Navin Varadarajan

University of Houston | U of H, UH · Department of Chemical & Biomolecular Engineering

Background Chimeric antigen receptor (CAR) T cell therapies show remarkable progress in treating liquid tumors, with a complete remission rate of over 57%.¹ Translating the success of CAR T cells to solid tumors will need an understanding of the key mechanisms responsible for the cytotoxicity of CAR T cells. The primary factors contributing to tumo...

Adoptive cell therapy (ACT) based on ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable antitumor responses even in heavily pretreated patients. However, only a subset of patients responds to ACT; efforts to identify correlates of response have focused on profiling the tumor or the TIL but rarely in an interactive...

Trogocytosis is an active process that transfers surface material from targeted to effector cells. Using multiple in vivo tumor models and clinical data, we report that chimeric antigen receptor (CAR) activation in natural killer (NK) cells promoted transfer of the CAR cognate antigen from tumor to NK cells, resulting in (1) lower tumor antigen den...

Extracellular vesicles (EVs) regulate the tumor microenvironment by facilitating transport of biomolecular cargo including RNA, protein, and metabolites. The biological effects of EV-mediated transport have been studied using supra-physiological concentrations of EVs, but the cells that are responsible for EV secretion and the mechanisms that suppo...

The in vivo persistence of adoptively transferred T cells is predictive of anti-tumor response. Identifying functional properties of infused T cells that lead to in vivo persistence and tumor eradication has remained elusive. We profiled CD19-specific CAR T cells that comprise the infusion products used to treat large B cell lymphomas using high-th...

Immunization programs against SARS-CoV-2 with commercial intramuscular (IM) vaccines prevent disease but not infections. The continued evolution of variants of concern (VOC) like Delta and Omicron has increased infections even in countries with high vaccination coverage. This is due to commercial vaccines being unable to prevent viral infection in...

Adoptive 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 potentia...

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 unders...

Respiratory viral infections, especially Influenza (endemic) or SARS-CoV-2 (pandemic since 2020), cause morbidity and mortality worldwide. Despite remarkable progress in the development and deployment of vaccines, they are clearly impacted by the rapid emergence of viral variants. The development of an off-the-shelf, effective, safe, and low-cost d...

Understanding immune response to infections and vaccines lags understanding humoral responses. While neutralizing antibody responses wane over time, T cells are instrumental in long-term immunity. We apply machine learning and time-lapse imaging microscopy in nanowell grids (TIMING) to study thousands of videos of T cells with specificity for SARS-...

Understanding the cellular immune response to infections, cancers and vaccines lags behind the investigation of humoral responses. While neutralizing antibody responses wane over time, the ability of T cells to recognize viruses including SARS-CoV-2 is instrumental to providing long-term immunity. Although T-cell receptor (TCR) repertoire screening...

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 hum...

Background Despite approval of immunotherapy for wide ranges of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response which is partially attributed to immunosuppression co-opted by tumor cells. However, it is challenging to utilize conventional methods to systematically evaluate the potential of tu...

Background Adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes (TIL) have shown durable responses in metastatic melanoma, yet these responses are unpredictable. Bioenergetics dictates the function and fate of adoptively transferred human T cells within the tumor microenvironment but the nature of metabolic competition leading to T-...

Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear lo...

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...

Despite remarkable progress in the development and authorization of vaccines against SARS-CoV-2, there is a need to validate vaccine platforms for broader application. The current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity in the nasal compartment, which is the first barrier that SARS-CoV-2 v...

Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response which may be attributed to immunosuppression co-opted by tumor cells. However, it is challenging to utilize conventional methods to systematically evaluate the potential of tumor intrinsic fa...

Background The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. Methods We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout ( CD8 and CD4 ) models and administered C...

Significance The mesoscopic p53-rich clusters that we discover represent a new class of biological condensate, distinct from amorphous and ordered aggregates and the dense liquids found with several physiologically active proteins. The demonstrated two-step mechanism of amyloid fibril nucleation, whereby the clusters host the nucleation of p53 amyl...

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...

Background Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the pot...

Microglia are ubiquitous CNS-resident macrophages that maintain homeostasis of neural tissues and protect them from pathogen attacks. Yet, their differentiation in different compartments remains elusive. We performed single cell RNA-seq (scRNA-seq) to compare microglial subtypes in the cortex and the spinal cord. A multi-way comparative analysis wa...

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly,...

Exosomes mediate intercellular communication in health and disease. Conventional assays are limited in profiling exosomes secreted from large populations of cells and are unsuitable for studying the functional consequences of individual cells exhibiting varying propensity for exosome secretion. In cancer, since exosomes can support the development...

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with fourth generation 'armored'...

Tools for tuning transcription in mammalian cells have broad applications, from basic biological discovery to human gene therapy. While precise control over target gene transcription via dosing with small molecules (drugs) is highly sought, the design of such inducible systems that meets required performance metrics poses a great challenge in mamma...

A safe and durable vaccine is urgently needed to tackle the COVID19 pandemic that has infected >15 million people and caused >620,000 deaths worldwide. As with other respiratory pathogens, the nasal compartment is the first barrier that needs to be breached by the SARS-CoV-2 virus before dissemination to the lung. Despite progress at remarkable spe...

Transgenic coexpression of a class I–restricted tumor antigen–specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4 ⁺ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4 ⁺ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function...

Immune checkpoint therapy has produced remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible SH2-containing (CIS) protein, a key negative regulator of interleukin (IL)-15 signaling, with chimeric antigen receptor (CAR...

Microglia are heterogeneous and ubiquitous CNS-resident macrophages that maintain homeostasis of neural tissues and protect them from pathogen attacks. Yet, their differentiation in different compartments remains elusive. We performed single cell RNA-seq (scRNA-seq) analysis to compare the transcriptomes of 32760 microglia in adult mouse (C57/Bl) b...

The oncogenic properties of mutant p53 have been ascribed to destabilization of the p53 conformation, followed by aggregation into insoluble fibrils. Here we combine immunofluorescent 3D confocal microscopy of breast cancer cells expressing the p53 mutant Arg248Gln (R248Q) with light scattering from solutions of the purified protein and molecular s...

Background The combination of the αPD-1 (nivolumab) and hypomethylating agent azacytidine demonstrated encouraging response rate and overall survival in relapsed/refractory acute myeloid leukemia (AML) patients, compared to azacytidine alone1 (NCT02397720). However, the percentage of the patient who achieved an IWG 2016 response to such therapy was...

We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn containing substrates....

Time lapse microscopy is essential for quantifying the dynamics of cells, subcellular organelles and biomolecules. Biologists use different fluorescent tags to label and track the subcellular structures and biomolecules within cells. However, not all of them are compatible with time lapse imaging, and the labeling itself can perturb the cells in un...

Decades of research on exosomes, nano-sized vesicles secreted by cells, have revealed novel roles of these vesicles in the formation of pre-metastasis niches that enhances the migration of tumor cells to those sites. Paradoxically, more recent work has suggested that these tumor-derived exosomes can also have an immunostimulatory role, depending on...

Metastatic breast cancer is the most dreadful malignant disease that accounts for the majority of cancer-related deaths worldwide among women. A number of studies have shown that the tumor microenvironment (TME) plays a crucial role in regulating metastasis. It is therefore imperative to understand the dynamic interactions between cancer cells and...

Metastatic breast cancer is the most dreadful malignant disease that accounts for the majority of cancer-related deaths worldwide among women. A number of studies have shown that the tumor microenvironment (TME) plays a crucial role in regulating metastasis. It is therefore imperative to understand the dynamic interactions between cancer cells and...

Decades of research on exosomes, nano-sized vesicles secreted by cells, have revealed novel roles of these vesicles in the formation of pre-metastasis niches that enhances the migration of tumor cells to those sites. Paradoxically, more recent work has suggested that these tumor-derived exosomes can also have an immunostimulatory role, depending on...

Novel single-cell profiling technologies have delineated the cellular and molecular landscapes that dominate the joints in rheumatoid arthritis and the skin and kidneys in systemic lupus erythematosus, shedding light on potential pathogenic mechanisms.

The challenge of predicting which patients with breast cancer will develop metastases leads to the overtreatment of patients with benign disease and to the inadequate treatment of aggressive cancers. Here, we report the development and testing of a microfluidic assay that quantifies the abundance and proliferative index of migratory cells in breast...

Over the last decade, both early diagnosis and targeted therapy have improved the survival rates of many cancer patients. Most recently, immunotherapy has revolutionized the treatment options for cancers such as melanoma. Unfortunately, a significant portion of cancers (including lung and breast cancers) do not respond to immunotherapy, and many of...

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 resoluti...

T cell-based therapies have shown promising results, but adoptive T cell therapy (ACT) for solid tumors like melanoma treatment has met limited success. The goal of this study was to identify attributes of T cell fitness important for optimal anti-tumor efficacy. This was accomplished by integrated profiling of expanded tumor infiltrating lymphocyt...

Objective To obtain the comprehensive transcriptome profile of human citrulline‐specific B cells from patients diagnosed with rheumatoid arthritis (RA). Methods Citrulline and hemagglutinin (HA) specific B cells were flow‐sorted using peptide‐streptavidin conjugates from peripheral blood of RA patients and healthy individuals respectively. Transcr...

B-acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19 have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that...

Immunotherapy relies on the reinvigoration of immune system to combat diseases and has transformed the landscape of cancer treatments. Clinical trials using immune checkpoint inhibitors (ICI), and adoptive transfer of genetically modified T cells have demonstrated durable remissions in subsets of cancer patients. A comprehensive understanding of th...

121 Background: Chimeric antigen receptor (CAR) T cells targeting CD19 have shown remarkable efficacy in treating relapsed B cell acute lymphoblastic leukemia (B-ALL). However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL...

List of reagents described in this manuscript. (XLSX)

The frequency of IFN-γ secreting NK cells under various cell density conditions. As the cell density in the nanowell increased, frequencies of IFN-γ secreting NK cells also increased as expected, however, there was no evidence for significant cooperation or synergistic effect. (XLSX)

Thin bottom nanowell arrays fabricated by spin-coating. Representative SEM images of top view (A) and side view (B) of nanowell arrays with indications of measured dimensions shown, and the summary of measurements listed (C). Scale bar = 100 μm. (TIF)

A representative example of an IFN-γ secreting NK cell. Green denotes IFN-γ (bead) and CD16 (cell). Time is displayed as hh: mm and movie is sped up 1800×. (MP4)

Distribution of functionalized beads and pre-stained NK cells in individual nanowell. Representative density matrix indicates the number of nanowells that contain 0–3 beads and 1–3 NK cells. Both numbers and frequency of wells are shown. (TIF)

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 attenuator) expression on transferred CD8(+) TIL was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTL...

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (Fc...

Although the contribution of B-cell derived autoreactive antibodies to rheumatoid arthritis (RA) has been studied extensively, the autoantibody-independent roles of B cells in the progression of the disease is not well-defined. Here we present the first comprehensive transcriptome profile of human autoreactive B cells in an autoimmune disease by pe...

Background Microsatellite instable (MSI) phenotype, as expression of mismatch-repair deficiency, characterizes tumors with high mutational load and consequently increased production of immunogenic neoantigens [1]. These characteristics have been correlated with susceptibility to immune checkpoint inhibitors (CKI) [2]. Nevertheless, MSI is rare (34%...

Adoptive cell therapy (ACT) based on the transfer of chimeric antigen receptor (CAR) T cells rendered specific for CD19 has demonstrated significant anti-tumor effects in patients with refractory CD19+ B-cell malignancies. There are however no biomarkers that can predict the potency of T-cell infusion products and thus the ability to enrich functio...

Poster presentation from the 31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer.

CD8(+) T cells develop increased sensitivity following Ag experience, and differences in sensitivity exist between T cell memory subsets. How differential TCR signaling between memory subsets contributes to sensitivity differences is unclear. We show in mouse effector memory T cells (TEM) that >50% of lymphocyte-specific protein tyrosine kinase (Lc...

Non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system has been successfully tested in humans to express a chimeric antigen receptor (CAR) to redirect T-cell specificity to CD19. This system has been modified to (i) improve the design of the CD19-specific CAR and (ii) reduce the time in culture to 14 days. Our previous...

Interferon-γ (IFNγ), a cytokine secreted by effector lymphocytes upon activation, is known to cause upregulation of MHC class I in tumor cells, which has been associated with natural killer (NK) cell inhibition. NK cells are very heterogeneous with respect to receptor expression and their function depends on an interplay between these inhibitory an...

CD8+ T cells develop increased sensitivity following antigen experience, and differences in sensitivity exist between T cell memory subsets. How differential T cell receptor (TCR) signaling between memory subsets contributes to sensitivity differences is unclear. We show that constitutive activity of lymphocyte-specific protein tyrosine kinase (Lck...

The origin and physiopathological relevance of catalytic antibodies is not well understood owing to the fact that catalytic antibodies have been studied in relatively small cohorts of patients with rare diseases and/or without systematic follow-up. In the present study, we have followed the evolution of the levels of catalytic IgG in a large cohort...

B cells are important in the pathogenesis of rheumatoid arthritis (RA). By utilizing a flow-cytometric assay to reliably detect the low frequency of autoreactive B cells (ABC) in the blood of RA patients, we performed RNA sequencing of citrulline-specific ABCs and compared them with hemaglutinin (HA) specific B cells. To the best of our knowledge,...

Microsystems and microfluidics technology have miniaturized complex analyses of biological systems and altered our scope of understanding cellular functions at the single-cell and single-molecule level. Studying intercellular communication facilitated either by secretion of peptides, proteins, or small molecules or by direct cell–cell contact has b...

Renal transplant is the treatment of choice for patients with terminal end-stage renal disease. We have previously identified low levels of catalytic IgG as a potential prognosis marker for chronic allograft rejection. The origin and physiopathological relevance of catalytic Abs is not well understood, owing to the fact that catalytic Abs have been...

T cells genetically modified to enforce expression of a chimeric antigen receptor (CAR) have shown considerable promise in clinical trials even in tumors refractory to all other treatment methods. In particular, the use of CAR T cells rendered specific for CD19 demonstrated significant anti-tumor effects in patients with CD19+ chronic lymphocytic l...

Iterative screening of expressed protein libraries using fluorescence-activated cell sorting (FACS) typically involves culturing the pooled clones after each sort. In these experiments, if cell viability is compromised by the sort conditions and/or expression of the target protein(s), rescue PCR provides an alternative to culturing but requires re-...

There is a need for effective automated methods for profiling dynamic cell-cell interactions with single-cell resolution from high-throughput time-lapse imaging data, especially, the interactions between immune effector cells and tumor cells in adoptive immunotherapy. Fluorescently labeled human T cells, Natural Killer cells (NK), and various targe...