Discovery Lab

Center for Immunotherapy & Precision Immuno-Oncology

About

Researchers in the Center for Immunotherapy & Precision Immuno-Oncology (CITI) Discovery Lab are dedicated to developing the latest state-of-the-art molecular and genomic discovery platforms for translational immunology. We innovate, develop and utilize large scale technologies to facilitate and accomplish discovery in the immunotherapy and immunogenomics space.

We collaborate with Cleveland Clinic investigators to support innovative high-throughput research, clinical trials and immunomonitoring assays. We are engaged with clinical departments to develop correlates for clinical trials and partner with industry collaborators on research and development efforts.

We utilize high-throughput genetic screening assays, cutting edge immunological assays, and genomics technologies to advance patient centric immunotherapy research. The scope of our immunotherapy research involves but is not limited to the fields of cancer, infectious diseases, metabolic diseases, transplantation, neurologic diseases, autoimmune disorders and vascular diseases.

The integrated CITI computational team works hand in hand with the Discovery Lab to provide world-class computational analysis and interpretation for projects.

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Who We Are

Raghvendra Srivastava, M.Sc, M.Tech, PhD

Project Staff & Discovery Lab Head
srivasr@ccf.org

Saurabh Srivastava

Lead Research Technologist
srivass3@ccf.org

Hana Husic

Bioinformatics Technician
husich@ccf.org

Nicole Osborne

Research Technician
osbornn@ccf.org

Shadi Swaidani, MD, PhD

Project Staff
swaidas@ccf.org

Publications

Raghvendra M. Srivastava Publications:


  1. Krishna C, Dinatale RG, Kuo F, Srivastava RM, , Vuong L, Chowell D, Gupta S, Purohit TA, Liu M, Kansler E, Nixon BG, Chen Y-B, Makarov V, Blum KA, Attalla K, Weng S, Salmans ML, Golkaram M, Liu L, Zhang S, Vijayraghavan R, Pawlowski T, Reuter V, Carlo MI, Voss MH, Coleman J, Russo P, Motzer RJ, Li MO, Leslie CS, Chan TA, Hakimi AA.

    2. Single cell sequencing links multiregional immune landscapes and tissue resident T cells in clear cell renal cell carcinoma to tumor topology and therapy efficacy.
     Cancer Cell. 2021. Apr 12; S1535-6108(21)00165-3. DOI: 10.1016/j.ccell.2021.03.007.

  2. Samstein RM, Krishna C, Ma Xiaoxiao, Xin P, Lee K-W, Makarov V, Kuo F, Chung J, Srivastava RM, Purohit TA, Chung J, Hoen DR, Mandal R, Setton J, Wu W, Shah R, Qeriqi B, Chang Q, Kendall S, Braunstein L, Weigelt B, Albornoz, PBC , Morris LGT, Mandelker DL, Reis-Filho JS, Stanchina E, Powell SN, Chan TA, Riaz  N.

    3. Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.
     Nature Cancer. 2020. Nov.16. https://doi.org/10.1038/s43018-020-00139-8.

  3. Yang W*, Lee K-W*, Srivastava RM*, Kuo F, Krishna C, Chowell D, Makarov V, Dalin MG, Wexler L, Ghossein R, Katabi N, Nadeem Z, Cohen MA, Tian SK, Robine N, Arora K, Geiger H, Bouvier N, Huberman K, Vanness K,  Havel JJ, Samstein RM, Mandal R, Tepe R, Ganly I, Ho AL, Riaz N, Wong RJ, Shukla N, Chan TA, Morris LGT.

    4.  Immunogenic neoantigens derived from gene fusions stimulate T cell responses
     Nature Medicine. 2019. May; 25 (5): 767-775. (*Joint 1st author)

    Nature Review Cancer research highlight: Hindson J. Gene-fusion neoantigens stimulate T cells. https://doi.org/10.1038/s41568-019-0160-6.

    Cancer Discovery research watch: Oncogenic gene fusion derived neoantigens elicit specific T- cell responses. DOI: 10.1158/2159-8290.CD-RW2019-065.

  4. Mandal R, Samstein RM, Lee Ken-Wing, Havel JJ, Wang H, Krishna C, Sabio EY, Makarov V, Kuo F, Blecua P, Ramaswamy AT, Durham JN, Bartlett B, Xiaoxiao Ma, Srivastava RM, Middha S, Zehir A, Hechtman JF, Morris LGT, Weinhold N, Riaz N, Le DT, Diaz Jr. LA, Chan TA.

    5. Genetic diversity of tumors with mismatch repair deficiency influences tumor evolution and response to PD-1 blockade.
    Science. 2019. May 3,364 (6439), 485-491.

  5. Srivastava RM,  Purohit TA, Chan TA ( Review)

    6. Diverse neoantigens and the Development of Cancer therapies.
    Seminars in Radiation Oncology. 2020. 30(2). 113-128

  6. Bessell CA, Isser A, Havel JJ, Lee S, Bell D, Hickey JW, Chaisawangwong W, Bieler JG, Srivastava RM, Kuo F, Purohit TA, Zhou R, Chan TA, Schneck JP.

    7.  Commensal bacteria stimulate anti-tumor responses via T cell cross-reactivity
    JCI Insight. 2020. Apr 23; 5(8): e135597.

  7. Srivastava RM, Marincola FM, Shanker A.

    8.  Editorial article: Lymphocyte functional crosstalk and regulation.
     Frontiers in Immunology. 2019. Dec 10; 10: 2916. doi: 10.3389/fimmu.2019.02916 

  8. Shanker A, Srivastava RM, Marincola FM, eds.

    9. eBook: Lymphocyte functional crosstalk and regulation. Lausanne: Frontiers Media SA.
    Frontiers in Immunology. 2020. doi:10.3389/978-2-88963-414-9

  9. Liu Z, Shayan G, McMichael E, Li J, Chen K, Srivastava RM, Kane L, Lu B, Ferris RL.

    10. Novel effector phenotype of Tim3+ regulatory T cells leads to enhanced suppressive function in head and neck cancer patients.
    Clinical Cancer Research. 2018. Sep 15; 24(18):4529-4538.

  10. Lu S, Concha-Benavente F, Shayan G, Srivastava RM, Gibson SP, Wang L, Gooding WE, Ferris RL.

    11. STING activation enhances cetuximab-mediated NK cell activation and DC maturation and correlates with HPV+ status in head and neck cancer.
    Oral Oncology. 2018.78:186-193.

  11. Shayan G, Kansy BA, Gibson SP, Srivastava RM, Bryan JK, Bauman JE, Ohr J, Kim S, Duvvuri U, Clump DA, Heron D, Johnson JT, Hershberg RM, Ferris R L.

    12. A Phase 1b study of immune biomarker modulation with neoadjuvant cetuximab and TLR8 stimulation in head and neck cancer to overcome suppressive myeloid signals.
    Clinical Cancer Research. 2018. January 2, 24(1):62-72.

  12. Kansy BA, Concha-Benavente F, Srivastava RM, Jie HB, Shayan G, Lei Y, Moskovitz J, Moy J, Li J, Brandau S, Lang S, Schmitt NC, Freeman GJ, Gooding WE, Clump DA, Ferris RL.

    13. PD-1 status in CD8+ T cells associates with survival and anti-PD-1 therapeutic outcomes in head and neck cancer.
    Cancer Research. 2017 Nov 15; 77(22): 6353-6364.

  13. Kikuchi M, Clump DA, Srivastava RM, Sun L, Zeng D, Diaz-Perez JA, Anderson CJ, Edward WB, Ferris RL.

    14. Preclinical immunoPET/CT imaging using Zr-89-labeled anti-PD-L1 monoclonal antibody for assessing radiation-induced PD-L1 upregulation in head and neck cancer and melanoma.
    Onco-Immunology. 2017. May 19; 6(7):6 1329071.

  14. Mazorra Z, Lavastida A, Concha-Benavente F, Valdés A, Srivastava RM, García-Bates T M, Hechavarría E,González Z, González A, Lugiollo M, Cuevas I, Frómeta C, Mestre BF, Barroso MC, Crombet T, Ferris RL.

    15. Nimotuzumab induces NK cell activation, cytotoxicity, dendritic cell maturation and   expansion of EGFR-specific T cells in head and neck cancer patients.
    Frontiers in Pharmacology. 2017. Jun 19;8:382.

  15. Jie H-B, Srivastava RM, Argiris A, Bauman J E, Kane L P, Ferris R L.

    16. Increased PD-1+ and TIM-3+ TIL during cetuximab therapy inversely correlate with response in head and neck cancer patients.
    Cancer Immunology Research. 2017; 5(5): 408-416.

  16.  Shayan G, Srivastava RM, Li J, Schmitt N, Kane LP, Ferris RL.

    17. Adaptive resistance to anti-PD-1 therapy by Tim-3 upregulation is mediated by PI3K-Akt pathway in head and neck cancer
    Onco-Immunology. 2016 Dec 23; 6(1):e1261779.

  17. Srivastava RM, Trivedi S, Concha-Benavente F, Gibson SP, Reeder C, Ferrone S, Ferris RL.

    18. CD137 stimulation enhances cetuximab induced natural killer (NK): dendritic cell (DC) priming of anti-tumor T cell immunity in head and neck cancer patients.
    Clinical Cancer Research. 2017;23(3):707-716. 

  18. Concha-Benavente F, Srivastava RM, Trivedi S, Yu Lei, Chandran U, Seethala RR, Freeman G, and Ferris RL.

    19. JAK2/STAT1 is a common mechanism of EGFR- and IFNγ-induced PD-L1 expression in  both HPV positive and negative head and neck cancer.
    Cancer Research. 2016. 76(5):1031-43.

  19. Trivedi S, Srivastava R M, Concha-Benevente F, Ferrone S, Gracia-Bates TM, Jing Li,

    20. Ferris RL. 
    Anti-EGFR targeted monoclonal antibody isotype influences anti-tumor cellular immunity in head and neck cancer patients.
    Clinical Cancer Research. 2016. DOI: 10.1158/1078-0432.CCR-15-2971.

  20. Concha-Benavente F, Srivastava RM, Ferrone S, Ferris RL. (Review)

    21. Immunological and clinical significance of HLA class I antigen processing machinery component defects in malignant cells.
    Oral Oncology. 2016; 58:52-8.

  21. Srivastava RM, Trivedi S, Concha-Benevente F, Jie HB, Wang L, Seethala RR, Branstetter FF, Ferrone S, Ferris RL.

    22. STAT1 Induced HLA class I Upregulation Enhances Immunogenicity and Clinical Response to anti-EGFR mAb Cetuximab Therapy in HNC Patients.
    Cancer Immunology Research.2015; 3(8):936-45.

  22.  Li J, Srivastava RM, Ettyreddy A, Ferris RL.

    23. Cetuximab ameliorates suppressive phenotypes of myeloid antigen presenting cells in head and neck cancer patients.
    Journal for ImmunoTherapy of Cancer. 2015 Nov 17;3:54.

  23. Jie HB, Schuler P, Lee S C, Srivastava RM, Argiris A, Ferrone S, Whiteside TL, Ferris RL.

    24. CTLA-4+ Regulatory T Cells are Increased in Cetuximab Treated Head and Neck Cancer Patients, Suppress NK Cell Cytotoxicity and Correlate with Poor Prognosis.
    Cancer Research. 2015; 75(11):2200-10.

  24. Srivastava RM, Lee SC, Andrade Filho PA, Lord CA, Jie HB, Davidson C, López-Albaitero A, Gibson SP, Gooding WE, Ferrone S, Ferris RL.

    25. Cetuximab-activated natural killer (NK) and dendritic cells (DC) collaborate to trigger tumor antigen-specific T cell immunity in head and neck cancer patients.
    Clinical Cancer Research. 2013 Apr 1; 19(7):1858-72.

  25. Trivedi S, Concha-Benavente F, Srivastava RM, Jie HB, Gibson SP and Ferris RL. (Review)

    26. Immune biomarkers of anti-EGFR monoclonal antibody therapy.
    Annals of oncology. 2015;26(1):40-7.

  26.  Srivastava RM*, Srivastava S*, Singh M, Bajpai VK, Ghosh JK.

    27. Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide.
    Journal of Biological Chemistry. 2012;287(3):1980-95. (*Joint 1st author)

  27.  Leibowitz MS, Srivastava RM, Andrade Filho PA, Egloff AM, Wang L, Seethala RR, Ferrone S, Ferris RL.

    28. SHP2 is overexpressed and inhibits pSTAT1-mediated APM component expression, T-cell attracting chemokine secretion, and CTL recognition in head and neck cancer cells.
    Clinical Cancer Research. 2013; 19(4):798-808.

  28. Jie H B, Gildener-leapman N, Lee J, Srivastava RM, Gibson SP, Whiteside TL, Ferris R L.

    29. Intratumoral regulatory T cells upregulate immunosuppressive molecules in head and neck cancer.
    British Journal of cancer. 2013. Nov 12;109(10):2629-35.

  29. Concha-Benavente F, Srivastava RM, Ferrone S, Ferris RL. (Review)

    30. EGFR-mediated tumor immunoescape: The imbalance between phosphorylated STAT1 and phosphorylated STAT3.
    Oncoimmunology. 2013;2(12):e27215.

  30. Lee SC*, Srivastava RM*, López-Albaitero A, Ferrone S, Ferris RL.

    31. Natural killer (NK): dendritic cell (DC) cross talk induced by therapeutic monoclonal antibody triggers tumor antigen-specific T cell immunity.
    Immunological Research. 2011;50 (2-3):248-54. (*Joint 1st author)

  31. Srivastava RM, Singh S, Dubey SK, Misra K, Khar A. (Review)

    32. Immunomodulatory and therapeutic activity of curcumin.
    International  Immunopharmacology. 2011 Mar;11(3):331-41.

  32. Pandey BK, Ahmad A, Asthana N, Azmi S, Srivastava RM, Srivastava S, Verma R, Vishwakarma AL, Ghosh JK.

    33. Cell-selective lysis by novel analogues of melittin against human red blood cells and Escherichia coli.
    Biochemistry. 2010;49(36):7920-9.

  33. Ahmad A, Asthana N, Azmi S, Srivastava RM, Pandey BK, Yadav V, Ghosh JK.

    34. Structure-function study of cathelicidin-derived bovine antimicrobial peptide BMAP-28: design of its cell-selective analogs by amino acid substitutions in the heptad repeat sequences.
    Biochimica et Biophys Acta. 2009;1788(11):2411-20.

  34. Ahmad A, Azmi S, Srivastava RM, Srivastava S, Pandey BK, Saxena R, Bajpai VK, Ghosh JK.

    35. Design of nontoxic analogues of cathelicidin-derived bovine antimicrobial peptide BMAP-27: the role of leucine as well as phenylalanine zipper sequences in determining its toxicity.
    Biochemistry. 2009;48(46):10905-17.

  35. Srivastava RM, Khar A. (Review)

    36. Dendritic cells and their receptors in antitumor immune response.
    Current Molecular Medicine. 2009;9(6):708-24.

  36. Srivastava RM, Varalakshmi Ch, Khar A.

    37. The ischemia-responsive protein 94 (irp94) activates dendritic cells through NK cell receptor protein-2/NK group
    2 member D (NKR-P2/NKG2D) leading to their maturation.
    The Journal of Immunology. 2008; 180(2):1117-30.

  37. Varalakshmi Ch, Ali AM, Pardhasaradhi BV, Srivastava RM, Singh S, Khar A.

    38. Immunomodulatory effects of curcumin: in-vivo.
    International  Immunopharmacology. 2008;8(5):688-700.

  38. Srivastava RM, Varalakshmi Ch, Khar A.

    39. Cross-linking a mAb to NKR-P2/NKG2D on dendritic cells induces their activation and maturation leading to enhanced anti-tumor immune response.

    International  Immunology. 2007;19(5):591-607.

Technologies

Interrogation of cancer cells, stromal cells and immune cells in the tumor microenvironment and blood is crucial to developing novel therapeutic strategies and resolve limitations in existing cancer therapies.

The 10X genomics platform is a facile and powerful platform to discover novel immune subsets and transform our understanding the tumor microenvironment. We apply this high throughput state-of-the-art 10X genomics technology to interrogate cellular heterogeneity, intrinsic variation in TCR repertoire, define cellular states, characterize HLA genotype, and examine functional dynamics across cell types in many disease states.

In the Discovery Lab, we utilize a variety of cancer tissues, core biopsies, and blood to prepare genomic libraries for gene expression, V(D)J (TCR and Ig). The Discovery Lab uses an Agilent Bioanalyzer system for genomic library quality control, and DNA/RNA quantitation.

We are using combined cellular indexing of transcriptomes and epitope by sequencing ( CITE-seq), which provides additional ability to resolve proteome and transcriptome in greater detail.

We utilize the MiSeq Illumina sequencing system perform next generation DNA sequencing. It integrates cluster generation, sequencing and data analysis. MiSeq workflow allows upto 30 million reads in single run. The MiSeq system also combines proven sequencing by synthesis (SBS) technology with a workflow from DNA to data analysis in few hours. The sequencing data has broad range of applications in immunotherapy.

This sequencing platform allows flexibility and quick access for targeted re-resequencing, 16S metagenomics, small genome sequencing.

We perform TCR sequencing and HLA typing for collaborative projects or as a Core service.

Multicolor flow cytometry is one of the most robust and powerful technologies for immunology. It simultaneously utilizes a diverse range of fluorescent markers to discover cellular subsets, regulatory proteins, and detects changes in cell surface and intracellular proteins in diverse range of cell types.

We utilize a new 5 LASER BD FACSymphonyTM, machine to detect up to 23 fluorescent color simultaneously across millions of cells. This resides in the Immunomonitoring laboratory of CITI. Discovery Lab collaborations center work that required non-standard panels not already offered by the Immunomoitoring laboratory.

Our FACSymphonyTM, is also equipped with a high-throughput sampler to efficiently run samples in 96 well plates.

We routinely utilize this technology to interrogate a range of cells, including rare neoantigen specific T cells, and develop novel immunomonitoring assays. We measure cell activation states in in-vitro experiments, and perform massive immuno-profiling of immuno-therapy treated cancer patients.

Cancer neoantigen-specific T cells play a dominant role in determining the efficacy of PD-1-, CTLA-4- and PD-L1-blocking therapeutic antibodies in several cancers. Due to neo-antigens’ potential to develop robust and specific T cell responses, they are considered high value targets of personalized vaccine candidate. In the Discovery Lab, we screen and validate neoantigens.

We utilize a tandem minigene approach to clone and prepare transcripts of all detected mutations in patients. We transfect RNA containing mutations into patients’ dendritic cells and detect the activation of patient’s mutation specific T cells in autologous settings. We also utilize mass spectrometry methods to detect and validate antigen specific T cells.

In several immunological diseases, cellular function is defined by cellular gene expression and by the location of immune cells within the tissue architecture. Spatial transcriptomics reveals the location of active genes in immune cells found in specific regions and disease tissue. In addition to single-cell-based RNA profiling in spatial transcriptomics, we interrogate simultaneously the expression of gene sets in distinct regions of clinical specimens (in development).

CD8 cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an important role in the control of microbial infections and tumors. Cytolysis of antigen-specific 51Cr-labeled targets in the standard assay help to quantify, using traditional gamma counters, 51Cr released into the supernatant and collected from CTL cultures.

Our lytic assay system reduces the amount of dry and mixed radioactive waste generated while using the same instrument for gamma- and beta-emitting isotopes. We utilize Lumaplates to substantially reduce radioactive waste for environmental concern. This high-throughput and safe assay methodology enables us to perform most critical immunological test in our cancer neoantigen vaccine discovery program.

Immunotherapy modulates immune cell types and several soluble factors. The immune cell activation states, cross-talk and cellular proliferation is highly dependent on soluble factors.

We utilize the robust Luminex™ 200™ Instrument System to analyze soluble factors in clinical specimens. The Luminex 200 Instrument System also sets the standard for multiplexing, providing the ability to perform up to 100 different tests in a single reaction volume on a flow cytometry-based platform. The Luminex 200 system is also compatible with Procarta Plex multiplex immunoassays and QuantiGene Plex multiplex gene expression assays.

The open architecture of Luminex xMAP technology uses imaging, microspheres, digital signal processing and traditional chemistry, combining proven technologies in a unique way.

Our Luminex platform utilizes small sample input and enables fast, reproducible results from favorable kinetics of the liquid bead array approach.

Our Luminex provides broad coverage of applications, including protein expression and gene expression profiling in clinical trial specimens and can reveal novel biomarkers to understand disease scenario and therapeutic outcomes.