Down-regulation of HLA in tumor cells, low numbers and dysfunctionality of NK cells are commonly observed in patients with end-stage cervical cancer. Adoptive transfer of high numbers of cytotoxic NK ...cells might be a promising treatment approach in this setting. Here, we explored the cytotoxic efficacy on ten cervical cancer cell lines of activated allogeneic NK cells from two sources, i.e., peripheral blood (PBNK) with and without cetuximab (CET), a tumor-specific monoclonal antibody directed against EGFR, or derived from umbilical cord blood (UCB-NK). Whereas CET monotherapy was ineffective against the panel of cervical cancer cell lines, irrespective of their EGFR expression levels and despite their
RAS
wt
status, it significantly enhanced the in vitro cytotoxic efficacy of activated PBNK (
P
= 0.002). Equally superior cytotoxicity over activated PBNK alone was achieved by UCB-NK (
P
< 0.001). Both PBNK- and UCB-NK-mediated cytotoxic activity was dependent on the NK-activating receptors natural killer group 2, member D receptor (NKG2D) and DNAX accessory molecule-1 (DNAM-1) (
P
< 0.05) and unrelated to expression levels of the inhibitory receptors HLA-E and/or HLA-G. Most strikingly, whereas the PBNK’s cytotoxic activity was inversely correlated with HLA-ABC levels (
P
= 0.036), PBNK + CET and UCB-NK cytotoxicity were entirely independent of HLA-ABC expression. In conclusion, this study provides a rationale to initiate a clinical trial for cervical cancer with adoptively transferred allogeneic NK cells, employing either UCB-NK or PBNK + CET for EGFR-expressing tumors. Adoptive transfer of UCB-NK might serve as a generally applicable treatment for cervical cancer, enabled by HLA-, histology- and HPV-independent killing mechanisms.
A number of studies point to an aberrant differentiation and accumulation of CD14
+
PD-L1
+
M2-macrophage-like cells in the microenvironment of cervical cancer, which promote immunosuppressive ...conditions and are associated with tumor invasion, angiogenesis and metastasis. Therapeutic targeting of these macrophages may tip the balance in favor of antitumor immunity. Cervical cancer is the fourth most common cancer among women worldwide and is caused by a persistent infection and subsequent integration of high-risk types of the human papillomavirus. Continuous expression of the viral oncoproteins E6 and E7 has been shown essential to maintain the transformed state of infected keratinocytes. As these non-self oncoproteins are immunogenic, cervical cancer requires a highly immune suppressed tumor microenvironment to metastasize through lymphovascular space invasion (LVSI) to the pelvic tumor-draining lymph nodes (TDLN). Unraveling the mechanisms underlying this immune suppression may uncover novel therapeutic targets aimed at loco-regional control of cervical cancer.
Checkpoint blockade immunotherapies have revolutionised cancer treatment in the last decade. Nevertheless, these are only beneficial for a small proportion of cancer patients. Important ...prognosticators for response to immunotherapy are the mutation burden of tumours as well as the quality and quantity of tumour‐infiltrating immune cells. High‐throughput multiplex immunophenotyping technologies have a central role in deciphering the complexity of anti‐tumour immune responses. Current techniques for the immunophenotyping of solid tumours are held back by the lack of spatial context, limitations in the number of targets that can be visualised simultaneously, and/or cumbersome protocols. We developed a tyramide signal amplification‐free method for the simultaneous detection of seven cellular targets by immunofluorescence. This method overcomes limitations posed by most widespread techniques and provides a unique tool for extensive phenotyping by multispectral fluorescence microscopy. Furthermore, it can be easily implemented as a high‐throughput technology for validation of discovery sets generated by RNA sequencing or mass cytometry and may serve in the future as a complementary diagnostic tool.
The host's immune system plays a pivotal role in many tumor types, including squamous cell carcinomas (SCCs). We aim to identify immunological prognosticators for lymph node metastases (LNM) and ...disease-specific survival (DSS) in penile SCC. For this retrospective observational cohort study, penile SCC patients (
= 213) treated in the Netherlands Cancer Institute, were selected if sufficient formalin-fixed, paraffin-embedded tumor material was available. Analysis included previously described high-risk human papilloma virus (hrHPV) status, immunohistochemical scores for classical and non-classical human leukocyte antigen (HLA) class I, programmed death ligand-1 (PD-L1) expression, and novel data on tumor-infiltrating macrophages and cytotoxic an regulatory T-cells. Clinicopathological characteristics and extended follow-up were also included. Regression analyses investigated relationships of the immune parameters with LNM and DSS. In the total cohort, diffuse PD-L1 tumor-cell expression, CD163
macrophage infiltration, non-classical HLA class I upregulation, and low stromal CD8
T-cell infiltration were all associated with LNM. In the multivariable model, only tumor PD-L1 expression remained a significant predictor for LNM (odds ratio (OR) 2.8,
= 0.05). hrHPV negativity and diffuse PD-L1 tumor-cell expression were significantly associated with poor DSS and remained so upon correction for clinical parameters hazard ratio (HR) 9.7,
< 0.01 and HR 2.8,
= 0.03. The only immune factor with different expression in HPV
and HPV
tumors was PD-L1, with higher PD-L1 expression in the latter (
= 0.03). In the HPV
cohort (
= 158), LNM were associated with diffuse PD-L1 tumor-cell expression, high intratumoral CD163
macrophage infiltration, and low number of stromal CD8
T-cells. The first two parameters were also linked to DSS. In the multivariable regression model, diffuse PD-L1 expression remained significantly unfavorable for DSS (HR 5.0,
< 0.01). These results emphasize the complexity of the tumor microenvironment in penile cancer and point toward several possible immunotherapy targets. Here described immune factors can aid risk-stratification and should be evaluated in clinical immunotherapy studies to ultimately lead to patient tailored treatment.
BackgroundTherapeutic immune intervention is highly dependent on the T-cell priming and boosting capacity of tumor-draining lymph nodes (TDLN). In vulvar cancer, in-depth studies on the immune status ...of (pre)metastatic TDLN is lacking.MethodsWe have phenotyped and enumerated various T-cell and myeloid subsets in tumor-free (LN−, n=27) and metastatic TDLN (LN+, n=11) using flow cytometry. Additionally, we studied chemokine and cytokine release profiles and assessed expression of indoleamine 2,3-dioxygenase (IDO) in relation to plasmacytoid dendritic cell (pDC) or myeloid subsets.ResultsMetastatic involvement of TDLN was accompanied by an inflamed microenvironment with immune suppressive features, marked by hampered activation of migratory DC, increased cytokine/chemokine release, and closely correlated elevations of pDC and LN-resident conventional DC (LNR-cDC) activation state and frequencies, as well as of terminal CD8+ effector-memory T-cell (TemRA) differentiation, regulatory T-cell (Treg) rates, T-cell activation, and expression of cytotoxic T-lymphocyte protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1) immune checkpoints. In addition, high indoleamine 2,3-dioxygenase (IDO) expression and increased frequencies of monocytic myeloid-derived suppressor cells (mMDSC) were observed. Correlation analyses with primary and metastatic tumor burden suggested respective roles for Tregs and suppression of inducible T cell costimulator (ICOS)+ T helper cells in early metastatic niche formation and for CD14+ LNR-cDC and terminal T-cell differentiation in later stages of metastatic growth.ConclusionsMetastatic spread in vulvar TDLN is marked by an inflamed microenvironment with activated effector T cells, which are likely kept in check by an interplay of suppressive feedback mechanisms. Our data support (neoadjuvant) TDLN-targeted therapeutic interventions based on CTLA-4 and PD-1 blockade, to reinvigorate memory T cells and curb early metastatic spread and growth.
A better understanding of the microenvironment in relation to lymph node metastasis is essential for the development of effective immunotherapeutic strategies against cervical cancer.In the present ...study, we investigated the microenvironment of tumor-draining lymph nodes of cervical cancer patients, by comprehensive flow cytometry-based phenotyping and enumeration of immune-cell subsets in tumor-negative (LN-, n = 20) versus tumor-positive lymph nodes (LN+, n = 8), and by the study of cytokine release profiles (n = 4 for both LN- and LN+).We found significantly lower CD4+ and higher CD8+ T-cell frequencies in LN+ samples, accompanied by increased surface levels of activation (HLA-DR and ICOS) and inhibitory markers (PD-1 and CTLA-4). Furthermore, in LN+ we found increased rates of a potentially regulatory antigen-presenting cell (APC) subset (CD11chiCD14+PD-L1+) and of myeloid-derived suppressor cell (MDSC) subsets, which in the case of the former correlated significantly with elevated frequencies of FoxP3+ Tregs. After in vitro stimulation with different TLR ligands (PGN; Poly-IC; R848), we observed higher production levels of IL-6, IL-10 and TNFα but lower levels of IFNγ in LN+.We conclude that, despite increased T-cell differentiation and activation, a striking switch to a profound immune suppressive microenvironment in LN+ of cervical cancer patients will enable immune escape. Our data point to the CD14+PD-L1+ APC/Treg axis as a particularly attractive and relevant therapeutic target to specifically tackle microenvironmental immune suppression and thus enhance the efficacy of immunotherapy in patients with metastasized cervical cancer.
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