Investigating genes and micro-RNAs that may predict clinical benefits of anti-CTLA-4 therapy.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3043-3043
Author(s):  
Jianjun Gao ◽  
Hong Chen ◽  
Derek Ng Tang ◽  
Padmanee Sharma
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Immune Modulation ◽  
Expression Profiles ◽  
Predictive Biomarkers ◽  
Rna Isolation ◽  
Complete Response ◽  
Micro Rnas ◽  
Clinical Benefits

3043 Background: Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, ipilimumab, has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of ipilimumab for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4 therapy. In order to identify genes, microRNAs, and signaling pathways that are modulated by anti-CTLA-4, which may be used for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4+T cells from patients treated with anti-CTLA-4 for changes in gene and microRNA expression profiles. Methods: On an IRB-approved phase Ia presurgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of ipilimumab at 10 mg/kg at weeks 1 and 4. Pre-therapy and post-therapy blood samples were collected for CD4+ T cell enrichment by using the T cell isolation kit from Miltenyi Biotec (Auburn, CA). RNA and microRNA were isolated from purified CD4+T cells using Qiagen RNA isolation kits for Affymetrix microarray and micoRNA array analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. Results: Anti-CTLA-4 treatment resulted in modulation of differentially expressed genes (DEGs). After two doses of treatment, anti-CTLA-4 significantly changed expression of a total of 289 DEGs. Further pathway analyses indicated that anti-CTLA-4 induced a variety of pathways involved in cell proliferation and immune modulation, including PI3K/AKT, MAP/ERK, and IFN/JAK-STAT pathways. We have also identified 9 microRNAs that potentially regulate the expression of genes changed by anti-CTLA-4 therapy. Conclusions: Anti-CTLA-4 treatment results in modulation of multiple genes, microRNAs, and pathways, which likely play important roles in anti-tumor immune responses. We are currently testing a number of these identified genes and microRNAs as potential predictive biomarkers for anti-CTLA-4 therapy in a small cohort of patients who had complete response vs. progression of disease after anti-CTLA-4 therapy.

A study of genes and microRNAs that may predict clinical responses to anti-CTLA-4 therapy.

2013 ◽  
Vol 31 (6_suppl) ◽  
pp. 285-285
Author(s):  
Jianjun Gao ◽  
Hong Chen ◽  
Derek Ng Tang ◽  
Padmanee Sharma
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Immune Modulation ◽  
Expression Profiles ◽  
Predictive Biomarkers ◽  
Rna Isolation ◽  
Complete Response ◽  
Expression Of Genes ◽  
Pathway Analyses

285 Background: Blockade of T cell co-inhibitory receptor CTLA-4 with a monoclonal antibody, Ipilimumab (BMS), has led to augmented anti-tumor immune responses, clinical benefit, and FDA approval of Ipilimumab for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4 therapy. In order to identify genes, microRNAs, and signaling pathways that are modulated by anti-CTLA-4, which may be used for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4+ T cells from patients treated with anti-CTLA-4 for changes in gene and microRNA expression profiles. Methods: On an IRB-approved Phase Ia presurgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of Ipilimumab at 10 mg/kg at weeks 1 and 4. Pre-therapy and post-therapy blood samples were collected for CD4+ T cell enrichment by using the T cell isolation kit from Miltenyi Biotec (Auburn, CA). RNA and microRNA were isolated from purified CD4+ T cells using Qiagen RNA isolation kits for Affymetrix microarray and micoRNA array analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. Results: Ipilimumab treatment resulted in modulation of differentially expressed genes (DEGs). After two doses of treatment, Ipilimumab significantly changed expression of a total of 289 DEGs. Further pathway analyses indicated that Ipilimumab induced a variety of pathways involved in cell proliferation and immune modulation, including PI3K/AKT, MAP/ERK, and IFN/JAK-STAT pathways. We have also identified 9 microRNAs that potentially regulate the expression of genes changed by anti-CTLA-4 therapy. Conclusions: Ipilimumab treatment results in modulation of multiple genes, microRNAs, and pathways, which likely play important roles in anti-tumor immune responses. We are currently testing a number of these identified genes and microRNAs as potential predictive biomarkers for anti-CTLA-4 therapy in a small cohort of patients who had complete response vs. progression of disease after anti-CTLA-4 therapy.

Micro-RNA Profiling in CD4+ T Cell Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3887-3887
Author(s):  
Arnob Banerjee ◽  
Felix Schambach ◽  
Scott Hammond ◽  
Steven Reiner
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd4 T Cells ◽  
Expression Profiles ◽  
T Cell Differentiation ◽  
Micro Rna ◽  
Cd4 T Cell ◽  
T Helper ◽  
Micro Rnas

Abstract Micro-RNAs comprise a class of small noncoding RNAs which have been found to be important regulators of cellular differentiation in multiple species. Previous analysis of micro-RNA expression in the murine hematopoietic system has suggested a role in cell differentiation and the maintenance of cell identity. Naïve progenitor CD4+ T cells respond to a combination of appropriate antigen and other specific signals by undergoing proliferation and further differentiation into one of at least two subsets. T helper 1 (TH1) cells produce high levels of the cytokine IFN-γ and T helper 2 (TH2) cells produce high levels of IL-4, optimizing them for control of intracellular and extracellular pathogens, respectively. It is currently not known whether micro-RNA molecules influence CD4+ T cell differentiation. We have used oligonucleotide arrays to analyze micro-RNA expression profiles of freshly isolated murine CD4+ T cells compared to cells differentiating into TH1 and TH2 subsets. Expression profiles were found to differ significantly between naïve and stimulated CD4+ cells, with fewer differences between TH1 and TH2 subsets. Promising candidate micro-RNAs are being further evaluated by northern blot and genetic studies. Micro-RNA-155 is upregulated on stimulation of CD4+ T cells in multiple oligonucleotide array assays. Micro-RNA-155 is encoded by the BIC oncogene and has been implicated in lymphomagenesis as well as in other malignancies. We have verified the induction of micro-RNA-155 in stimulated helper T cells by northern blot and are studying the effects of this micro-RNA on CD4+ T cell differentiation. Our observations support a role for micro-RNAs in helper T cell differentiation during the immune response.

scholarly journals Enhancing the in vivo expansion of adoptively transferred EBV-specific CTL with lymphodepleting CD45 monoclonal antibodies in NPC patients

Blood ◽  
2009 ◽  
Vol 113 (11) ◽  
pp. 2442-2450 ◽  
Author(s):  
Chrystal U. Louis ◽  
Karin Straathof ◽  
Catherine M. Bollard ◽  
Claudia Gerken ◽  
M. Helen Huls ◽  
...  
Keyword(s):  
T Cells ◽  
Monoclonal Antibodies ◽  
T Cell ◽  
Cytotoxic T Cells ◽  
Complete Response ◽  
Barr Virus ◽  
Significant Toxicity ◽  
Clinical Benefits ◽  
Epstein Barr

Treatment of Epstein-Barr virus (EBV)–positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of auto-logous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at http://www.clinialtrials.gov as NCT00608257.

Chronic Lymphocytic Leukemia Cells Acquire a Regulatory B-Cell Phenotype Modified By PD1 Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3319-3319 ◽  
Author(s):  
Shimrit Ringelstein-Harlev ◽  
Irit Avivi ◽  
Shoham Shivtiel-Arad ◽  
Tami Katz
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Immune Modulation ◽  
Lymphocytic Leukemia ◽  
T Cell Proliferation ◽  
Cell Contact

Abstract Introduction: Chronic lymphocytic leukemia (CLL) cells utilize several mechanisms of survival, some propagating proliferation and preventing apoptosis through intrinsic cell cycle signals, and others suppressing anti-tumor immune responses. Patients often present with a predominant population of regulatory T-cells (Tregs), and general features of T-cell exhaustion. Given the unique phenotype of CLL cells and the observed T-cell abnormalities we hypothesized that these cells function as regulatory B-cells (Bregs). Bregs, mostly explored in the autoimmune disease setting, produce interleukin-10 (IL10), which mediates attenuation of effector T-cell responses and enhances regulatory activity. These features have also been suggested to be responsible for weakening of anti-tumor immune responses. Breg activation requires stimulation of various combinations of Toll-like receptors (TLRs), the B-cell receptor (BCR) and CD40. Our previous studies have demonstrated that TLR9-stimulated CLL cells "acquire" Breg markers as well as PD1 and PDL1, which, while not being classic Breg discriminators, are established players in immune modulation. Moreover, such stimulation resulted in inhibition of proliferation of autologous T-cells. The current study aimed to further explore the regulatory characteristics of CLL cells focusing on additional suppressive mechanisms that may have a role in CLL immune evasion, particularly, the PD1/PDL1 axis. Methods: B-cells were isolated from peripheral blood mononuclear cells (PBMCs) of untreated CLL patients (Rai stages 0-IV). These B-CLL cells were stimulated with TLR-9 agonist (ODN) or CD40 ligand (CD40L) followed by their co-culture with isolated autologous CD4+ T cells. The regulatory features of B-CLL cells were studied by testing their effect on T cells. Their proliferation was evaluated using the CFSE method following stimulation with anti-CD3/CD28 antibodies and IL2; induction of Tregs (CD4+CD25highFoxp3+ population) was assessed by FACS analysis. The involvement of the PD1/PDL1 axis was examined by incubating B-cells with antiPD1 neutralizing antibodies prior to co-culture. Cell contact dependence was evaluated by plating B-cells in hanging cell culture inserts denying B and T cell contact while allowing flow of small soluble molecules. Results: CLL cells stimulated with ODN or CD40L, induced a significant increase in Tregs: 1.35±0.1-fold (p=0.03, N=12) for ODN and 1.7±0.2-fold (p=0.008, N=14) for CD40L, occurring in 68% and 80% of patients, respectively, while co-culture with unstimulated B-CLL cells did not result in the expansion of the Treg population. Treg induction was observed only under contact conditions (N=5), suggesting that this regulatory function requires cell-to-cell contact and cannot be carried out solely by secreted factors like IL10. Neutralization of PD1 on CLL B-cells affects both Treg induction and T-cell proliferation. Following CD40L stimulation, a 1.3-fold reduction in Treg percentage was observed when PD1 signaling was blunted (N=10). In contrast, PD1 blockage of ODN-stimulated CLL cells did not reduce Treg induction; however, it did adversely affect inhibition of T-cell proliferation (10%-decrease in inhibited T-cells; N=6). Conclusions: CLL cells "acquire" a Breg phenotype and function, inhibiting T-cell proliferation and inducing Tregs. These properties, while working together to promote immune regulation and cancer evasion, are elicited by different ligands in the cell environment and are likely to be mediated via separate pathways. The involvement of B-cell-associated PD1 in the induction of Tregs and inhibition of T-cell proliferation suggests a biologic role of PD1 signaling in CLL cells, strengthening the Breg phenotype. The current study has shown that CLL cells recruit several mechanisms operating cooperatively to support immune modulation and promote their survival. Disclosures No relevant conflicts of interest to declare.

Investigating genes and pathways that play a role in immune responses mediated by anti-CTLA-4 therapy.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 2520-2520
Author(s):  
Jianjun Gao ◽  
Hong Chen ◽  
Derek Ng Tang ◽  
Padmanee Sharma
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Immune Modulation ◽  
Cell Cycle Control ◽  
Control Cell ◽  
Tumor Tissues ◽  
Pathway Analyses ◽  
Microarray Analyses

2520^ Background: Blockade of the inhibitory T cell molecule CTLA-4 with a monoclonal antibody has led to enhanced anti-tumor immune responses and clinical benefit. Ipilimumab, an anti-CTLA-4 antibody (BMS), was recently FDA-approved for the treatment of metastatic melanoma. Only a subset of patients benefit from anti-CTLA-4. In order to identify genes and pathways that are induced by anti-CTLA-4, which may be used in future studies for potential correlation with clinical outcomes or provide additional targets for therapy, we purified and analyzed CD4 T cells from patients treated with anti-CTLA-4 for changes in gene expression profile. We also obtained tumor tissues from treated patients for similar studies. Methods: On an IRB-approved Phase Ia pre-surgical clinical trial, 6 patients with localized bladder cancer were treated with two doses of Ipilimumab at 10 mg/kg at weeks 1 and 4. Blood was collected pre-therapy and post-therapy (3 weeks after each dose). CD4 T cells were enriched from peripheral blood by using the CD4 T cell isolation kit from Miltenyi Biotec (Auburn, CA). Total RNA was isolated from purified CD4 T cells using Qiagen RNeasy kit for Affymetrix microarray analyses. Microarray data were then analyzed using Ingenuity iReport (Redwood City, CA). RT-PCR, RPPA and Western blot were used to confirm significant changes in genes or pathways identified in microarray analyses. Results: Ipilimumab treatment resulted in modulation of differentially expressed genes (DEGs). After dose #1, Ipilimumab only modulated 16 DEGs >2-fold (p<0.05) in CD4 T cells. After two doses of treatment, Ipilimumab significantly changed expression of a total of 100 DEGs. Further pathway analyses indicated that Ipilimumab induced a variety of pathways involved in cell cycle control, cell proliferation, apoptosis, and immune modulation. Specifically, these pathways include PI3K/AKT, MAP/ERK, IFN/JAK-STAT, granzyme, and protein ubiquination. Conclusions: Ipilimumab treatment results in modulation of multiple genes and pathways, which likely play important roles in anti-tumor immune responses and need to be considered for future optimization of anti-CTLA-4 therapy and design of combination immunotherapy strategies.

scholarly journals Contribution of innate and adaptive immune cells to the elimination of Salmonella enterica serotype Enteritidis infection in young broiler chickens

2021 ◽  
Author(s):  
Nathalie Meijerink ◽  
Robin H.G.A. van den Biggelaar ◽  
Daphne A. van Haarlem ◽  
J. Arjan Stegeman ◽  
Victor P.M.G. Rutten ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Nk Cells ◽  
Salmonella Enterica ◽  
Broiler Chickens ◽  
Immune Modulation ◽  
Severe Disease ◽  
Antibody Responses ◽  
Adaptive Immune

AbstractSalmonella enterica serotype Enteritidis (SE) is a zoonotic pathogen which causes foodborne diseases in humans through contaminated poultry products, as well as severe disease symptoms in young chickens. More insight in innate and adaptive immune responses of chickens to SE infection is needed to understand elimination of SE. Seven-day-old broiler chickens were experimentally challenged with SE and numbers and responsiveness of innate immune cells including natural killer (NK) cells, macrophages and dendritic cells (DCs) were assessed during 21 days post-infection (dpi). In parallel, numbers and function of γδ T cells, CD8+ and CD4+ T cells as well as antibody titres were determined. SE was observed in the intestine and spleen of SE-infected chickens at 7 dpi. NK and T cells responded first to SE at 1 and 3 dpi as indicated by increased numbers of intestinal IL-2Rα+ and 20E5+ NK cells, in addition to enhanced activation of intestinal and splenic NK cells. At 7 dpi in the spleen, the presence of macrophages and the expression of activation markers on DCs was increased. At 21 dpi, an increase in intestinal γδ and CD8+ T cell numbers was observed. Furthermore, SE-specific proliferation of splenic CD4+ and CD8+ T cells was observed and SE-specific antibodies were detected in all blood samples of SE-infected chickens. In conclusion, SE results in enhanced numbers and activation of innate cells during early stages of infection and it is hypothesized that in concert with subsequent specific T cell and antibody responses, reduction of SE in infected chickens is achieved. A better understanding of innate and adaptive immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance and prevent SE infection and colonization in young broiler chickens and hence increase food safety for humans.Author summarySalmonella enterica serotype Enteritidis (SE) causes foodborne zoonotic diseases in humans, as well as a severe disease in young chickens. As a consequence of which health and welfare of humans and chickens are affected, resulting in substantial economic losses. To enable development of immune-mediated prevention strategies in chickens, more insight in the immune responses to SE is needed to understand how the infection is eliminated. For this purpose, we investigated non-specific and specific immune responses upon experimental SE infection in young broiler chickens. In this study, we found SE in the intestine and spleen of SE-infected chickens at 7 days post-infection (dpi). We show that natural killer (NK) cells respond first by enhanced presence and activation, followed by increased presence of macrophages and activation of dendritic cells. These early responses are hypothesized to stimulate the observed subsequent specific T cell and antibody responses. Better understanding of immune responses important in the elimination of SE will aid in developing immune-modulation strategies, which may increase resistance and prevent SE infection and colonization in young chickens and hence reduce SE-related foodborne illness in humans.

Aryloxy Triester Phosphonamidates of Phosphoantigens Exhibit Favorable Stability and Potent Activation of Vγ9/Vδ2 T‐Cells

2018 ◽  
Author(s):  
Hachemi Kadri ◽  
Taher E. Taher ◽  
Qin Xu ◽  
Richard T. Bryan ◽  
Benjamin E. Willcox ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Serum Stability ◽  
T Cell Immune Responses ◽  
Further Development

We previously reported the application of the aryloxy triester phosphoramidate prodrug technology to the phosphoantigen (E)-4-hydroxybut-2-enyl phosphate (HMBP). Although these prodrugs exhibited potent activation of Vγ9/Vδ2 T‐cell immune responses, their stability was low due to the rapid cleavage of the -O-P- bond. To address this, we herein report the application of the same prodrug strategy to two HMBP phosphonates, which have stable -CH2-P- or -CF2-P- bonds. These HMBP phosphonate prodrugs, phosphonamidates, exhibited excellent serum stability and potent activation of Vgama9/Vdelta2 T‐cells making them attractive compounds for further development as potential immunotherapeutics.

Aryloxy Triester Phosphonamidates of Phosphoantigens Exhibit Favorable Stability and Potent Activation of Vγ9/Vδ2 T‐Cells

2018 ◽  
Author(s):  
Hachemi Kadri ◽  
Taher E. Taher ◽  
Qin Xu ◽  
Richard T. Bryan ◽  
Benjamin E. Willcox ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Serum Stability ◽  
T Cell Immune Responses ◽  
Further Development

We previously reported the application of the aryloxy triester phosphoramidate prodrug technology to the phosphoantigen (E)-4-hydroxybut-2-enyl phosphate (HMBP). Although these prodrugs exhibited potent activation of Vγ9/Vδ2 T‐cell immune responses, their stability was low due to the rapid cleavage of the -O-P- bond. To address this, we herein report the application of the same prodrug strategy to two HMBP phosphonates, which have stable -CH2-P- or -CF2-P- bonds. These HMBP phosphonate prodrugs, phosphonamidates, exhibited excellent serum stability and potent activation of Vgama9/Vdelta2 T‐cells making them attractive compounds for further development as potential immunotherapeutics.

scholarly journals Immunomodulating nano-adaptors potentiate antibody-based cancer immunotherapy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cheng-Tao Jiang ◽  
Kai-Ge Chen ◽  
An Liu ◽  
Hua Huang ◽  
Ya-Nan Fan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cancer Immunotherapy ◽  
Tumor Cells ◽  
Noncovalent Interactions ◽  
Killer Cell ◽  
Antibody Immobilization ◽  
Effector Cells ◽  
Nanoparticle Surface

AbstractModulating effector immune cells via monoclonal antibodies (mAbs) and facilitating the co-engagement of T cells and tumor cells via chimeric antigen receptor- T cells or bispecific T cell-engaging antibodies are two typical cancer immunotherapy approaches. We speculated that immobilizing two types of mAbs against effector cells and tumor cells on a single nanoparticle could integrate the functions of these two approaches, as the engineered formulation (immunomodulating nano-adaptor, imNA) could potentially associate with both cells and bridge them together like an ‘adaptor’ while maintaining the immunomodulatory properties of the parental mAbs. However, existing mAbs-immobilization strategies mainly rely on a chemical reaction, a process that is rough and difficult to control. Here, we build up a versatile antibody immobilization platform by conjugating anti-IgG (Fc specific) antibody (αFc) onto the nanoparticle surface (αFc-NP), and confirm that αFc-NP could conveniently and efficiently immobilize two types of mAbs through Fc-specific noncovalent interactions to form imNAs. Finally, we validate the superiority of imNAs over the mixture of parental mAbs in T cell-, natural killer cell- and macrophage-mediated antitumor immune responses in multiple murine tumor models.

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