Ex vivo isolation of RCC-reactive CD8+ CTL clones from HLA-identical allogeneic donor T cell lines stimulated by CD80-transfected tumor cells

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15625-15625 ◽  
Author(s):  
S. S. Tykodi ◽  
J. A. Thompson ◽  
B. M. Sandmaier ◽  
M. B. Maris ◽  
R. Storb ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Ex Vivo ◽  
Unrelated Donor ◽  
T Cell Antigens ◽  
Ifn Γ ◽  
T Cell Lines ◽  
Anti Tumor Activity

15625 Background: Regression of metastatic renal cell carcinoma (mRCC) is observed in a minority of patients treated by immunotherapies such as interleukin-2 (IL-2), interferon-a (IFN-a), or reduced-intensity allogeneic hematopoietic cell transplantation. However, the development of specific cellular immunotherapies for mRCC has been hindered by the lack of molecularly characterized T cell antigens with preferential expression on RCC cells. We have developed an ex vivo strategy for the isolation of RCC-reactive CD8+ CTL clones that may facilitate the identification of novel RCC-associated T cell antigens. Methods: RCC tumor lines were established from two patients with mRCC presenting to our institution for allogeneic HCT received from either an HLA-matched sibling or volunteer unrelated donor. Irradiated RCC tumor lines that were unmodified or transfected with a cDNA for human CD80 were used to stimulate responder CD8+ T cells isolated from pretransplant patient (autologous) or donor-derived (allogeneic) blood samples in mixed lymphocyte/tumor cell (MLTC) cultures supplemented with recombinant human IL-7 and IL-12 (stimulation #1) or IL-2 (2nd and subsequent stimulations). T cell lines with anti-tumor activity measured by IFN-γ ELISA were then cloned by limiting dilution. Results: After two or more in vitro stimulations, allogeneic CD8+ T cell lines stimulated by CD80- transfected RCC tumor cells, but not the other MLTC culture combinations tested demonstrated tumor-specific IFN-γ release. CD3+/CD8+/TCRaβ+ CTL clones with potent in vitro anti-tumor activity for unmodified RCC tumor were isolated from both sibling- and unrelated- donor derived T cell lines. Three such clones with unique specificities for allogeneic targets recognized the unmodified RCC tumor but not LCL or fibroblast target cells isolated from the same patient suggesting tumor-restricted expression of the target antigens. Conclusions: Ex vivo MLTC culture utilizing CD80-transfected RCC tumor and HLA- matched allogeneic responder CD8+ T cells warrants further study as a strategy to isolate CTL clones that may be used to identify novel RCC-associated T cell antigens. No significant financial relationships to disclose.

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

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.

scholarly journals An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

1998 ◽  
Vol 66 (10) ◽  
pp. 4981-4988 ◽  
Author(s):  
Irina Lyadova ◽  
Vladimir Yeremeev ◽  
Konstantin Majorov ◽  
Boris Nikonenko ◽  
Sergei Khaidukov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antimycobacterial Activity ◽  
Enzymatic Digestion ◽  
Interleukin 4 ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

Generation of Cytomegalovirus (CMV)-Specific CD4 and CD8 T Cell Lines Using Protein-Spanning Pools of pp65 and IE1 Derived Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 477-477
Author(s):  
Erica Dander ◽  
Giuseppina Li Pira ◽  
Ettore Biagi ◽  
Fabrizio Manca ◽  
Andrea Biondi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cell ◽  
Effector Memory ◽  
Target Cells ◽  
Pulsed Dc ◽  
Ifn Γ ◽  
T Cell Lines

Abstract BACKGROUND: Reactivation of latent CMV in immunocompromised recipients of allogeneic stem cell transplantation remains a major cause of morbidity and mortality. Reconstitution of immunity by CMV specific immunotherapy is an attractive alternative to drugs currently used, which show high toxicity and are sometimes ineffective. It has been demonstrated that CD4 helper T-cell function is crucial for the persistence of in vivo transferred CD8 CMV-specific CTL. Based on this finding, we have explored the feasibility of generating both anti-CMV CD4 and anti-CMV CD8 T-cell lines. METHODS: Dendritic Cells (DC) were generated from donor peripheral blood (PB) monocytes after a 7-day culture in the presence of GM-CSF plus IL-4 and matured with TNF-α, IFN-α, IFN-γ, IL1-β, POLI I:C. Matured-DC were then pulsed with a pool of 50 peptides spanning pp65 and IE1 proteins which are recognised by both CD4 and CD8 T lymphocytes. Donor T cells were stimulated three times at a T cell/DC ratio of 1:6 on day 0, +7 and +14 with mature peptide pulsed-DC. At the end of the culture the specificity of generated T cells was determined as percentage of pentamer-positive cells and intracellular IFN-γ production after incubation with peptide pulsed-DC. Cultured T cells were also analysed for their ability to proliferate in response to peptide pulsed-target cells, to kill them in a standard citotoxicity assay and to migrate in response to inflammatory (CXCL9, CCL3 and CCL5) and constitutive (CXCL12) chemokines. RESULTS: CMV-specific T cell lines were generated from five CMV seropositive donors. In four cases CD4 and CD8 CMV-specific T cell lines were expanded successfully. Cultured T cells expressed CD8 (mean= 70%, range 60–81%) and CD4 (mean= 20%, range 15–28%) and showed a CD45RA- CCR7- Effector Memory phenothype (mean=26%, range 19–30%) or a CD45RA+ CCR7- T Effector Memory RA-Positive phenothype (mean=67%, range 59–77%). An enriched CMV-specific T cell population was observed after staining with pentamers (7–45% pentamer-positive T cells). Furthermore, 90% of CD8+ and 40% of CD4+ T cells expressed high levels of intracytoplasmatic perforin and granzyme. In 4/5 cases tested, cutured T cells showed a cytolitic activity against CD8-peptide pulsed target cells (average lysis=50%, range 40–55%) and to a lesser extent against CD4-peptide pulsed target cells (average lysis=35%, range 30–40%). In addition, cultured T lymphocytes were able to proliferate and to produce intracytoplasmic IFN-γ (average production=50%, range 35–60%) after exposure to peptide-pulsed DC. Finally, Cultured T cells strongly migrated in response to chemokines (CXCL9, CCL3 and CCL5) involved in the recruitment of effector cells during viral infection. DISCUSSION: In conclusion, a great advantage of this method is represented by the possibility to generate anti-CMV CD4+ T cells, which could support in vivo the persistence of re-infused CMV-specific CTL. Moreover, the possibility of generating peptides under GMP conditions would facilitate the translation of this approach into clinical intervention.

scholarly journals CD8 T cell clones from young nonobese diabetic (NOD) islets can transfer rapid onset of diabetes in NOD mice in the absence of CD4 cells.

1996 ◽  
Vol 183 (1) ◽  
pp. 67-76 ◽  
Author(s):  
F S Wong ◽  
I Visintin ◽  
L Wen ◽  
R A Flavell ◽  
C A Janeway
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Nod Mice ◽  
Cd8 T Cell ◽  
Rapid Onset ◽  
Cytotoxic T Cell ◽  
Nonobese Diabetic ◽  
T Cell Lines

T cells play an important role in the pathogenesis of diabetes in the nonobese diabetic (NOD) mouse. CD8 cytotoxic T cell lines and clones were generated from the lymphocytic infiltrate in the islets of Langerhans of young (7-wk-old). NOD mice by growing them on (NOD x B6-RIP-B7-1)F1 islets. These cells proliferate specifically to NOD islets and kill NOD islets in vitro. The cells are restricted by H-2Kd, and all bear T cell antigen receptor encoded by V beta 6. When these CD8 T cell lines and clones are adoptively transferred to irradiated female NOD, young NOD-SCID, and CB17-SCID mice, diabetes occurs very rapidly, within 10 d of transfer and without CD4 T cells.

Rapid generation of combined CMV-specific CD4+ and CD8+ T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants

Blood ◽  
2004 ◽  
Vol 103 (9) ◽  
pp. 3565-3572 ◽  
Author(s):  
Georg Rauser ◽  
Hermann Einsele ◽  
Christian Sinzger ◽  
Dorothee Wernet ◽  
Gabriele Kuntz ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Lines ◽  
Adoptive Transfer ◽  
Cd8 T Cell ◽  
Cell Transplants ◽  
Ifn Γ ◽  
Rapid Generation ◽  
T Cell Lines

Abstract Adoptive transfer of cytomegalovirus (CMV)-specific T cells can restore long-lasting, virus-specific immunity and clear CMV viremia in recipients of allogeneic stem cell transplants if CD4+ and CD8+ CMV-specific T cells are detected in the recipient after transfer. Current protocols for generating virus-specific T cells use live virus, require leukapheresis of the donor, and are time consuming. To circumvent these limitations, a clinical-scale protocol was developed to generate CMV-specific T cells by using autologous cellular and serum components derived from a single 500-mL blood draw. CMV-specific T cells were stimulated simultaneously with CMV-specific major histocompatibility complex class I (MHC I)- restricted peptides and CMV antigen. Activated T cells were isolated with the interferon-γ (IFN-γ) secretion assay and expanded for 10 days. In 8 randomly selected, CMV-seropositive donors, 1.34 × 108 combined CD4+ and CD8+ CMV-specific T cells, on average, were generated, as determined by antigen-triggered IFN-γ production. CMV-infected fibroblasts were efficiently lysed by the generated T cells, and CMV-specific CD4+ and CD8+ T cells expanded if they were stimulated with natural processed antigen. On the other hand, CD4+ and CD8+ T cell-mediated alloreactivity of generated CMV-specific T-cell lines was reduced compared with that of the starting population. In conclusion, the culture system developed allowed the rapid generation of allodepleted, highly enriched, combined CD4+ and CD8+ CMV-specific T cells under conditions mimicking good manufacturing practice. (Blood. 2004; 103:3565-3572)

scholarly journals T cells specific for alpha-beta interface regions of hemoglobin recognize the isolated subunit but not the tetramer and indicate presentation without processing

1989 ◽  
Vol 86 (17) ◽  
pp. 6729-6733 ◽  
Author(s):  
M Z Atassi ◽  
M Yoshioka ◽  
G S Bixler
Keyword(s):  
T Cells ◽  
Major Histocompatibility Complex ◽  
T Cell ◽  
Cell Lines ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Oligomeric Protein ◽  
T Cell Lines ◽  
Antigen Presenting

Processing of a protein antigen into fragments is believed to be a prerequisite for its presentation by the antigen-presenting cell to the T cell. This model would predict that, in oligomeric proteins, T cells prepared with specificity for regions that are buried within subunit association surfaces should recognize the respective regions in vitro equally well on the isolated subunit or on the oligomer. Three hemoglobin (Hb) alpha-chain synthetic peptides, corresponding to areas that are situated either completely [alpha-(31-45)] or partially [alpha-(41-45) and alpha-(81-95)] within the interface between the alpha and beta subunits of Hb, and a fourth peptide representing a completely exposed area in tetrameric Hb were used as immunogens in SJL/J (H-2s) mice. Peptide-primed T cells were passaged in vitro with the respective peptide to obtain peptide-specific T-lymphocyte lines. T-cell clones were isolated from these lines by limiting dilution. T-cell lines and clones that were specific for buried regions in the subunit association surfaces recognized the free peptide and the isolated subunit but not the Hb tetramer. On the other hand, T cells with specificity against regions that are not involved in subunit interaction and are completely exposed in the tetramer recognized the peptide, the isolated subunit, and the oligomeric protein equally well. The responses of the T-cell lines and clones were major histocompatibility complex-restricted. Since the same x-irradiated antigen-presenting cells were employed, the results could not be attributed to differences or defects in Hb processing. The findings indicate that in vitro the native (unprocessed and undissociated) oligomeric protein was the trigger of major histocompatibility complex-restricted T-cell responses.

Naturally Processed Peptides Eluted from Acute Myeloid Leukemia Cells : A Potent Antigen Source for Immunotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1801-1801
Author(s):  
Stephanie Delluc ◽  
Lea Tourneur ◽  
Charlotte Boix ◽  
Anne-Sophie Michallet ◽  
Bruno Varet ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Immune Recognition ◽  
T Cell Lines ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogenous group of diseases characterized by a clonal proliferation of myeloid progenitors. Its poor prognosis with conventional chemotherapy justifies seeking for adjuvant immunotherapeutic approaches to eliminate minimal residual disease. We evaluated an immunotherapeutic strategy that bypass the need for epitope identification and the limitation due to HLA restriction. Naturally processed peptides were extracted by acid elution from AML cells at diagnosis, and loaded on mature dendritic cells (mDCs) derived from autologous monocytes obtained when the patients were in complete remission (CR). We evaluated i) the feasibility to elute naturally processed peptides from AML cells at diagnosis, ii) the capacity of mDCs loaded with eluted peptides (mDC/EP) to stimulate specific T cell lines in vitro. We showed that stimulation by mDC/EP was able to generate anti-leukemic T cells lines from PBMC of 6 AML patients in CR. CD4+ and CD8+ T cells were isolated from T cell lines of 5 patients and analyzed for their proliferation, INF-γ production and cytotoxicity in response to autologous or allogeneic AML targets, or to normal autologous PBMC. We showed that both CD4+ and CD8+ leukemia-specific T cells were generated in vitro by mDC/EP stimulations since proliferation of CD4+ T cells, IFN-γ secretion by CD4+ and CD8+ T cells and cytotoxicity mediated by CD8+ T cells were induced in response to stimulation with autologous AML cells. Furthermore, we could not detect auto-immune recognition of autologous normal PBMC, consistent with the specificity of the T cell response induced by mDC/EP. These results provide the proof of concept for using mDC/EP to vaccinate patients with poor-risk AML, and will soon be evaluated in a phse I/II clinical trial.

Development of Chronic Lymphocytic Leukemia (CLL) Reactive Cytotoxic T Lymphocytes after Non-Myeloablative Hematopoietic Stem Cell Transplant Correlates with Anti-Leukemia Response.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 413-413
Author(s):  
Tetsuya Nishida ◽  
Ana Kostic ◽  
David G. Maloney ◽  
Rainer F. Storb ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Histocompatibility Antigens ◽  
Antitumor Response ◽  
Hematopoietic Stem ◽  
Minor Histocompatibility Antigens ◽  
Cell Responses ◽  
T Cell Lines

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) following non-myeloablative (NM) conditioning is a promising approach for treating patients with advanced fludarabine refractory CLL. In this setting, a graft versus leukemia (GVL) effect mediated by donor T cells is critical for tumor eradication. We have evaluated the development of alloreactive and CLL-reactive cytotoxic T lymphocyte (CTL) responses in patients after NM-HSCT to determine if the generation of detectable T cell responses was associated with an antitumor response. Seven patients with fludarabine refractory CLL were conditioned with fludarabine (30mg/m2 x 3 doses) and total body irradiation (2 Gy) prior to receiving G-CSF mobilized peripheral blood stem cells from an HLA matched donor. Peripheral blood mononuclear cells (PBMC) were obtained from the recipient pretransplant and at intervals after NM-HSCT. When chimerism showed a major proportion of donor CD3+ T cells, the postransplant PBMC were stimulated in vitro with recipient CLL cells from the pretransplant collections. CLL cells lack or express low levels of co-stimulatory and adhesion molecules, and are poor stimulators of T cells in vitro. Thus, prior to their use as stimulators and targets, the CLL cells were activated with CD40 ligand (CD40L), which upregulates costimulatory, adhesion, and MHC molecule expression, and turns CLL cells into effective antigen presenting cells. The cultures were stimulated weekly and supplemented with IL2 and IL7. After two stimulations, the T cell lines were tested for cytotoxicity against donor and recipient target cells including recipient CLL. T cell lines generated from four patients with a good antitumor response after NM-HSCT exhibited cytotoxicity against recipient CLL and EBV transformed B cells (B-LCL), but not against donor B-LCL. By contrast, T cell lines generated from three patients with persistent or progressive disease after NM-HSCT did not have cytotoxicity against recipient CLL, despite the development of GVHD in all patients. Multiparameter flow cytometry and IFN-g secretion assay of T cell lines from patients with an antitumor response showed that both CD8+ and CD4+ T cells produced INF-g in response to recipient CLL. We sorted and expanded CD8+ INF-g+ and CD4+ IFN-g+ T cells and both subsets were able to lyse CLL cells. The cytotoxicity of CD4+ and CD8+ T cells was inhibited completely by concanamycin A, suggesting perforin is the major mechanism for leukemia cell lysis. Twenty-one CD8+ T cell clones specific for distinct minor histocompatibility antigens expressed on CLL were isolated from T cell lines of the four responding patients. Multiple specificities were recognized in three of the four patients. Screening a cDNA expression library has identified the genes encoding two minor histocompatibility antigens recognized by CD8+ T cells, and their characterization is in progress. These findings suggest that the development after NM-HSCT of early, diverse, alloreactive T cell responses specific for antigens expressed by CLL may be an important predictor of outcome. The identification of the antigens recognized may facilitate the development of strategies to evoke an effective antitumor response in a larger fraction of patients.

Adenovirus Specific T-Cell Lines Devoid of Alloreactivity Against Haploidentical Recipients Can Be Obtained Using a Set of Adenovirus Hexon Peptides.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3273-3273
Author(s):  
Patrizia Comoli ◽  
Marco W. Schilham ◽  
Sabrina Basso ◽  
Tamara van Vreeswijk ◽  
Rita Maccario ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Peptide Pool ◽  
Specific Lysis ◽  
Hematopoietic Stem ◽  
Hexon Protein ◽  
T Cell Lines

Abstract Human Adenovirus (HAdV) infection/reactivation may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in pediatric recipients of a T-cell depleted allograft from haploidentical family donor. The effectiveness of pharmacological therapy for HAdV infection is still suboptimal. It has been recently demonstrated that cell therapy may offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of HAdV-specific T-cells in the haplo-HSCT cohort poses the concern that GVHD may ensue as a consequence of T-cell transfer. We have conducted scale-up experiments to validate a method of in vitro culture to expand T-cells specific for HAdV, based on stimulation of donor peripheral blood mononuclear cells (PBMC) with a pool of 5 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT (Veltrop-Duits et al, Eur J Immunol36, p2410; 2006). A total of 20 T-cell lines were generated, starting from a median of 20 × 106 donor PBMC, that yielded a median of 80 × 106 cells. Most of the cell lines obtained included a majority of CD4+ T-lymphocytes, with a lower % CD8+ cells (median and range: 78, 19–94 and 18, 5–58, respectively) but 5/20 lines contained a high number of CD8+ T cells (ranging between 43% and 58%), which were CD56+ and/or TCRγδ+, and in 1 case also 44% NK cells. Eighteen of the 20 T-cell lines were HAdV-specific, since they showed a median proliferation to the HAdV hexon peptide pool and inactivated HAdV of 14615 (95%CI 8924–31532) and 11103 (95%CI 8805–30174) cpm/105 cells after subtraction of background (responders+irradiated autologous PBMC), respectively. HAdV-specific lysis >10% at a 2:1 effector to target (E:T) ratio was observed in 50% of the T-cell lines. The 2 non-specific, as well as the 3 T-cell lines with lower specific activity, included >40% CD8+ T-cells. Production of IFNγ in an ELIspot assay to HAdV hexon peptide pool above 40 SFU/105 cells was observed in 9 out of 13 tested T-cell lines. Evaluation of specific response to hexon peptides in showed a majority of responses to II42 (80%), with 50–60% responses to II50, II57, II61, and II64. Only 2 out of the 20 T-cell lines tested were prevalently alloreactive against the recipient. Of the 18 HAdV-specific lines, 1 showed higher proliferation to patient PBMC than to HAdV (13518 vs 11717 mean cpm), and would have thus been discarded as unsuitable for in vivo use, while the other 17 showed no alloreactivity (14) or alloreactivity between 10 and 23% of specific proliferation (3). None of these 18 T-cell lines showed lysis >5% against recipient PHA blasts in the cytotoxicity assay. Our data show that PBMC stimulation with HAdV hexon protein-derived 30-mer peptides is able to reproducibly induce the generation of HAdV-specific CD4+ T-cell lines with efficient in vitro antiviral response in most HLA-mismatched HSCT donors. The majority of these T-cell lines show low/undetectable alloreactivity against recipient targets, and could therefore be safely employed for adoptive treatment of HAdV complications developing after HSCT from a HLA-haploidentical donor.

CMV-Specific T Cell Immunotherapy Promotes Restoration of Durable Functional Immunity in High Risk Patients Following Allogeneic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 352-352
Author(s):  
Karl S Peggs ◽  
Stephanie Verfuerth ◽  
Arnold Pizzey ◽  
Christine Chow ◽  
Kirsty Thomson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Risk ◽  
Viral Infection ◽  
Cell Lines ◽  
Immune Reconstitution ◽  
Cellular Therapy ◽  
Ex Vivo ◽  
T Cell Lines ◽  
Cmv Disease

Abstract Adoptive cellular therapy with virus-specific T-cells offers the potential for accelerating pathogen-specific immune reconstitution, thus limiting the morbidity and mortality of viral infections following allogeneic haematopoietic stem cell transplantation (aHSCT). However, the logistics of production and the risk of inducing graft-versus-host disease (GvHD) secondary to the infusion of alloreactive clones have restricted their application. We examined the use of polyclonal cytomegalovirus (CMV)-specific mixed CD4+ and CD8+ T-cell lines, generated by short-term ex vivo culture of donor lymphocytes with donor monocyte-derived dendritic cells pulsed with virus-lysate. Following initial evaluation in a phase I study of pre-emptive cellular therapy in mainly matched related donor transplant recipients, we performed a phase I-II study including a higher risk population routinely receiving CMV-specific T cells 28 days following transplantation. Thirty patients received CMV-specific T cells in this study. The majority were at high risk of viral infection, being CMV seropositive recipients (24/30), having received a T-cell depleted graft (24/30), and/or having an unrelated or mismatched donor (14/30). T cell lines were administered prior to viral DNA detection (prophylaxis) in 10 patients, following a single viral DNA detection episode (pre-emptive) in 10 and concurrent with antiviral drug therapy in 10. There were no significant immediate toxicities. Acute GvHD > Grade I occurred in 2/24 T-cell depleted transplants (1 Grade II, 1 Grade III), and 2/6 T-cell replete transplants (2 Grade III), but in 0/14 unrelated or mismatched donor transplants, suggesting no excess of GvHD associated with cellular therapy. Only 3/10 treated prophylactically developed CMV infection requiring therapy (2 following introduction of systemic steroids). All 10 receiving cells pre-emptively required antiviral drugs. CMV-specific immune reconstitution was monitored using class I HLA-pentamers restricted by HLA-A*0201 (the NLV epitope of pp65, and the VLE epitope of IE-1) and HLA-B*0702 (the TPR and RPH epitopes of pp65). In vivo expansions of hCMV-specific T-lymphocytes were observed within days to weeks of adoptive transfer and temporally associated with periods of viral replication. Peak pp65-directed responses following infection ranged between 6.7–43% of CD8+ T cells, with absolute levels of 60.2–371.5 × 106/l, well in excess of those previously reported to offer protection against CMV (10–20 × 106/l), whilst those in patients not experiencing viral DNAemia reached maximal levels of 0.5–0.8% of CD8+ T cells equivalent to absolute levels of 0.2–0.8 × 106/l. Assuming transfused cells are largely responsible for the increase in CMV-specific T cell numbers, at least in the T-cell depleted unrelated donor setting which is associated with markedly impaired immune reconstitution within the first 100 days following transplantation, we estimate that NLVspecific T cells were expanded up to 5 log over a period as short as 10 days following transfer (assuming a 5 litre circulating volume and that 2% of the lymphocyte pool is located in the peripheral blood) with lymphocyte doubling times possibly as low as every 12 hours. Expanding populations maintained functional competence in terms of ability to produce IFNγ in ex vivo restimulation assays (up to 77% of pentamer-labelling cells secreted IFNγ). Following a primary post-transplantation treatment episode 20 patients were evaluable for subsequent viral infection. With a median follow-up of 953 days (range 250–1590) none developed a second episode requiring therapy (compared to 45/72 historical controls, P < 0.0001) and there were no cases of CMV disease. Combined with the absolute circulating levels attained and the functional competence of CMV-specific T cells following transfer, these data indicate that infusion of cell lines containing both CD4+ and CD8+ virus-specific T cells promotes reconstitution of durable functional CMV-specific immunity, effectively preventing recurrent viral infection and late CMV disease in a group of high risk aHSCT recipients.

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