-tumoral expression of these immune inhibiting molecules was observed in the majority of HCC patients. Low expression of PD-L1 and Galectin-9 and low CD8+TIL count are associated with poor HCC-specific survival. Combining immune biomarkers leads to superior predictors of HCC mortality
Blockade of LAG3 enhances responses of tumor-infiltrating T cells in mismatch repair-proficient liver meta
Purpose: Liver metastasis develops in >50% of patients with colorectal cancer (CRC), and is a leading cause of CRC-related mortality. We aimed to identify which inhibitory immune checkpoint pathways can be targeted to enhance functionality of intra-tumoral T-cells in mismatch repair-proficient liver metastases of colorectal cancer (LM-CRC). Methodology: Intra-tumoral expression of multiple inhibitory molecules was compared among mismatch repair-proficient LM-CRC, peritoneal metastases of colorectal cancer (PM-CRC) and primary CRC. Expression of inhibitory molecules was also analyzed on leukocytes isolated from paired resected metastatic liver tumors, tumor-free liver tissues, and blood of patients with mismatch repair-proficient LM-CRC. The effects of blocking inhibitory pathways on tumor-infiltrating T-cell responses were studied in ex vivo functional assays. Results: Mismatch repair-proficient LM-CRC showed higher expression of inhibitory receptors on intra-tumoral T-cells and contained higher proportions of CD8+ T-cells, dendritic cells and monocytes than mismatch repair-proficient primary CRC and/or PM-CRC. Inhibitory receptors LAG3, PD-1, TIM3 and CTLA4 were higher expressed on CD8+ T-cells, CD4+ T-helper and/or regulatory T-cells in LM-CRC tumors compared with tumor-free liver and blood. Antibody blockade of LAG3 or PD-L1 increased proliferation and effector cytokine production of intra-tumoral T-cells isolated from LM-CRC in response to both polyclonal and autologous tumor-specific stimulations. Higher LAG3 expression on intra-tumoral CD8+ T-cells associated with longer progression-free survival of LM-CRC patients. Conclusion: Mismatch repair-proficient LM-CRC may be more sensitive to immune checkpoint inhibitors than mismatch repair-proficient primary CRC. Blocking LAG3 enhances tumor-infiltrating T-cell responses of mismatch repair-proficient LM-CRC, and therefore may be a new promising immunotherapeutic target for LM-CRC
FcγRIIB engagement drives agonistic activity of Fc-engineered αOX40 antibody to stimulate human tumor-infiltrating T cells
Background OX40 (CD134) is a costimulatory molecule
of the tumor necrosis factor receptor superfamily that
is currently being investigated as a target for cancer
immunotherapy. However, despite promising results in
murine tumor models, the clinical efficacy of agonistic αOX40
antibodies in the treatment of patients with cancer has fallen
short of the high expectation in earlier-stage trials.
Methods Using lymphocytes from resected tumor, tumorfree (TF) tissue and peripheral blood mononuclear cells
(PBMC) of 96 patients with hepatocellular and colorectal
cancers, we determined OX40 expression and the in vitro
T-cell agonistic activity of OX40-targeting compounds.
RNA-Seq was used to evaluate OX40-mediated
transcriptional changes in CD4+ and CD8+
Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions
Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (Treg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibite
Detection of oncogenic mutations in paired circulating tumor DNA and circulating tumor cells in patients with hepatocellular carcinoma
Background and aims: Circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) may be used for diagnostic or prognostic purposes in patients with hepatocellular carcinoma (HCC). We aim to determine whether CTCs or ctDNA are suitable to determine oncogenic mutations in HCC patients. Methods: Twenty-six mostly advanced HCC patients were enrolled. 30 mL peripheral blood from each patient was obtained. CellSearch system was used for CTC detection. A sequencing panel covering 14 cancer-relevant genes was used to identify oncogenic mutations. TERT promoter C228T and C250T mutations were determined by droplet digital PCR. Results: CTCs were detected in 27% (7/26) of subjects but at low numbers (median: 2 cells, range: 1–15 cells) and ctDNA in 77% (20/26) of patients. Mutations in ctDNA were identified in several genes: TERT promoter C228T (77%, 20/26), TP53 (23%, 6/26), CTNNB1 (12%, 3/26), PIK3CA (12%, 3/26) and NRAS (4%, 1/26). The TERT C228T mutation was present in all patients with one or more ctDNA mutations, or detectable CTCs. The TERT C228T and TP53 mutations detected in ctDNA were present at higher levels in matched primary HCC tumor tissue. The maximal variant allele frequency (VAF) of ctDNA was linearly correlated with largest tumor size and AFP level (Log10). CtDNA (or TERT C228T) positivity was associated with macrovascular invasion, and positivity of ctDNA (or TERT C228T) or CTCs (≥ 2) correlated with poor patient survival. Conclusions: Oncogenic mutations could be detected in ctDNA from advanced HCC patients. CtDNA analysis may serve as a promising liquid biopsy to identify druggable mutations.</p
GITR engagement in combination with CTLA-4 blockade completely abrogates immunosuppression mediated by human liver tumor-derived regulatory T cells ex vivo
In liver cancer tumor-infiltrating regulatory T cells (Ti-Treg) are potent suppressors of tumor-specific T-cell responses and express high levels of the Treg-associated molecules cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR). In this study, we have evaluated the capacity of GITR-ligation, CTLA-4-blockade and a combination of both treatments to alleviate immunosuppression mediated by Ti-Treg. Using ex vivo isolated cells from individuals with hepatocellular carcinoma (HCC) or liver metastases from colorectal cancer (LM-CRC) we show that treatment with a soluble form of the natural ligand of GITR (GITRL), or with blocking antibodies to CTLA-4, reduces the suppression mediated by human liver tumor-infiltrating CD4+Foxp3+ Treg, thereby restoring proliferation and cytokine production by effector T cells. Importantly, combined treatment with low doses of both molecules exhibited stronger recovery of T cell function compared with either treatment alone. Our data suggest that in patients with primary and secondary liver cancer both GITR-ligation and anti-CTLA-4 mAb can improve the antitumor immunity by abrogating Ti-Treg mediated suppression
Tumor-infiltrating plasmacytoid dendritic cells promote immunosuppression by Tr1 cells in human liver tumors
CD4+ type 1 T regulatory (Tr1) cells have a crucial role in inducing tolerance. Immune regulation by these cells is mainly mediated through the secretion of high amounts of IL-10. Several studies have suggested that this regulatory population may be involved in tumor-mediated immune-suppression. However, direct evidence of a role for Tr1 cells in human solid tumors is lacking. Using ex vivo isolated cells from individuals with hepatocellular carcinoma (HCC; n = 39) or liver metastases from colorectal cancer (LM-CRC; n = 60) we identify a CD4+FoxP3-IL-13-IL-10+ T cell population in tumors of individuals with primary or secondary liver cancer that is characterized as Tr1 cells by the expression of CD49b and the lymphocyte activation gene 3 (LAG-3) and strong suppression activity of T cell responses in an IL-10 dependent manner. Importantly, the presence of tumor-infiltrating Tr1 cells is correlated with tumor infiltration of plasmacytoid dendritic cells (pDCs). pDCs exposed to tumor-derived factors enhance IL-10 production by Tr1 cells through up-regulation of the inducible co-stimulatory ligand (ICOS-L). These findings suggest a role for pDCs and ICOS-L in promoting intra-tumoral immunosuppression by Tr1 cells in human liver cancer, which may foster tumor progression and which might interfere with attempts of immunotherapeutic intervention