Tumor-Derived Extracellular Vesicles Impair CD171-Specific CD4+ CAR T Cell Efficacy

Chimeric antigen receptor (CAR) T cell efficacy against solid tumors is currently limited by several immune escape mechanisms, which may include tumor-derived extracellular vesicles. Advanced neuroblastoma is an aggressive childhood tumor without curative treatment options for most relapsed patients today. We here evaluated the role of tumor-derived extracellular vesicles on the efficacy of CAR T cells targeting the neuroblastoma-specific antigen, CD171. For this purpose, CAR T cell activation, cytokine production, exhaustion, and tumor cell-directed cytotoxicity upon co-culture was evaluated. Tumor-derived extracellular vesicles isolated from SH-SY5Y neuroblastoma cells neither affected CAR T cell activation nor expression of inhibitory markers. Importantly, exposure of CD4+ CD171-specific CAR T cells to tumor-derived extracellular vesicles significantly impaired tumor cytotoxicity of CAR T cells. This effect was independent of neurotrophic receptor tyrosine kinases 1 or 2 (NTRK1, NTRK2) expression, which is known to impact immune responses against neuroblastoma. Our results demonstrate for the first time the impact of tumor-derived extracellular vesicles and non-cell-mediated tumor-suppressive effects on CD4+ CAR T cell efficacy in a preclinical setting. We conclude that these factors should be considered for any CAR T cell-based therapy to make CAR T cell therapy successful against solid tumors

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients

IntroductionTriple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets.Materials and methodsTo follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3’ UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival.ResultssHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3’ UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy responseConclusionThe results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets

Vesicular-Bound HLA-G as a Predictive Marker for Disease Progression in Epithelial Ovarian Cancer

Extracellular vesicles (EV) and their tumor-supporting cargos provide a promising translational potential in liquid biopsies for risk assessment of epithelial ovarian cancer (EOC) patients frequently relapsing, despite initial complete therapy responses. As the immune checkpoint molecule HLA-G, which is operative in immune-escape, can be released by EV, we evaluate the abundance of EV and its vesicular-bound amount of HLA-G (HLA-GEV) as a biomarker in EOC. After enrichment of EV from plasma samples, we determined the EV particle number and amount of HLA-GEV by nanoparticle tracking analysis or ELISA. The association of results with the clinical status/outcome revealed that both, EV particle number and HLA-GEV were significantly elevated in EOC patients, compared to healthy females. However, elevated levels of HLA-GEV, but not EV numbers, were exclusively associated with a disadvantageous clinical status/outcome, including residual tumor, presence of circulating tumor cells, and disease progression. High HLA-GEV status was an independent predictor of progression, besides residual tumor burden and platinum-sensitivity. Especially among patients without residual tumor burden or with platinum-sensitivity, HLA-GEV identified patients with high risk of progression. Thus, this study highlights HLA-GEV as a potential novel biomarker for risk assessment of EOC patients with a rather beneficial prognosis defined by platinum-sensitivity or lack of residual tumor burden

The Inner and Outer Qualities of Extracellular Vesicles for Translational Purposes in Breast Cancer

Breast cancer (BC) is the second most common cause of cancer mortality of women worldwide. BC is a systemic disease with a highly heterogeneous course of disease. Therefore, prognostic and diagnostic biomarkers are required to improve the clinical risk management. Cancer-derived or cancer-associated extracellular vesicles (EVs) procured from the bloodstream of BC patients offer a novel platform for the qualitative and quantitative screening and establishment of biomarkers. Here, we focus on common aspects of EVs, on the function of BC-derived EVs and their translational potential considering the EV abundancy, intravesicular as well as outer membrane-anchored composition and current challenges of implementation in clinical practice

Individual Immune-Modulatory Capabilities of MSC-Derived Extracellular Vesicle (EV) Preparations and Recipient-Dependent Responsiveness

Treatment with extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been suggested as novel therapeutic option in acute inflammation-associated disorders due to their immune-modulatory capacities. As we have previously observed differences in the cytokine profile of independent MSC-EV preparations, functional differences of MSC-EV preparations have to be considered. To evaluate the immune-modulatory capabilities of specific MSC-EV preparations, reliable assays are required to characterize the functionality of MSC-EV preparations prior to administration to a patient. To this end, we established an in vitro assay evaluating the immune-modulatory capacities of MSC-EV preparations. Here, we compared the efficacy of four independent MSC-EV preparations to modulate the induction of T cell differentiation and cytokine production after phorbol 12-myristate 13-acetate (PMA)/Ionomycin stimulation of peripheral blood mononuclear cells (PBMC) derived from six healthy donors. Flow cytometric analyses revealed that the four MSC-EV preparations differentially modulate the expression of surface markers, such as CD45RA, on CD4+ and CD8+ T cells, resulting in shifts in the frequencies of effector and effector memory T cells. Moreover, cytokine profile in T cell subsets was affected in a MSC-EV-specific manner exclusively in CD8+ naïve T cells. Strikingly, hierarchical clustering revealed that the T cell response towards the MSC-EV preparations largely varied among the different PBMC donors. Thus, besides defining functional activity of MSC-EV preparations, it will be crucial to test whether patients intended for treatment with MSC-EV preparations are in principal competent to respond to the envisioned MSC-EV therapy

HLA-G 3′ untranslated region variants +3187G/G, +3196G/G and +3035T define diametrical clinical status and disease outcome in epithelial ovarian cancer

Abstract Expression of the non-classical human leukocyte antigen-G (HLA-G) promotes cancer progression in various malignancies including epithelial ovarian cancer (EOC). As single nucleotide polymorphisms (SNPs) in the HLA-G 3′ untranslated region (UTR) regulate HLA-G expression, we investigated HLA-G 3′UTR haplotypes arranged by SNPs in healthy controls (n = 75) and primary EOC patients (n = 79) and determined soluble HLA-G (sHLA-G) levels. Results were related to the clinical status and outcome. Although haplotype frequencies were similar in patients and controls, (i) sHLA-G levels were increased in EOC independent of the haplotype, (ii) homozygosity for UTR-1 or UTR-2 genotypes were significantly associated with metastases formation and presence of circulating tumor cells before therapy, whereas (iii) the UTR-5 and UTR-7 haplotypes were significantly associated with a beneficial clinical outcome regarding negative nodal status, early FIGO staging, and improved overall survival. Lastly, (iv) the ambivalent impact on clinical EOC aspects could be deduced to specific SNPs in the HLA-G 3′UTR: +3187G, +3196G and +3035T alleles. Our results give evidence that even if the genetic background of the HLA-G 3′UTR is identical between patients and controls, certain SNPs have the potential to contribute to diametrical clinical status/outcome in EOC

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image_2_The Donor Major Histocompatibility Complex Class I Chain-Related Molecule A Allele rs2596538 G Predicts Cytomegalovirus Viremia in Kidney Transplant Recipients.jpeg

<p>The interaction of major histocompatibility complex class I chain-related protein A (MICA) and its cognate activating receptor natural killer (NK) group 2 member D (NKG2D) receptor plays a significant role in viral immune control. In the context of kidney transplantation (KTx), cytomegalovirus (CMV) frequently causes severe complications. Hypothesizing that functional polymorphisms of the MICA/NKG2D axis might affect antiviral NK and T cell responses to CMV, we explored the association of the MICA-129 Met/Val single nucleotide polymorphism (SNP) (affecting the binding affinity of MICA with the NKG2D receptor), the MICA rs2596538 G/A SNP (influencing MICA transcription), and the NKG2D rs1049174 G/C SNP (determining the cytotoxic potential of effector cells) with the clinical outcome of CMV during the first year after KTx in a cohort of 181 kidney donor-recipients pairs. Univariate analyses identified the donor MICA rs2596538 G allele status as a protective prognostic determinant for CMV disease. In addition to the well-known prognostic factors CMV high-risk sero-status of patients and the application of lymphocyte-depleting drugs, the donor MICA rs2596538 G allele carrier status was confirmed by multivariate analyses as novel-independent factor predicting the development of CMV infection/disease during the first year after KTx. The results of our study emphasize the clinical importance of the MICA/NKG2D axis in CMV control in KTx and point out that the potential MICA transcription in the donor allograft is of clinically relevant importance for CMV immune control in this allogeneic situation. Furthermore, they provide substantial evidence that the donor MICA rs2596538 G allele carrier status is a promising genetic marker predicting CMV viremia after KTx. Thus, in the kidney transplant setting, donor MICA rs2596538 G may help to allow the future development of personal CMV approaches within a genetically predisposed patient cohort.</p