Irfan Bandey | University of Houston (original) (raw)

Papers by Irfan Bandey

Research paper thumbnail of Overcoming migratory exhaustion: Expression of Matrix metalloproteinase-8 (MMP-8) promotes tumor infiltration and anti-tumor activity of CAR-T cells

Despite the encouraging success of chimeric antigen receptor (CAR) T-cell therapy in treating hem... more Despite the encouraging success of chimeric antigen receptor (CAR) T-cell therapy in treating hematological malignancies, the translation of adoptive cell therapies to solid tumors remains a challenge. Several studies have attributed the inability of tumor-infiltrating T cells to traffic to solid tumors, primarily to the presence of the extracellular matrix (ECM) and immunosuppressive environment of solid tumors. The ability of the transferred T cells to infiltrate the tumor is an essential prerequisite for anti-tumor activity. We show here that upon activation and expansion, T cells quickly lose their migratory capacity, leading to migratory exhaustion. At the molecular level, migratory exhaustion could be attributed to the downregulation of matrix metalloproteinase 8 (MMP8). To overcome this, we hypothesized that T cells genetically modified to secrete the mature form of matrix metalloproteinase 8 (mMMP8) would facilitate migration across matrix barriersin vitroandin vivo. We demo...

Research paper thumbnail of Integrated profiling of T cells and tumor cells demonstrates metabolic adaptation essential for melanoma cell therapy

Journal of Immunology, May 1, 2019

Research paper thumbnail of Multiomic dynamic single-cell profiling of CAR T cell populations associated with efficacy

The Journal of Immunology

T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown ef... more T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown efficacy for the treatment of hematologic malignancies. Although treatment with CAR T cell can result in high response rates, the properties of the cells that comprise the cellular infusion product, associated with clinical benefit are incompletely understood. We utilized a suite of high-throughput single-cell assays including single-cell RNA-sequencing (scRNA-seq); confocal microscopy; and Timelapse Imaging Microscopy In Nanowell Grids (TIMING). TIMING profiling of a cohort of 16 patients showed that persistent motility of T cells in the presence of tumor cells was associated with both serial killing capacity and polyfunctionality. Confocal microscopy on these same T cells revealed that persistent motility is linearly correlated with both mitochondrial volume and lysosomal number. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell proliferative...

Research paper thumbnail of CAR+ T cell anti-tumor efficacy revealed by multi-dimensional single-cell profiling

The Journal of Immunology

T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising... more T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising clinical responses in patients with certain hematologic malignancies, however, it is desirable to be able to enrich cells with enhanced anti-tumor efficacy prior to infusion. We utilized a suite of high-throughput technologies with single-cell resolution, including Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates cytokine profiling to reveal that persistent motility of CD19- specific CAR T cells is correlated to desirable polyfunctionality (elimination of tumor cells and cytokine secretion), contributing to anti-tumor effects. We implemented a marker-free Boyden chamber-based method to enrich CAR+ T cells with persistent motility (motile cell). Integration of transcriptomic profiling, immune phenotyping and metabolism demonstrated that motile cells are more naïve-like with higher oxidative metabolism and spare respiratory capacity. Our result also revealed that th...

Research paper thumbnail of Engineering non-cytotoxic delivery of proteins by T cells via fusion to NPC2

Engineering cellular therapeutics by programming T cells has great potential in immunology. The p... more Engineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells...

Research paper thumbnail of 402 T cell killing is facilitated by multiple cytotoxic pathways

Regular and Young Investigator Award Abstracts

Research paper thumbnail of Multidimensional single-cell analysis identifies a role for CD2-CD58 interactions in clinical antitumor T cell responses

Journal of Clinical investigation, 2022

Multidimensional single-cell analysis identifies a role for CD2-CD58 interactions in clinical ant... more Multidimensional single-cell analysis identifies a role for CD2-CD58 interactions in clinical antitumor T cell responses

Research paper thumbnail of T-cell potential for CD19-expressing malignancies revealed by multi-dimensional single-cell profiling

BioRxv, 2022

ABSTRACTAdoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-ce... more ABSTRACTAdoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-cell malignancies serves as a model for identifying subsets with superior clinical activity. We profiled the infusion products (IP) of 16 patients with large B-cell lymphoma (LBCL) using an integrated suite of single-cell assays to reveal the therapeutic potential of CD19-specific CAR+ T cells. Timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed migration (persistent motion for at least one body length), and migration was associated with serial killing capacity. In addition, confocal microscopy revealed that migration is linearly correlated with both mitochondrial volume and lysosomal volume; and scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering. A marker-free sorting strategy enriched T cells with migratory capacity and vali...

Research paper thumbnail of Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal for ImmunoTherapy of Cancer, 2021

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells ... more BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimula...

Research paper thumbnail of Glucuronoxylomannan in theCryptococcusspecies capsule as a target for CAR+T-cell therapy

The genusCryptococcuscomprises two major fungal species that cause clinical infections in humans:... more The genusCryptococcuscomprises two major fungal species that cause clinical infections in humans:C. gattiiandC. neoformans. To establish invasive human disease, inhaled Cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases ofCryptococcusinfection are reported worldwide, and the infection’s mortality rate ranges from 20% to 70%. HIV+/AIDS patients are highly susceptible toCryptococcusinfection. Therefore, we hypothesized that CD8+T cells could be redirected to target glucuronoxylomannan (GXM), a sugar present in theCryptococcusspecies capsule, via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR) for treatment of cryptococcosis. GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk, a CD28 transmembrane domain, and CD3-ς and CD28 signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their su...

Research paper thumbnail of Design and characterization of a salicylic acid-inducible gene expression system for Jurkat cells

Journal of Biotechnology, 2022

With continued progress in cell and gene therapies, there is an immediate need for exogenously tu... more With continued progress in cell and gene therapies, there is an immediate need for exogenously tunable gene expression systems with safe and predictable behavior in specific human cell types. Here, we demonstrate the ability of the salicylic acid (SA)-inducible MarR repressor protein from Escherichia coli to regulate target gene expression in a human T lymphocyte cell line. Two lentiviral vectors, one encoding an enhanced green fluorescent protein (EGFP) reporter cassette and the other a repressor cassette, were sequentially transduced into Jurkat cells, using fluorescence-activated cell sorting (FACS) to isolate stable Jurkat progeny. As a result, EGFP expression was repressed by MarR and was inducible upon the addition of SA (~1.3 fold). This represents the first example of functional expression of bacterial MarR in mammalian cells, and opens the possibility for further development of regulated, SA-tunable gene expression system for T-cells.

Research paper thumbnail of Rho-dependent transcription termination: more questions than answers

Journal of Microbiology, 2006

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an... more Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain ob...

Research paper thumbnail of Design and characterization of a salicylic acid inducible gene expression system for Jurkat cells

Journal of Biotechnology, 2022

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells

Biotechnology and Bioengineering

Research paper thumbnail of TIMING 2.0: High-throughput single-cell profiling of dynamic cell-cell interactions by time-lapse imaging microscopy in nanowell grids

Bioinformatics (Oxford, England), 2018

Automated profiling of cell-cell interactions from high-throughput time-lapse imaging microscopy ... more Automated profiling of cell-cell interactions from high-throughput time-lapse imaging microscopy data of cells in nanowell grids (TIMING) has led to fundamental insights into cell-cell interactions in immunotherapy. This application note aims to enable widespread adoption of TIMING by (i) enabling the computations to occur on a desktop computer with a graphical processing unit (GPU) instead of a server; (ii) enabling image acquisition and analysis to occur in the laboratory avoiding network data transfers to/from a server; and (iii) providing a comprehensive graphical user interface. On a desktop computer, TIMING 2.0 takes 5 sec/block/image frame, 4 times faster than our previous method on the same computer, and twice as fast as our previous method (TIMING) running on a Dell PowerEdge server. The cell segmentation accuracy (f-number = 0.993) is superior to our previous method (f-number 0.821). A graphical user interface provides the ability to inspect the video analysis results, mak...

Research paper thumbnail of Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal of Immunotherapy of Cancer, 2021

Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells... more Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.

Methods To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.

Results With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.

Conclusions Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

Research paper thumbnail of Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy

Research paper thumbnail of Glucuronoxylomannan in the Cryptococcus species capsule as a target for CAR + T-cell therapy

bioRxiv, 2020

The genus Cryptococcus comprises two major fungal species that cause clinical infections in 22 hu... more The genus Cryptococcus comprises two major fungal species that cause clinical infections in 22 humans: C. gattii and C. neoformans. To establish invasive human disease, inhaled 23

Research paper thumbnail of Bioimage informatics TIMING 2.0: high-throughput single-cell profiling of dynamic cell-cell interactions by time-lapse imaging microscopy in nanowell grids

Bioimage informatics, 2018

Motivation: Automated profiling of cell-cell interactions from high-throughput time-lapse imaging... more Motivation: Automated profiling of cell-cell interactions from high-throughput time-lapse imaging microscopy data of cells in nanowell grids (TIMING) has led to fundamental insights into cell-cell interactions in immunotherapy. This application note aims to enable widespread adoption of TIMING by (i) enabling the computations to occur on a desktop computer with a graphical processing unit instead of a server; (ii) enabling image acquisition and analysis to occur in the laboratory avoiding network data transfers to/from a server and (iii) providing a comprehensive graphical user interface. Results: On a desktop computer, TIMING 2.0 takes 5 s/block/image frame, four times faster than our previous method on the same computer, and twice as fast as our previous method (TIMING) running on a Dell PowerEdge server. The cell segmentation accuracy (f-number = 0.993) is superior to our previous method (f-number ¼ 0.821). A graphical user interface provides the ability to inspect the video analysis results, make corrective edits efficiently (one-click editing of an entire nanowell video sequence in 5-10 s) and display a summary of the cell killing efficacy measurements. Availability and implementation: Open source Python software (GPL v3 license), instruction manual , sample data and sample results are included with the Supplement (https://github.com/ RoysamLab/TIMING2).

Research paper thumbnail of Enzymatic immunomodulation of solid tumors and uses thereof

Patents, 2016

In some embodiments the present disclosure pertains to a method of activating an anti-tumor immu... more In some embodiments the present disclosure pertains to a method of activating an anti-tumor immune response for the treatment of a cancer. In some embodiments, such a method comprises detecting CD26 expression in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising adenosine deaminase. In some embodiments, the adenosine deaminase stimulates T cell proliferation and activates maturation of macrophages or dendritic cells. In some embodiments, the present disclosure pertains to a method for targeted reduction of adenosine or deoxyadenosine in a tumor microenvironment of a solid tumor.

Research paper thumbnail of Overcoming migratory exhaustion: Expression of Matrix metalloproteinase-8 (MMP-8) promotes tumor infiltration and anti-tumor activity of CAR-T cells

Despite the encouraging success of chimeric antigen receptor (CAR) T-cell therapy in treating hem... more Despite the encouraging success of chimeric antigen receptor (CAR) T-cell therapy in treating hematological malignancies, the translation of adoptive cell therapies to solid tumors remains a challenge. Several studies have attributed the inability of tumor-infiltrating T cells to traffic to solid tumors, primarily to the presence of the extracellular matrix (ECM) and immunosuppressive environment of solid tumors. The ability of the transferred T cells to infiltrate the tumor is an essential prerequisite for anti-tumor activity. We show here that upon activation and expansion, T cells quickly lose their migratory capacity, leading to migratory exhaustion. At the molecular level, migratory exhaustion could be attributed to the downregulation of matrix metalloproteinase 8 (MMP8). To overcome this, we hypothesized that T cells genetically modified to secrete the mature form of matrix metalloproteinase 8 (mMMP8) would facilitate migration across matrix barriersin vitroandin vivo. We demo...

Research paper thumbnail of Integrated profiling of T cells and tumor cells demonstrates metabolic adaptation essential for melanoma cell therapy

Journal of Immunology, May 1, 2019

Research paper thumbnail of Multiomic dynamic single-cell profiling of CAR T cell populations associated with efficacy

The Journal of Immunology

T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown ef... more T-cell therapy with specificity redirected through chimeric antigen receptors (CARs) has shown efficacy for the treatment of hematologic malignancies. Although treatment with CAR T cell can result in high response rates, the properties of the cells that comprise the cellular infusion product, associated with clinical benefit are incompletely understood. We utilized a suite of high-throughput single-cell assays including single-cell RNA-sequencing (scRNA-seq); confocal microscopy; and Timelapse Imaging Microscopy In Nanowell Grids (TIMING). TIMING profiling of a cohort of 16 patients showed that persistent motility of T cells in the presence of tumor cells was associated with both serial killing capacity and polyfunctionality. Confocal microscopy on these same T cells revealed that persistent motility is linearly correlated with both mitochondrial volume and lysosomal number. ScRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell proliferative...

Research paper thumbnail of CAR+ T cell anti-tumor efficacy revealed by multi-dimensional single-cell profiling

The Journal of Immunology

T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising... more T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising clinical responses in patients with certain hematologic malignancies, however, it is desirable to be able to enrich cells with enhanced anti-tumor efficacy prior to infusion. We utilized a suite of high-throughput technologies with single-cell resolution, including Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates cytokine profiling to reveal that persistent motility of CD19- specific CAR T cells is correlated to desirable polyfunctionality (elimination of tumor cells and cytokine secretion), contributing to anti-tumor effects. We implemented a marker-free Boyden chamber-based method to enrich CAR+ T cells with persistent motility (motile cell). Integration of transcriptomic profiling, immune phenotyping and metabolism demonstrated that motile cells are more naïve-like with higher oxidative metabolism and spare respiratory capacity. Our result also revealed that th...

Research paper thumbnail of Engineering non-cytotoxic delivery of proteins by T cells via fusion to NPC2

Engineering cellular therapeutics by programming T cells has great potential in immunology. The p... more Engineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells...

Research paper thumbnail of 402 T cell killing is facilitated by multiple cytotoxic pathways

Regular and Young Investigator Award Abstracts

Research paper thumbnail of Multidimensional single-cell analysis identifies a role for CD2-CD58 interactions in clinical antitumor T cell responses

Journal of Clinical investigation, 2022

Multidimensional single-cell analysis identifies a role for CD2-CD58 interactions in clinical ant... more Multidimensional single-cell analysis identifies a role for CD2-CD58 interactions in clinical antitumor T cell responses

Research paper thumbnail of T-cell potential for CD19-expressing malignancies revealed by multi-dimensional single-cell profiling

BioRxv, 2022

ABSTRACTAdoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-ce... more ABSTRACTAdoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) for B-cell malignancies serves as a model for identifying subsets with superior clinical activity. We profiled the infusion products (IP) of 16 patients with large B-cell lymphoma (LBCL) using an integrated suite of single-cell assays to reveal the therapeutic potential of CD19-specific CAR+ T cells. Timelapse imaging microscopy in nanowell grids (TIMING) profiling revealed that T cells from responders showed migration (persistent motion for at least one body length), and migration was associated with serial killing capacity. In addition, confocal microscopy revealed that migration is linearly correlated with both mitochondrial volume and lysosomal volume; and scRNA-seq demonstrated that T cells from responders were enriched in pathways related to T-cell killing, migration and actin cytoskeleton, and TCR clustering. A marker-free sorting strategy enriched T cells with migratory capacity and vali...

Research paper thumbnail of Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal for ImmunoTherapy of Cancer, 2021

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells ... more BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimula...

Research paper thumbnail of Glucuronoxylomannan in theCryptococcusspecies capsule as a target for CAR+T-cell therapy

The genusCryptococcuscomprises two major fungal species that cause clinical infections in humans:... more The genusCryptococcuscomprises two major fungal species that cause clinical infections in humans:C. gattiiandC. neoformans. To establish invasive human disease, inhaled Cryptococci must penetrate the lung tissue and reproduce. Each year, about 1 million cases ofCryptococcusinfection are reported worldwide, and the infection’s mortality rate ranges from 20% to 70%. HIV+/AIDS patients are highly susceptible toCryptococcusinfection. Therefore, we hypothesized that CD8+T cells could be redirected to target glucuronoxylomannan (GXM), a sugar present in theCryptococcusspecies capsule, via expression of a GXM-specific chimeric antigen receptor (GXMR-CAR) for treatment of cryptococcosis. GXMR-CAR has an anti-GXM single-chain variable fragment followed by an IgG4 stalk, a CD28 transmembrane domain, and CD3-ς and CD28 signaling domains. After lentiviral transduction of human T cells with the GXMR-CAR construct, flow cytometry demonstrated that 82.4% of the cells expressed GXMR-CAR on their su...

Research paper thumbnail of Design and characterization of a salicylic acid-inducible gene expression system for Jurkat cells

Journal of Biotechnology, 2022

With continued progress in cell and gene therapies, there is an immediate need for exogenously tu... more With continued progress in cell and gene therapies, there is an immediate need for exogenously tunable gene expression systems with safe and predictable behavior in specific human cell types. Here, we demonstrate the ability of the salicylic acid (SA)-inducible MarR repressor protein from Escherichia coli to regulate target gene expression in a human T lymphocyte cell line. Two lentiviral vectors, one encoding an enhanced green fluorescent protein (EGFP) reporter cassette and the other a repressor cassette, were sequentially transduced into Jurkat cells, using fluorescence-activated cell sorting (FACS) to isolate stable Jurkat progeny. As a result, EGFP expression was repressed by MarR and was inducible upon the addition of SA (~1.3 fold). This represents the first example of functional expression of bacterial MarR in mammalian cells, and opens the possibility for further development of regulated, SA-tunable gene expression system for T-cells.

Research paper thumbnail of Rho-dependent transcription termination: more questions than answers

Journal of Microbiology, 2006

Escherichia coli protein Rho is required for the factor-dependent transcription termination by an... more Escherichia coli protein Rho is required for the factor-dependent transcription termination by an RNA polymerase and is essential for the viability of the cell. It is a homohexameric protein that recognizes and binds preferably to C-rich sites in the transcribed RNA. Once bound to RNA, it utilizes RNA-dependent ATPase activity and subsequently ATPase-dependent helicase activity to unwind RNA-DNA hybrids and release RNA from a transcribing elongation complex. Studies over the past few decades have highlighted Rho as a molecule and have revealed much of its mechanistic properties. The recently solved crystal structure could explain many of its physiological functions in terms of its structure. Despite all these efforts, many of the fundamental questions pertaining to Rho recognition sites, differential ATPase activity in response to different RNAs, translocation of Rho along the nascent transcript, interactions with elongation complex and finally unwinding and release of RNA remain ob...

Research paper thumbnail of Design and characterization of a salicylic acid inducible gene expression system for Jurkat cells

Journal of Biotechnology, 2022

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells

Biotechnology and Bioengineering

Research paper thumbnail of TIMING 2.0: High-throughput single-cell profiling of dynamic cell-cell interactions by time-lapse imaging microscopy in nanowell grids

Bioinformatics (Oxford, England), 2018

Automated profiling of cell-cell interactions from high-throughput time-lapse imaging microscopy ... more Automated profiling of cell-cell interactions from high-throughput time-lapse imaging microscopy data of cells in nanowell grids (TIMING) has led to fundamental insights into cell-cell interactions in immunotherapy. This application note aims to enable widespread adoption of TIMING by (i) enabling the computations to occur on a desktop computer with a graphical processing unit (GPU) instead of a server; (ii) enabling image acquisition and analysis to occur in the laboratory avoiding network data transfers to/from a server; and (iii) providing a comprehensive graphical user interface. On a desktop computer, TIMING 2.0 takes 5 sec/block/image frame, 4 times faster than our previous method on the same computer, and twice as fast as our previous method (TIMING) running on a Dell PowerEdge server. The cell segmentation accuracy (f-number = 0.993) is superior to our previous method (f-number 0.821). A graphical user interface provides the ability to inspect the video analysis results, mak...

Research paper thumbnail of Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal of Immunotherapy of Cancer, 2021

Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells... more Background Adoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.

Methods To identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.

Results With the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.

Conclusions Broadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

Research paper thumbnail of Glucuronoxylomannan in the Cryptococcus species capsule as a target for Chimeric Antigen Receptor T-cell therapy

Research paper thumbnail of Glucuronoxylomannan in the Cryptococcus species capsule as a target for CAR + T-cell therapy

bioRxiv, 2020

The genus Cryptococcus comprises two major fungal species that cause clinical infections in 22 hu... more The genus Cryptococcus comprises two major fungal species that cause clinical infections in 22 humans: C. gattii and C. neoformans. To establish invasive human disease, inhaled 23

Research paper thumbnail of Bioimage informatics TIMING 2.0: high-throughput single-cell profiling of dynamic cell-cell interactions by time-lapse imaging microscopy in nanowell grids

Bioimage informatics, 2018

Motivation: Automated profiling of cell-cell interactions from high-throughput time-lapse imaging... more Motivation: Automated profiling of cell-cell interactions from high-throughput time-lapse imaging microscopy data of cells in nanowell grids (TIMING) has led to fundamental insights into cell-cell interactions in immunotherapy. This application note aims to enable widespread adoption of TIMING by (i) enabling the computations to occur on a desktop computer with a graphical processing unit instead of a server; (ii) enabling image acquisition and analysis to occur in the laboratory avoiding network data transfers to/from a server and (iii) providing a comprehensive graphical user interface. Results: On a desktop computer, TIMING 2.0 takes 5 s/block/image frame, four times faster than our previous method on the same computer, and twice as fast as our previous method (TIMING) running on a Dell PowerEdge server. The cell segmentation accuracy (f-number = 0.993) is superior to our previous method (f-number ¼ 0.821). A graphical user interface provides the ability to inspect the video analysis results, make corrective edits efficiently (one-click editing of an entire nanowell video sequence in 5-10 s) and display a summary of the cell killing efficacy measurements. Availability and implementation: Open source Python software (GPL v3 license), instruction manual , sample data and sample results are included with the Supplement (https://github.com/ RoysamLab/TIMING2).

Research paper thumbnail of Enzymatic immunomodulation of solid tumors and uses thereof

Patents, 2016

In some embodiments the present disclosure pertains to a method of activating an anti-tumor immu... more In some embodiments the present disclosure pertains to a method of activating an anti-tumor immune response for the treatment of a cancer. In some embodiments, such a method comprises detecting CD26 expression in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising adenosine deaminase. In some embodiments, the adenosine deaminase stimulates T cell proliferation and activates maturation of macrophages or dendritic cells. In some embodiments, the present disclosure pertains to a method for targeted reduction of adenosine or deoxyadenosine in a tumor microenvironment of a solid tumor.