P38 MAPK expression and activation predicts failure of response to CHOP in patients with Diffuse Large B-Cell Lymphoma

The p38 MAPK is constitutively activated in B-NHL cell lines and regulates chemoresistance. Accordingly, we hypothesized that activated p38 MAPK may be associated with the in vivo unresponsiveness to chemotherapy in B-NHL patients.Tissue microarrays generated from eighty untreated patients with Diffused Large B Cell Lymphoma (DLBCL) were examined by immunohistochemistry for the expression of p38 and phospho p38 (p-p38) MAPK. In addition, both Bcl-2 and NF-κB expressions were determined. Kaplan Meier analysis was assessed.Tumor tissues expressed p38 MAPK (82 %) and p-p38 MAPK (30 %). Both p38 and p-p38 MAPK expressions correlated with the high score performance status. A significant correlation was found between the expression p-p38 and poor response to CHOP. The five year median follow-up FFS was 81 % for p38(-) and 34 % for p38(+) and for OS was 83 % for p38(-) and 47 % for p38(+). The p-p38(+) tissues expressed Bcl-2 and 90 % of p-p38(-) where Bcl-2(-). The coexpression of p-p38 and Bcl-2 correlated with pool EFS and OS. There was no correlation between the expression of p-p38 and the expression of NF-κB.The findings revealed, for the first time, that a subset of patients with DLBCL and whose tumors expressed high p-p38 MAPK responded poorly to CHOP therapy and had poor EFS and OS. The expression of p38, p-p38, Bcl2 and the ABC subtype are significant risk factors both p38 and p-p38 expressions remain independent prognostic factors

Multifaceted role of BTLA in the control of CD8+ T cell fate after antigen encounter

Purpose: Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40%–50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte associated) expression on transferred CD8+ TILs was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8+BTLA+TIL subset and define the contribution of the Grb2 motif of BTLA in T-cell costimulation. Experimental Design: We determined the functional role and downstream signal of BTLA in both human CD8+ TILs and mouse CD8+ T cells. Functional assays were used including single-cell analysis, reverse-phase protein array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as patient-derived xenograft (PDX) model using immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TILs. Results: CD8+BTLA? TILs could not control tumor growth in vivo as well as their BTLA+ counterpart and antigen-specific CD8+BTLA? T cells had impaired recall response to a vaccine. However, CD8+BTLA+ TILs displayed improved survival following the killing of a tumor target and heightened “serial killing” capacity. Using mutants of BTLA signaling motifs, we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation. Conclusions: Our data portrays BTLA as a molecule with the singular ability to provide both costimulatory and coinhibitory signals to activated CD8+ T cells, resulting in extended survival, improved tumor control, and the development of a functional recall response. Clin Cancer Res; 23(20); 6151–64. ©2017 AACR

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa

BackgroundBacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance.MethodsIn this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains.ResultsPhage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia.ConclusionsWe show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy’s evolutionary impact that may be exploited to generate robust therapy schemes

Identifying signatures of EV secretion in metastatic breast cancer through functional single-cell profiling

Summary: Extracellular vesicles (EVs) regulate the tumor microenvironment by facilitating transport of biomolecules. Despite extensive investigation, heterogeneity in EV secretion among cancer cells and the mechanisms that support EV secretion are not well characterized. We developed an integrated method to identify individual cells with differences in EV secretion and performed linked single-cell RNA-sequencing on cloned single cells from the metastatic breast cancer cells. Differential gene expression analyses identified a four-gene signature of breast cancer EV secretion: HSP90AA1, HSPH1, EIF5, and DIAPH3. We functionally validated this gene signature by testing it across cell lines with different metastatic potential in vitro. Analysis of the TCGA and METABRIC datasets showed that this signature is associated with poor survival, invasive breast cancer types, and poor CD8+ T cell infiltration in human tumors. We anticipate that our method for directly identifying the molecular determinants of EV secretion will have broad applications across cell types and diseases

Single-Cell Cloning of Breast Cancer Cells Secreting Specific Subsets of Extracellular Vesicles

Extracellular vesicles (EVs) mediate communication in health and disease. Conventional assays are limited in profiling EVs secreted from large populations of cells and cannot map EV secretion onto individual cells and their functional profiles. We developed a high-throughput single-cell technique that enabled the mapping of dynamics of EV secretion. By utilizing breast cancer cell lines, we established that EV secretion is heterogeneous at the single-cell level and that non-metastatic cancer cells can secrete specific subsets of EVs. Single-cell RNA sequencing confirmed that pathways related to EV secretion were enriched in the non-metastatic cells compared with metastatic cells. We established isogenic clonal cell lines from non-metastatic cells with differing propensities for CD81+CD63+EV secretion and showed for the first time that specificity in EV secretion is an inheritable property preserved during cell division. Combined in vitro and animal studies with these cell lines suggested that CD81+CD63+EV secretion can impede tumor formation. In human non-metastatic breast tumors, tumors enriched in signatures of CD81+CD63+EV have a better prognosis, higher immune cytolytic activity, and enrichment of pro-inflammatory macrophages compared with tumors with low CD81+CD63+EVs signatures. Our single-cell methodology enables the direct integration of EV secretion with multiple cellular functions and enables new insights into cell/disease biology

P38 MAPK expression and activation predicts failure of response to CHOP in patients with Diffuse Large B-Cell Lymphoma.

BackgroundThe p38 MAPK is constitutively activated in B-NHL cell lines and regulates chemoresistance. Accordingly, we hypothesized that activated p38 MAPK may be associated with the in vivo unresponsiveness to chemotherapy in B-NHL patients.MethodsTissue microarrays generated from eighty untreated patients with Diffused Large B Cell Lymphoma (DLBCL) were examined by immunohistochemistry for the expression of p38 and phospho p38 (p-p38) MAPK. In addition, both Bcl-2 and NF-κB expressions were determined. Kaplan Meier analysis was assessed.ResultsTumor tissues expressed p38 MAPK (82 %) and p-p38 MAPK (30 %). Both p38 and p-p38 MAPK expressions correlated with the high score performance status. A significant correlation was found between the expression p-p38 and poor response to CHOP. The five year median follow-up FFS was 81 % for p38(-) and 34 % for p38(+) and for OS was 83 % for p38(-) and 47 % for p38(+). The p-p38(+) tissues expressed Bcl-2 and 90 % of p-p38(-) where Bcl-2(-). The coexpression of p-p38 and Bcl-2 correlated with pool EFS and OS. There was no correlation between the expression of p-p38 and the expression of NF-κB.ConclusionThe findings revealed, for the first time, that a subset of patients with DLBCL and whose tumors expressed high p-p38 MAPK responded poorly to CHOP therapy and had poor EFS and OS. The expression of p38, p-p38, Bcl2 and the ABC subtype are significant risk factors both p38 and p-p38 expressions remain independent prognostic factors

P38 MAPK expression and activation predicts failure of response to CHOP in patients with Diffuse Large B-Cell Lymphoma.

The p38 MAPK is constitutively activated in B-NHL cell lines and regulates chemoresistance. Accordingly, we hypothesized that activated p38 MAPK may be associated with the in vivo unresponsiveness to chemotherapy in B-NHL patients.Tissue microarrays generated from eighty untreated patients with Diffused Large B Cell Lymphoma (DLBCL) were examined by immunohistochemistry for the expression of p38 and phospho p38 (p-p38) MAPK. In addition, both Bcl-2 and NF-κB expressions were determined. Kaplan Meier analysis was assessed.Tumor tissues expressed p38 MAPK (82 %) and p-p38 MAPK (30 %). Both p38 and p-p38 MAPK expressions correlated with the high score performance status. A significant correlation was found between the expression p-p38 and poor response to CHOP. The five year median follow-up FFS was 81 % for p38(-) and 34 % for p38(+) and for OS was 83 % for p38(-) and 47 % for p38(+). The p-p38(+) tissues expressed Bcl-2 and 90 % of p-p38(-) where Bcl-2(-). The coexpression of p-p38 and Bcl-2 correlated with pool EFS and OS. There was no correlation between the expression of p-p38 and the expression of NF-κB.The findings revealed, for the first time, that a subset of patients with DLBCL and whose tumors expressed high p-p38 MAPK responded poorly to CHOP therapy and had poor EFS and OS. The expression of p38, p-p38, Bcl2 and the ABC subtype are significant risk factors both p38 and p-p38 expressions remain independent prognostic factors

Single-cell profiling of dynamic cytokine secretion and the phenotype of immune cells

<div><p>Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled <i>ex vivo</i> revealed that CD56^dimCD16⁺ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.</p></div

NK cells that secrete IFN-γ early have higher CD16 surface expression.

<p>(AB): The distributions (black columns) of t_Secrete of single-NK cells were positively skewed, indicating the existence of a faster secretor subpopulation within the population of NK cells that secrete IFN-γ. The corresponding normal distributions (red curve) were plotted using the same mean and standard deviation of t_Secrete of single-NK cells. The relative comparison of CD16 (C) or CD56 (D) surface expression of early secretors (t_Secrete < population mean) and late secretors (t_Secrete > population mean). (E) The amount of IFN-γ secreted by NK cells during the 6 h period of observation was statistically different across two donors. The amount of IFN-γ secreted is inferred from the ratio of fluorescent intensities (ratio of maximum and minimum value) from the fitting curve; (F) The relative IFN-γ secretion rate was a donor-dependent parameter. The rate of secretion of IFN-γ was inferred from the Hill slope (MFI versus time) obtained from curve fit on two different donors (donor 1: light red; donor 2: dark red). Error bar: mean and 95% confidence intervals are shown. Mann-Whitney test was performed, ns: not significant, ***: <i>p</i>-value < 0.001, ****: <i>p</i>-value < 0.0001.</p