High throughput analysis of TCR-β rearrangement and gene expression in single T cells

Zhou, Dun; Srivastava, Rajneesh; Grummel, Verena; Cepok, Sabine; Hartung, Hans‐Peter; Hemmer, Bernhard

doi:10.1038/labinvest.3700381

Cited by 19 publications

(19 citation statements)

References 29 publications

(28 reference statements)

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“…41 In addition to the single V gene segment-specific primer approach, 2 degenerate primers covering 98% of the 55 V␤ region gene segments were used for analysis of TCR ␤-chains. 42 …”

Section: Tcr Analysismentioning

confidence: 99%

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies

Schuster¹,

Busch²,

Eppinger³

et al. 2007

Blood

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Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm re- IntroductionAlthough it is controversially discussed whether adaptive immune responses in tumor-bearing hosts play a role in controlling growth and recurrence of human tumors, 1,2 T cells can be converted to highly efficient killers of tumors. Donor leukocyte infusions (DLIs) are responsible for a graft-versus-leukemia effect (GvL) after allogeneic stem-cell transplantation (SCT), representing a therapeutic option with curative potential in different diseases at advanced stages. 3 Although GvL responses have also been shown for low-grade lymphomas, improvement of long-term survival after allogeneic transplantation has not been demonstrated in these patients. [4][5][6][7][8] This can be attributed primarily to the high treatmentassociated mortality due to transfer of T cells recognizing allogeneic minor histocompatibility (MHC) antigens, thereby causing potentially life-threatening graft-versus-host disease (GvHD). For broader applicability of this therapeutic option, it is therefore essential to reduce the risk of detrimental GvHD.One approach to gain a tumor-specific effect while significantly reducing the risk of GvHD is to generate allorestricted T cells with specificity for epitopes derived from tumor-associated antigens (TAAs). 9 Such allorestricted peptide-specific T cells may display high avidity toward MHC-presented TAAs, because they have not been negatively selected in the thymus. Moreover, they can be isolated by tetramers and cloned by limiting dilution to identify the specific TCR responsible for tumor-selective killing. 10 The isolation of a TCR with defined specificity for TAAs facilitates genetic TCR transfer into T-cell lines, 11-14 allowing major expansion of tumor-specific T cells.The choice of an appropriate target antigen is of fundamental importance for the success of an antitumor immunotherapeutic approach. So far, there is no universal antigen that serves as a target antigen in a broad range of malignancies. Many tumor-specific antigens are derived from aberrant expression of cell type-specific proteins and are expressed only in a restricted number of tumor types, limiting the tumors that can be treated. However, even the presence of a suitable antigen does not guarantee effective antigen recognition, because tumor cells evade immune recognition by down-regulation of the specific target or by inhibition of MHC class I-restricted antigen presentation. Thus, the level of epitope presentation in the context of classical or nonclassical MHC...

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“…41 In addition to the single V gene segment-specific primer approach, 2 degenerate primers covering 98% of the 55 V␤ region gene segments were used for analysis of TCR ␤-chains. 42 …”

Section: Tcr Analysismentioning

confidence: 99%

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies

Schuster¹,

Busch²,

Eppinger³

et al. 2007

Blood

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“…14 After an initial amplification with CH-specific and VH family-specific FR1 primers, 11 IGH cDNA was reamplified by seminested PCR with the same VH familyspecific primers and a JH consensus primer (5Јctga(ag)gagac(ag)gtgacc3Ј). Resulting products were sequenced.…”

Section: Single-cell Igh Variable Region Gene Sequencesmentioning

confidence: 99%

A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease

Bagnara

Kaufman

Calissano

et al. 2011

Blood

188

217

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Chronic lymphocytic leukemia (CLL) is IntroductionThe most common leukemia among white adults, B-cell chronic lymphocytic leukemia (CLL), remains incurable and its pathogenesis poorly defined. 1 Currently no system permits differentiation and long-term growth of CLL cells in vitro; therefore, an in vivo animal model that reproducibly supports engraftment and growth of human CLL cells would help elucidate key features of CLL cell biology and lead to better treatments.Previous attempts to engraft human CLL cells into mice have been hampered for 2 reasons. First, xenogeneic recipients were not sufficiently immune deficient to prevent human cell rejection. [2][3][4][5] Although Dü rig et al 5 successfully transferred CLL cells into nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mice, apparently the level of CLL cell growth was not sufficient to correlate kinetics with essential interactions with different cell subpopulations. Second, optimal engraftment and growth may have been impaired by the inability of a murine microenvironment to support CLL cells in vivo. Indeed, in vitro studies suggest at least 3 cell lineages are involved in CLL survival and growth: lymphoid (T cells 6,7 ), myeloid (monocytes and monocyte-derived nurse-like cells 8 ), and mesenchymal ("stromal cells" 9,10 ).To provide a more physiologic microenvironment for CLL cells within highly immune incompetent recipients, we introduced precursors of human hematopoietic and mesenchymal lineages into NOD/Shi-scid,␥c null (NSG) mice, a NOD/SCID-derived strain that lacks the IL-2 family common cytokine receptor gamma chain gene (␥c), rendering animals completely deficient in lymphocytes, including natural killer (NK) cells. We found activated autologous T cells were essential for leukemia cells to successfully engraft, survive, and proliferate in vivo and to recapitulate cardinal features of human CLL cells: kinetics, CD38 expression, and growth in secondary lymphoid tissues. This adoptive transfer model may facilitate the definition of leukemic and nonleukemic elements involved in the interactions and kinetics of CLL cells in patients. Methods Patients and samplesThe Institutional Review Board and the Institutional Animal Care and Utilization Committee of the North Shore-LIJ Health System sanctioned these studies. After obtaining informed consent, in accordance with the Submitted December 10, 2010; accepted February 17, 2011. Prepublished online as Blood First Edition paper, March 8, 2011 DOI 10.1182 DOI 10. /blood-2010 An Inside Blood analysis of this article appears at the front of this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on June 7, 2019. by guest www.bloodjournal.org From Declaration of Helsinki, we collected blood from 37 CLL patients for whom clinical information, la...

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“…Subfamily-specific PCR of the TCR ␣-chain was performed with 34 different V␣ primers (36). The TCR ␤-chain was analyzed by PCR with degenerated V␤ primers as previously described (37). Specifically amplified gene products were analyzed by DNA sequencing (Sequiserve).…”

Section: Tcr Analysismentioning

confidence: 99%

CMV-Specific TCR-Transgenic T Cells for Immunotherapy

Schub

Schuster

Hammerschmidt

et al. 2009

The Journal of Immunology

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Reactivation of CMV can cause severe disease after allogeneic hemopoietic stem cell transplantation. Adoptive T cell therapy was successfully used for patients who had received transplants from CMV-positive donors. However, patients with transplants from CMV-negative donors are at highest risk, and an adoptive therapy is missing because CMV-specific T cells are not available from such donors. To address this problem, we used retroviral transfer of CMV-specific TCR genes. We generated CMV-specific T cell clones of several HLA restrictions recognizing the endogenously processed Ag pp65. The genes of four TCRs were cloned and transferred to primary T cells from CMV-negative donors. These CMV-TCR-transgenic T cells displayed a broad spectrum of important effector functions (secretion of IFN-γ and IL-2, cytotoxicity, proliferation) in response to endogenously processed pp65 and could be enriched and expanded by strictly Ag-specific stimulation. Expansion of engineered T cells was accompanied by an increase in specific effector functions, indicating that the transferred specificity is stable and fully functional. Hence, we expect these CMV-TCR-transgenic T cells to be effective in controlling acute CMV disease and establishing an antiviral memory.

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High throughput analysis of TCR-β rearrangement and gene expression in single T cells

Cited by 19 publications

References 29 publications

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies

A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease

CMV-Specific TCR-Transgenic T Cells for Immunotherapy

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