T-Cell Immunity to Two HLA-A2-Restricted Self-Determinants of Cyclin E May Contribute to Remission After Stem Cell Transplantation.

Abstract Abstract 686 Cyclin E1 (CCNE1) and cyclin E2 (CCNE2) are tightly regulated cell cycle genes in normal cells but are over-expressed and constitutively active in breast cancer and in the majority of hematological malignances. To validate CCNE as a potential target antigen for T-cells in leukemia, we first confirmed aberrant CCNE1 and CCNE2 protein in PBMC from 26 (93%) of 28 patients (CML = 16; AML = 7; ALL =2; NHL = 3) by Western Blot compared to 4 (33%) of 12 healthy controls (p < 0.0005). Next, we screened the sequences of CCNE1 and CCNE2 for HLA-A*0201 binding motifs and identified a pair of homologous nonameric peptides with highest predicted binding to HLA-A*0201 using an NCBI algorithm. The peptides, denoted CCNE1M (144ILLDWLMEV152) and CCNE2L (144ILLDWLLEV152), differed at P7 (M or L), and both differed from mouse sequence at P1 (V). Synthetic mouse and human peptides were used to confirm high affinity HLA-A2 binding on T2 cells by FACS analysis and peptide-pulsed T2 were used to elicit peptide-specific CTLs from healthy HLA-A2+ PBMC in vitro. CCNE1M-CTL lines specifically lysed both CCNE1M-loaded and CCNE2L-loaded T2 cells, while no CTL could be elicited with mouse peptide. Similarly, CCNE2L-stimulated CTL lines killed CCNE1M-loaded and CCNE2L-loaded T2 cells but not non-loaded T2 cells. Using CCNE1M and CCNE2L HLA-A2 tetramers, we found that either tetramer could bind equally to either the CCNE1M- or CCE2L-derived CTL lines, suggesting that both peptides could be cross-recognized by CTL lines elicited with either peptide. To further study the cross-recognition and potential immune dominance of both peptides and to determine their potential anti-leukemia activity, CCNE1M- and CCNE2L-CTL clones were derived by limiting dilution assay. Two peptide-specific CTL clones from each of the lines showed 25% and 26% specific lysis, respectively, of leukemia cells at E:T 10:1. Neither CCNE-specific CTLs showed lysis of BM cells that were obtained from the same patient during remission, nor HLA-A2+ BM cells from a healthy donor. Next, we compared the T-cell antigen receptor (TCR) avidity of these CCNE1M- and the CCNE2L-CTL clones by measuring tetramer dissociation half-times (t1/2) at 25°C using CCNE1M/HLA-A2 and CCNE2L/HLA-A2 (and control pp65/HLA-A2) tetramers analyzed by flow cytometry. The decay of normalized (to time = 0) tetramer-bound fluorescence versus time was linear for each clone with either tetramer (R2 = 0.85 to 0.91), showing that tetramer binding avidity could be used to proportionally determine TCR affinity. Furthermore, first order kinetics could be used to determine the t1/2 of each of the clones. The t1/2 of CCNE1M/HLA-A2 tetramer was 85 min and 25 min, respectively, while the t1/2of CCNE1L/HLA-A2 was 30 min and 11 min, respectively, for the CCNE1M-CTL and the CCNE2L-CTL. This suggests that while both peptides were cross recognized by unique T-cell clones (with unique TCR, determined by TCR-Vβ sequence comparisons), CCNE1M appeared to be immunodominant. To determine whether immune response (IR) to either peptide occurred in leukemia patients, we studied PBMC from 18 patients (10 CML; 8 ALL) before and 3–6 mo after SCT with CCNE1M/HLA-A2- and CCNE2L/HLA-A2-tetramer assay. The mean number of CCNE1M-CTL and CCNE2L-CTL cells increased after SCT (p< 0.002 in CCNE1M-CTL and CCNE2L-CTL) compared to no change in mean number of pp65-CTL before/after SCT. IR (defined as ≥ 20% increase of specific CTL after SCT) to either CCNE1M or CCNE2L did not correlate with type of leukemia, donor-recipient HLA disparity (matched or mismatched), or disease status prior to SCT by Fisher's exact test. However, in 8 CML patients not in remission prior to SCT, IR to either CCNE1 or CCNE2 occurred more frequently in patients who achieved CR compared to those that did not achieve CR after SCT (100% vs. 33%, respectively; p < 0.04). These findings were confirmed in an additional 25 AML patients with active disease at SCT. To study whether the peptide-specific CTL were functional, we measured IFN-γ and TNF-αa production after peptide stimulation by Luminex bead assay and by intracellular cytokine flow cytometry (CFC). The assays showed production of IFN-γ and TNF-αa cytokines by T-cells after stimulation with CCNE1M or CCNE2Lpeptides. Taken together, these results show that CCNE1M and CCNE2Lself-peptides from constitutively active cell cycle proteins are novel leukemia-associated antigens that could be studied in immunotherapy strategies. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

Frontiers in Immunology ◽

10.3389/fimmu.2021.577766 ◽

2021 ◽

Vol 12 ◽

Author(s):

Darina Ocadlikova ◽

Mariangela Lecciso ◽

Javier Martin Broto ◽

Katia Scotlandi ◽

Michele Cavo ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cell Lines ◽

Effector T Cells ◽

Sarcoma Cell ◽

Ifn Γ ◽

Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

Download Full-text

Abstract 001: Axl Expression by CD4+ T Lymphocytes Promotes Salt-Dependent Hypertension

Hypertension ◽

10.1161/hyp.64.suppl_1.001 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Kristine M Wadosky ◽

Sri N Batchu ◽

Angie Hughson ◽

Kathy Donlon ◽

Craig N Morrell ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cd4 T Cells ◽

White Blood Cells ◽

Interleukin 4 ◽

Th2 Cells ◽

Salt Hypertension ◽

Ifn Γ

Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10 9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

Download Full-text

CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia

Blood ◽

10.1182/blood-2002-01-0163 ◽

2002 ◽

Vol 100 (6) ◽

pp. 2132-2137 ◽

Cited By ~ 192

Author(s):

Carmen Scheibenbogen ◽

Anne Letsch ◽

Eckhard Thiel ◽

Alexander Schmittel ◽

Volker Mailaender ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Acute Myeloid Leukemia ◽

T Cell ◽

Myeloid Leukemia ◽

Wilms Tumor ◽

Proteinase 3 ◽

Ifn Γ ◽

Tumor Gene ◽

Acute Myeloid

Abstract Wilms tumor gene product WT1 and proteinase 3 are overexpressed antigens in acute myeloid leukemia (AML), against which cytotoxic T lymphocytes can be elicited in vitro and in murine models. We performed this study to investigate whether WT1- and proteinase 3-specific CD8 T cells spontaneously occur in AML patients. T cells recognizing HLA-A2.1-binding epitopes from WT1 or proteinase 3 could be detected ex vivo in 5 of 15 HLA-A2–positive AML patients by interferon-γ (IFN-γ) ELISPOT assay and flow cytometry for intracellular IFN-γ and in 3 additional patients by flow cytometry only. T cells producing IFN-γ in response to proteinase 3 were further characterized in one patient by 4-color flow cytometry, identifying them as CD3+CD8+CD45RA+ CCR7−T cells, resembling cytotoxic effector T cells. In line with this phenotype, most of the WT1- and proteinase-reactive T cells were granzyme B+. These results provide for the first time evidence for spontaneous T-cell reactivity against defined antigens in AML patients. These data therefore support the immunogenicity of WT1 and proteinase 3 in acute leukemia patients and the potential usefulness of these antigens for leukemia vaccines.

Download Full-text

Functional Comparison and Longitudinal Assessment of Tri-Functional T-Cells Recognizing CMV pp65 and IE-1 Polypeptides in Hematopoietic Stem Cell and Solid Organ Transplant Recipients.

Blood ◽

10.1182/blood.v108.11.2936.2936 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2936-2936

Author(s):

Don J. Diamond ◽

Simon F. Lacey ◽

Corinna La Rosa ◽

Wendy Zhou ◽

Ghislaine Gallez-Hawkins ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Solid Organ ◽

Transplant Recipients ◽

Hematopoietic Stem ◽

Specific Ctls ◽

Tnf Α ◽

Ifn Γ ◽

Cmv Disease

Abstract Reconstitution of adaptive T-cell responses to human cytomegalovirus (CMV) is critical to protection from CMV disease following hematopoietic stem cell (HSCT) or solid organ transplantation (SOT). However, there is an incomplete understanding of which CMV antigens and epitopes are most crucial to providing protective responses. The functional status of cytotoxic T-lymphocyte (CTL) populations recognizing cytomegalovirus IE-1 and pp65 polypeptides was investigated in PBMC from either HSCT or SOT recipients. Our previous finding of differing levels of degranulation between CMV IE1 and pp65/pp50 specific T-cells was complicated by the possibility that differences were epitope and/or HLA-specific. We generalized the approach using a combined flow-based CD107a/b degranulation/mobilization and intracellular cytokine (ICC) assays using peptide libraries as antigens. These assays indicated that a significantly higher proportion of pp65-specific CTLs were in a more mature functional state compared to IE-1-specific CTLs. Degranulation/multicytokine ICC assays also indicated that a significantly higher proportion of the pp65-specific versus IE-1-specific CTLs secreted both IFN-γ and TNF-α, in addition to possessing greater cytotoxic potential. These results support our earlier findings of functional differences between CTLs recognizing individual epitopes within the IE-1 and pp65 antigens in HSCT recipients, and extend them to a broader array of HLA-restricted responses to those antigens. A report that a subset of HIV-1 specific CTLs capable of producing both IFN-γ and TNF-α was associated with improved cytotoxic activity prompted us to investigate whether degranulation, a functional correlate of cytotoxicity, was positively associated with dual cytokine production and predicted differences between IE1 and pp65-specific CD8+ T-cells. A higher proportion of pp65-specific compared to IE1-specific T-cells were present in the trifunctional IFN-γ+,TNF-α+, CD107+ population (p=0.008) in HSCT recipients. We have extended these findings to investigate the role of donor CMV status in terms of functional maturity of CMV-specific T cell response in transplant recipients. T cell maturation/function may act as a mechanistic correlate to the survival advantage of recipients receiving a stem-cell graft from CMV sero-positive donors. These principles have also been applied to investigations of a high risk population of sero-negative recipients of a sero-positive liver allograft. Data from this study will also be reviewed in the context of the model of trifunctional T cells being indicative of enhanced protective capacity against CMV disease and associated with survival.

Download Full-text

PD-1/PD-1-Ligand Interaction Contributes to Immunosuppressive Microenvironment of Hodgkin Lymphoma.

Blood ◽

10.1182/blood.v110.11.379.379 ◽

2007 ◽

Vol 110 (11) ◽

pp. 379-379

Author(s):

Ryo Yamamoto ◽

Momoko Nishikori ◽

Toshio Kitawaki ◽

Tomomi Sakai ◽

Masakatsu Hishizawa ◽

...

Keyword(s):

Gene Expression ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Hodgkin Lymphoma ◽

Cell Lines ◽

Signaling System ◽

Peripheral T Cells ◽

Ifn Γ ◽

Immunosuppressive Microenvironment

Abstract Programmed death-1 (PD-1), a member of the CD28 costimulatory receptor superfamily, inhibits T cell activity by providing a second signal to T cells in conjunction with signaling through the T-cell receptor. PD-1/PD-1 ligand (PD-L) signaling system is indicated to be involved in the functional impairment of T cells such as in chronic viral infection or tumor immune evasion. We hypothesized that this signaling system is also involved in the pathogenesis of Hodgkin lymphoma (HL). We examined expression of B7-H1 and B7-DC, two known PD-Ls, in lymphoid cell lines using RT-PCR and flow cytometry. They were expressed in HL and several T-cell lines, whereas most B-NHL lines lacked their expression. Immunohistochemical staining of HL tissues demonstrated that PD-Ls were also expressed in primary H/RS cells. As gene expression of B7-H1 and B7-DC was increased in Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell lines, we examined the effect of EBV latent membrane proteins on their gene regulation. By luciferase reporter assay, both LMP1 and LMP2A were shown to enhance promoter activity of B7-H1 and B7-DC genes. This finding implies that in cases of EBV-positive HL, latent membrane proteins may help H/RS cells escape from host immune surveillance by upregulating PD-L gene expression. We next analyzed PD-1 expression of tumor-infiltrating T cells of HL tissue samples by flow cytometry, and found that PD-1+ cells were elevated markedly in these cells. As HL patients are well recognized as having defective cellular immunity, we compared PD-1 expression level in peripheral blood T cells of HL patients with those of healthy volunteers and B-NHL patients. PD-1 was significantly elevated in peripheral T cells of HL patients compared to the other two groups. PD-1+ T cells were highest in patients with active disease, and tended to decline along with treatment. Although regulatory T cells are reported to play a part in the pathogenesis of HL, FOXP3+ T cells were not significantly elevated in peripheral T cells of HL patients, and PD-1+ T cells did not overlap with these regulatory population. To elucidate whether the PD-1/PD-L signaling pathway is functional in the immunosuppressive microenvironment of HL, we finally examined the effect of blockade of this pathway. After culturing bulk HL tumor cells with anti-PD-L blocking antibodies, IFN-γ production was measured by ELISA. Blockade of PD-Ls augmented IFN-γ production of HL-infiltrating T cells. We concluded that anti-tumor activity of HL-infiltrating T cells was inhibited via the PD-1/PD-L pathway, and this inhibition could be successfully relieved by PD-L blockade. Taken together, our observations indicate that “T-cell exhaustion” is essential to the pathogenesis of HL, and tumor-infiltrating T cells around H/RS cells seem to be kept in balance by this inhibitory signaling. Our findings provide a potentially effective and clinically applicable strategy for the immunotherapy of HL.

Download Full-text

PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity

Frontiers in Immunology ◽

10.3389/fimmu.2021.695947 ◽

2021 ◽

Vol 12 ◽

Author(s):

Stephanie A. Amici ◽

Wissam Osman ◽

Mireia Guerau-de-Arellano

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Disease Severity ◽

Cell Cycle Progression ◽

T Cell Proliferation ◽

Cycle Progression ◽

Cyclin E1 ◽

Relapsing Remitting

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5’s link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4+ T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.

Download Full-text

Induction, Selection and Expansion of Acute Myeloid Leukemia Reactive Autologous T Cells for Adoptive Immunotherapy.

Blood ◽

10.1182/blood.v106.11.1061.1061 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1061-1061

Author(s):

Rui-kun Zhong ◽

Edward D. Ball

Keyword(s):

T Cells ◽

T Cell ◽

Growth Factor ◽

Adoptive Immunotherapy ◽

Cell Number ◽

Cell Killing ◽

Specific Lysis ◽

Gm Csf ◽

Highly Active ◽

Ifn Γ

Abstract To generate highly AML reactive autologous CTL, the optimal protocol of growth factor combination and sequence of administration were screened and compared in an AML-DC culture system aimed to induce AML cell DC differentiation, activate and expand naïve T cells against AML; and simultaneously activate and expand possible rare existing AML reactive memory T cells. Primary AML peripheral blood or marrow cells containing more than 90% AML blasts were cultured in 96 well plates. Every single well was regarded and treated as an independent culture. Various growth factor combinations of GM-CSF, IL2, IL-3, IL-4, IL7, IL12, CD40L, TNF-α, anti-CTLA-4 mAb were compared for efficiency of AML cell DC differentiation induction and autologous T cell activation in 7 day culture. Significantly enhanced CD80/CD86 expression and total cell number (include both AML-DC and T cells, see attached figure) in the culture was observed when GM-CSF/IL-4 combined with IL-12. The T cells in each well were than expanded with high dose IL-2 (6000 u/ml) and CTL activity against autologous AML were examined by both 51Cr release assay and culture supernatant IFN-γ concentration assay. Multiple wells of AML cell culture from the same patient with the same cell number and culture condition exhibited substantial functional variation demonstrated by MHC-restricted autologous AML cell killing (3 - 70% specific lysis) and IFN-γ secretion (25 - 2994 pg/ml), indicating that the T cells randomly put in the independent wells vary greatly in efficiency of AML antigen recognition and activation. IL-12 alone exerted significant enhanced IFN-γ secretion in 4 of 5 patients. IL-12 combined with GM-CSF/IL-2/IL-4/IL-7 enhanced IFN-γ secretion compared with IL-12 alone in 3 of 5 patients. CTLA-4 blockade further significantly enhanced T cell activity in 5 of 5 patients. Highly active CTL lines were selected from high IFN-γ secretion wells and rapidly expanded using OKT-3/CD28 coated, mitomycin-c treated autologous AML cells. After expansion, the highly active lines still maintained high autologous AML cell killing compared to the low active lines. A therapeutic quantity of AML reactive autologous T cells (20.2±30.1)x109, (n=5) can be obtained after 6 weeks of culture from (6.4±2.9)x107 AML PBMC. This study demonstrated AML cells cultured in IL-12 or IL-12 combined with GM-CSF/IL-2/IL-4/IL-7/anti-CTLA4 mAb activated the potential of the patient immune system and may maximize the possibility of AML reactive CTL generation for adoptive immunotherapy. Figure Figure

Download Full-text

Generation of CMV Specific T Cells From CMV Seronegative Donors by T Cell Receptor RNA Transfer.

Blood ◽

10.1182/blood.v114.22.3577.3577 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3577-3577

Author(s):

Simone Thomas ◽

Sebastian Klobuch ◽

Mirjam H.M. Heemskerk ◽

Diana Stolle ◽

Katrin Besold ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Human Fibroblasts ◽

Expression Level ◽

Specific Lysis ◽

Hematopoietic Stem ◽

Peptide Epitope ◽

Retroviral Transduction ◽

Ifn Γ

Abstract Abstract 3577 Poster Board III-514 Reactivation of latent human cytomegalovirus (CMV) infection is a frequent complication in CMV seropositive patients after allogeneic hematopoietic stem cell transplantation (HSCT). Although antiviral drug therapy is successfully used to reduce the risk of CMV disease, long-term virus control requires the re-establishment of protective antiviral T cell immunity in the host. The latter is challenging, particularly if the donor is CMV seronegative and thus, no CMV specific T cells are being transferred from donor to recipient during HSCT. Grafting nonreactive T cells of CMV seronegative donors by virus-antigen specific T cell receptors (TCR) may be an efficient means to transfer CMV specific T cell function into allogeneic HSCT recipients. In this study, we intended to reprogram T cells of CMV seronegative donors with human TCR recognizing the immunodominant HLA-A*0201 binding peptide epitope NLVPMVATV (495-503) derived from the CMV pp65 protein. A common approach for TCR gene transfer into T cells is retroviral transduction bearing the risk of insertional mutagenesis which hampers clinical translation. In addition, heterologous recombination between introduced and naturally expressed TCR chains might lead to the induction of harmful autoimmunity. Herein we used in vitro transcribed RNA encoding the CMV pp65/HLA-A*0201-specific TCR for electroporation of anti-CD3 stimulated T cells in peripheral blood mononuclear cells (PBMC) of CMV seronegative donors. This procedure resulted in transient surface expression of the introduced TCR chains up to 5 days as shown by flow cytometry. Maximum expression level was observed at 4 to 24 h after electroporation, with up to 70% of total CD8+ and CD4+ T cells staining positive for the vβ13.1 subfamily domain of the TCRβ chain. After introduction of TCR RNA, the intensity of CMV pp65/HLA-A*0201 tetramer staining was 60% and 50% of total CD8+ and CD4+ cells, respectively. In IFN-γ ELISPOT and 51Chromium-release assays, TCR RNA transfected T cells recognized HLA-A*0201 expressing T2 cells pulsed with titrated amounts of CMV pp65 (495-503) peptide. Minimal peptide concentration triggering specific lysis was 0.1 nM to 1 nM at a CD8+ to target (CD8+:T) ratio of 2:1. The EC50 value (0.2 nM) was in the same range of avidity compared to that of a retrovirally transduced counterpart construct of this TCR. Most importantly, TCR recipient CD8+ T cells gained the ability to lyse HLA-A*0201 positive human fibroblasts upon infection with CMV. Specific lysis between 20% and 100% was observed at a CD8+:T ratio of 1:1 or higher. We next sorted CD8+ T cells from PBMC of CMV seronegative donors into naïve and memory cells according to expression of the differentiation markers CD45RA and CD45RO. Although 90% of naïve CD8+ T cells stained positive for the CMV pp65/HLA-A*0201 tetramer after electroporation of TCR RNA, they mediated only marginal lysis toward CMV infected fibroblasts. In contrast, TCR RNA transfected memory CD8+ T cells showed strong lysis against CMV infected fibroblasts at a CD8+:T ratio of 0.7:1 or higher. Specific lysis was detected for at least 3 days after electroporation. In summary, our data demonstrate that nonreactive human T cells can be successfully redirected with CMV pp65 TCR RNA. The expression level of the introduced TCR is sufficient to trigger IFN-γ production and cytolytic activity toward CMV infected human fibroblasts. Electroporation of TCR RNA is comparably easy and eliminates the risk of retroviral transduction. We therefore believe that CMV pp65 TCR RNA has the potential to be further developed as a therapeutic “off-the-shelf” reagent for patients who undergo drug-resistant CMV reactivation after HSCT. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Generation of Tumor-Specific CD8+ Cytotoxic T Lymphocytes and CD4+ Th1 Helper T Cells by Transfer of T-Cell Receptor Genes Isolated from a WT1-Specific CD8+ Cytotoxic T-Lymphocyte Clone.

Blood ◽

10.1182/blood.v104.11.2523.2523 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2523-2523

Author(s):

Masaki Yasukawa ◽

Hironari Niiya ◽

Taichi Azuma ◽

Naoyuki Uchida ◽

Yoshihiro Yakushijin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cd8 T Cells ◽

Human Tumor ◽

Th1 Cells ◽

Human Tumor Cells ◽

Specific Ctls ◽

Ifn Γ ◽

Β Chain

Abstract Background: Cytotoxic T lymphocytes (CTLs) and T-helper type 1 (Th1) cells undoubtedly play a crucial role in the eradication of tumors in vivo. However, the production of Th1 cytokines such as IL-2 and IFN-γ is markedly suppressed in the majority of tumor-bearing hosts. Such defects in Th1-mediated immunity in cancer patients have made it difficult to induce tumor-specific CTLs that promote tumor rejection. Adoptive transfer of tumor-specific CTLs and Th1 cells can overcome the difficulty to induce tumor-specific immune response in cancer patients; however, the generation and expansion of tumor-specific CTLs and Th1 cells in vitro are not easy. In the present study, to overcome this problem, we isolated TCR-α and -β chain genes from a WT1-specific CD8+ CTL clone, which had been shown to exert strong cytotoxicity against hematopoietic malignancies and solid tumors in an HLA-A24-restricted manner, and transduced them into nonspecifically activated human CD8+ and CD4+ T cells. Consequently, both CD8+ and CD4+ T cells appeared to acquire WT1-specific function in an HLA-A24-restricted manner. Methods: A WT1 peptide (CMTWNQMNL)-specific CD8+ CTL clone, TAK-1, was established as reported previously (Blood95:286,2000). TAK-1 exerts cytotoxicity against variety of tumor cells including leukemia, myeloma, and lung cancer cells but not against normal cells in an HLA-A24-restricted manner. cDNAs encoding TCR-α and -β chain genes were amplified from cDNA of TAK-1 by RT-PCR. TCR-α and -β chain cDNAs were inserted into the plasmid vector. Preparation of lentiviral vectors for transduction of TCR-α and -β chain cDNAs was performed as described previously (Cancer Res64:1490,2004). Peripheral blood CD4+ and CD8+ T cells isolated from healthy individuals were cultured with anti-CD3 mAb and retronectin and then infected twice with lentivirus vectors. The infected cells were expanded by culture in the presence of IL-2, IL-12, IFN-γ and anti-IL-4 mAb. Cytotoxicity of CTLs against WT1-peptide-loaded cells and various human tumor cells was examined by a standard 51Cr-release assay. Recognition of tumor cells by Th1 cells was examined by measuring IFN-γ production by ELISA. Results: CD4+ T-cell line (CD4-TCR) and CD8+ T cell line (CD8-TCR) expressing TCR-α and -β chains of TAK-1 were established. Both CD4-TCR and CD8-TCR cells exerted cytotoxicity against WT1 peptide-loaded HLA-A24-positive but not -negative cells. CD8-TCR cells appeared to be cytotoxic against human tumor cells including leukemia, myeloma, and lung cancer cells in an HLA-A24-restricted manner, but did not show any cytotoxicity against HLA-A24-positive normal cells. CD4-TCR cells produced IFN-γ in response to stimulation with HLA-A24-positive but not -negative leukemia cells. Conclusion: The present data demonstrate the functional reconstitution of CD4+ as well as CD8+ T cells by transfer of the αβ TCR complex of a WT1-specific CD8+ CTL clone. Since WT1 is a universal tumor-associated antigen, transfer of TCR genes of WT1-specific CTLs into CD4+ and CD8+ T cells would be useful for Th1-based immunotherapy of various malignancies.

Download Full-text

Cytotoxic T Lymphocytes (CTL) Specific for CMV, Adenovirus, and EBV Can Be Generated From Naive T Cells for Adoptive Immunotherapy.

Blood ◽

10.1182/blood.v114.22.504.504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 504-504

Author(s):

Patrick Hanley ◽

Barbara Savoldo ◽

Conrad Russell Young Cruz ◽

Ann M. Leen ◽

Jeffrey J. Molldrem ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Adoptive Immunotherapy ◽

Peripheral Blood ◽

T Cell Responses ◽

Cell Transplant ◽

Cell Responses ◽

Specific Ctls ◽

Ifn Γ

Abstract Abstract 504 Allogeneic stem cell transplantation is the treatment of choice for patients with high-risk hematologic malignancies. Umbilical cord blood (CB) has emerged as an important source of stem cells for allotransplant patients lacking human leukocyte antigen (HLA)-matched donors–a significant problem for minorities. T cells in UCB grafts are, however, virus-naïve, leading to higher mortality rates due to infections with CMV, EBV, adenovirus (Ad) and other viruses. Peripheral Blood Stem Cell Transplant (PBSCT) from CMV-seronegative (CMVneg) donors to CMV-seropositive (CMVpos) recipients produces a similarly high incidence of CMV infection since donor T cells are naïve to this virus. Adoptive immunotherapy with peripheral blood (PB)-derived CMV/Ad-specific CTL generated from CMVpos donors effectively prevents CMV clinical disease after PBSCT, but this option has not been feasible when the donor cells are naive, irrespective of whether they are sourced from CB or CMV-PBS, since CTL generation from these donors has been unsuccessful. We have now overcome this problem and can routinely generate CMV, Ad and EBV-specific CTLs from CB and CMV specific CTLs from seronegative PBSC. We used an Ad5f35vector carrying the CMVpp65 transgene to transduce CB-derived or PB-derived dendritic cells and stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 stimulations with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector. The 9 CB-derived CTL lines we made in this way contained a mean of 87% (range 81-94) CD8+ and 26% (range 12-40) CD4+T cells, and exhibited significant cytotoxicity in 51Cr release assays against CMVpp65, Adhexon, and LCL targets. In IFN-γ ELISPOT assays there was a mean of 209 (range 45-694), 74 (range 0-128), and 157 (range 23-291) SFC following incubation with CMVpp65, Adhexon peptides and LCL, respectively. In addition, we generated CMVpp65, Adhexon, and LCL-specific responses from the peripheral blood of 4 CMVneg adult donors, which produced a mean of 92 (range 50-126), 163 (range 69-293), and 62 (range 37-86) SFC to CMVpp65 respectively. Neither CB- nor CMVneg-derived CTL responded to irrelevant peptides. Of note, the virus-specific T cells that we expanded from both CB and CMVneg donors derived only from T cells with a naive phenotype (CD45RA+/CCR7+). Moreover, both CB and CMVneg-derived CTL recognized ‘unconventional‘ CMV pp65 epitopes, as identified by overlapping pp65 peptide pools and confirmed by IFN-γ ELISPOT as well as multimer analysis (Table 1). In HLA-A2+ subjects, these naive-derived CTLs did not recognize conventional HLA-A2-associated CMV pp65 epitopes such as NLV, suggesting an inherent difference between naïve and memory T cell responses to CMV. In summary, virus-specific responses T cell responses can be obtained even from CB and virus-naive adult donors and may allow prevention and treatment of viral disease in the recipients of these allografts. Disclosures: No relevant conflicts of interest to declare.

Download Full-text