scholarly journals Limiting dilution comparison of the repertoires of high and low responder MHC-restricted T cells.

1988 ◽  
Vol 167 (3) ◽  
pp. 1100-1113 ◽  
Author(s):  
M Kojima ◽  
K B Cease ◽  
G K Buckenmeyer ◽  
J A Berzofsky
Keyword(s):  
T Cells ◽  
Cell Lines ◽  
Sperm Whale ◽  
Cell Repertoire ◽  
Restriction Element ◽  
Low Responder ◽  
Low X ◽  
Limiting Dilution ◽  
Immunodominant Epitopes

To approach the mechanism that determines Ir gene-controlled high or low responsiveness to whole proteins, such as sperm whale myoglobin (SWMb), we compared the repertoires of high and low responder haplotype-restricted T cells for different myoglobin epitopes by limiting dilution frequency analysis. Poisson analysis was performed using long-term limiting dilution cell lines of (B10.BR [low] X B10.D2[high])F1 T cells maintained on high or low responder APCs. The cell lines were tested with SWMb peptides and fragments for T cell repertoire fine specificities and Ia restrictions. The frequency of SWMb-specific F1 T cells responsive on B10.BR (H-2k) APCs was 2.5-3.6-fold lower than on B10.D2 (H-2d) APCs. Strikingly, all of the H-2k-restricted T cells used I-Ek as a restriction element, whereas both I-Ad- and I-Ed-restricted T cells were found among the H-2d-restricted lines. The I-Ad-restricted T cells were dominant, and the majority was specific for the synthetic peptide 102-118. T cells specific for peptide 132-146, dominant in association with I-Ed, were less frequent. However, no detectable H-2k-restricted T cells were specific for either of these peptides, but instead they were specific for fragment 1-55 or peptide 59-80. Fragment 1-55 also stimulated a similar number of H-2d-restricted T cells. Therefore, the low response of F1 T cells on H-2k-presenting cells may be due to the failure to see myoglobin plus I-Ak, in particular the immunodominant site around Glu 109, in contrast to the dominant response of high responder mice (both H-2d and H-2s) focused on the I-A molecule and the site around residue Glu 109. The I-E- low responder B10 strain also failed to respond to peptide 102-118, supporting the idea that the low responder status results from a limited repertoire lacking response to 102-118 plus I-A. In those strains that respond to the immunodominant site 102-118, the frequency of T cells in the repertoire specific for this site was always considerably greater than that for other sites. These results suggest that there is an important difference between immunodominant epitopes and minor epitopes and that Ir gene-controlled low responsiveness to a natural whole protein may be due primarily to the failure to respond to a single immunodominant site, even though a number of other epitopes can be recognized.

scholarly journals Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominanttrans-Sialidase-Specific CD8+T Cells

2016 ◽  
Vol 84 (9) ◽  
pp. 2627-2638 ◽  
Author(s):  
Charles S. Rosenberg ◽  
Weibo Zhang ◽  
Juan M. Bustamante ◽  
Rick L. Tarleton
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Trypanosoma Cruzi ◽  
T Cell Responses ◽  
Content Type ◽  
Cell Responses ◽  
Pathogen Control ◽  
Immunodominant Epitopes

Trypanosoma cruziinfection drives the expansion of remarkably focused CD8+T cell responses targeting epitopes encoded by varianttrans-sialidase (TS) genes. Infection of C57BL/6 mice withT. cruziresults in up to 40% of all CD8+T cells committed to recognition of the dominant TSKB20 and subdominant TSKB18 TS epitopes. However, despite this enormous response, these mice fail to clearT. cruziinfection and subsequently develop chronic disease. One possible reason for the failure to cureT. cruziinfection is that immunodomination by these TS-specific T cells may interfere with alternative CD8+T cell responses more capable of complete parasite elimination. To address this possibility, we created transgenic mice that are centrally tolerant to these immunodominant epitopes. Mice expressing TSKB20, TSKB18, or both epitopes controlledT. cruziinfection and developed effector CD8+T cells that maintained an activated phenotype. Memory CD8+T cells from drug-cured TSKB-transgenic mice rapidly responded to secondaryT. cruziinfection. In the absence of the response to TSKB20 and TSKB18, immunodominance did not shift to other known subdominant epitopes despite the capacity of these mice to expand epitope-specific T cells specific for the model antigen ovalbumin expressed by engineered parasites. Thus, CD8+T cell responses tightly and robustly focused on a few epitopes within variant TS antigens appear to neither contribute to, nor detract from, the ability to controlT. cruziinfection. These data also indicate that the relative position of an epitope within a CD8+immunodominance hierarchy does not predict its importance in pathogen control.

Production of Lymphokines by Murine T Cells Grown in Limiting Dilution and Long-Term Cultures

1982 ◽  
pp. 253-262 ◽  
Author(s):  
Peter H. Krammer ◽  
Michael Dy ◽  
Lothar Hultner ◽  
Peter Isakson ◽  
Ursula Kees ◽  
...  
Keyword(s):  
T Cells ◽  
Limiting Dilution

scholarly journals The isolation and sequence of a novel gene from a human functional T cell line.

1987 ◽  
Vol 165 (3) ◽  
pp. 601-614 ◽  
Author(s):  
J Jongstra ◽  
T J Schall ◽  
B J Dyer ◽  
C Clayberger ◽  
J Jorgensen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cdna Clone ◽  
Cell Lineage ◽  
Subtractive Hybridization ◽  
Protein Product ◽  
Novel Gene ◽  
Human T Cell

Using a subtractive hybridization procedure we have constructed a cDNA library enriched for sequences present in functional human T cell lines, but not in human EBV-transformed B cell lines. We have isolated a cDNA clone, AH2-519, representing a novel gene, designated 519. This novel gene is expressed in functional human cytolytic and Th cell lines but not in a variety of other cell lines, including several long-term human T cell tumor lines. The expression of gene 519 is inducible in cultures of normal human PBL using antigenic or mitogenic stimulation. Neither the DNA sequence determined from a full-length cDNA clone overlapping with clone AH2-519 nor the amino acid sequence of its predicted protein product has significant homology to published sequences in the GenBank or NBRF databases. The restricted expression of gene 519 suggests that its gene product is involved in the growth and/or differentiation of normal T cells. The data also show that normal, nontransformed, functional T cells express gene products that can not be readily identified in long-term tumor lines of the same cell lineage.

Reconstitution of the T Cell Repertoire Following Treatment with Alemtuzumab in Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2986-2986
Author(s):  
Mohammad R. Rezvany ◽  
Mahmood J. Tehrani ◽  
Claes Karlsson ◽  
Jeanette Lundin ◽  
Hodjattallah Rabbani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Long Term Follow Up

Abstract Background and Methods: B-cell chronic lymphocytic leukemia (B-CLL) occurs as a result of clonal accumulation of functionally abnormal B cells. Alemtuzumab is a humanized monoclonal antibody specific for the CD52 antigen, which is highly expressed on both B-CLL cells and normal lymphocytes, but not on hematopoietic (CD34) stem cells. Alemtuzumab has been shown to effectively deplete the blood and bone marrow of lymphocytes, including CD4 and CD8 T cells, which may lead to profound immunosuppression and make patients more susceptible to infections. We and others have previously shown that the CD4 T cells in B-CLL patients may be clonally distinct from the normal population in that they present a more clonal pattern of the T-cell receptor (TCR) repertoire (Rezvany et al, Blood2003;101:1063–1070). It is therefore of interest to study the T cell repertoire following alemtuzumab administration as well as factors affecting T cell reconstitution following CD52 targeted therapy. In this study, we evaluated in depth the T-cell receptor-beta-variable sequence (TCR BV) in CD4 and CD8 T cells by real-time PCR, before and repeatedly after/during long term follow-up, in 5 B-CLL patients who had received alemtuzumab as first-line therapy (Lundin et al, Blood2002;100:768–773). Also, an analysis was conducted of CDR3 length polymorphism to describe changes in the clonality pattern. Results: A decline in most of BV genes either in CD4 or CD8 T cells was observed shortly after alemtuzumab treatment, which was followed by a gradual increase in most of the BV genes during long-term follow up. CDR3 length polymorphism analysis shortly after treatment revealed an even more highly restricted pattern in CD4 T cells compared to baseline with a shift towards a monoclonal/oligoclonal pattern regardless of increased or decreased BV usage. Furthermore, in the analysis of the clonal spectrum that was expressed shortly after alemtuzumab therapy, the number of peaks was significantly reduced in CD4 (P <0.01) but not in CD8 T cells, which was followed by a gradual increase in diversity towards a polyclonal repertoire during long-term follow up. Conclusions: These results indicate that perturbations in the T cell repertoire following alemtuzumab are complex, and are not reflected by changes in CD4/CD8 T cell numbers only. The restricted CDR3 pattern present prior to therapy became even more restricted after end of treatment, followed by a normalization of CDR3 patterns in CD4 T-cells during long-term follow-up. These results further suggest a regulatory role for T cells in relation to the malignant B cell clone in patients with B-CLL.

Generation of Acute Myeloid Leukemia (AML)-Specific Autologous CD4 and CD8 T Cell Lines by Limiting Dilution AML Dendritic Cell Culture.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2018-2018
Author(s):  
Rui-kun Zhong ◽  
Thomas A. Lane ◽  
Edward D. Ball
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Cd8 T Cell ◽  
Ifn Gamma ◽  
T Cell Lines ◽  
Limiting Dilution ◽  
Acute Myeloid

Naturally occurring cytotoxic T cells directed against various leukemia associated antigens (LAA) expressed by acute myeloid leukemia (AML) cells have been described. However, these LAA-specific T cells are rare and obviously unable to initiate effective anti-leukemia responses. The challenge is how to investigate, select, activate and expand the rare LAA-specific T cells from the vast population of blood cells in patients with AML for immunotherapy. Based on our studies of inducing AML dendritic cell (AMLDC) differentiation and priming in situ AML-reactive T cells, we have developed a novel method of generating multiple autologous AML reactive T cell lines by limiting dilution AMLDC (LD-AMLDC) culture. The principle of LD-AMLDC is based on the assumption that autologous AML-reactive T cells or precursors are randomly distributed in the AML PBMC suspension, and that each one has an equal opportunity to respond to AML cells in the 96-well plates under optimized culture condition. By culturing AML PBMC (>90% blasts) in culture medium supplemented with GM-CSF/IL4/IL2/IL7/IL12 to induce AML DC differentiation and activate in situ autologous T cells, highly reactive anti-AML T cell lines (both CD4+ and CD8+ lines) were selected and expanded from LD-AMLDC culture using the appropriate numbers of AML PBMC in each culture well by the criterion of release of IFN-gamma in response to autologous AML blasts. By maximum likelihood solution, the estimated average frequency of AML reactive T cells or precursors is 6±3/1,000,000 AML PBMC (n=8). Strong intracellular IFN-gamma release of T cell lines obtained in LD-AMLDC was demonstrated by flow cytometry analysis after stimulation by autologous AML cells but not autologous B-lymphoblastoid cell line (LCL) (Figure). Effective specific lysis (up to 70% at E:T=20:1) of autologous AML cells but not autologous LCL or allogeneic AML cells by these T cell lines was observed. Two PR1 specific T cell lines were obtained by screening 39 AML reactive HLA-A2+ CD8+ T cell lines generated from 5 LD-AMLDC cultures, suggesting that other unidentified CD4 or CD8 lines with strong autologous AML responses may be reactive to known or unknown LAAs. These results encourage continued efforts to induce, activate and select T cells lines with high autologous AML reactivity using LD-AMLDC culture and to expand multi-LAA reactive T cell lines acquired from limiting dilution AML-DC culture for AML immunotherapy. Figure Figure

Exploiting Endogenous Micro-RNAs to Avoid off-Target Transgene Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3296-3296
Author(s):  
Raul Teruel Montoya ◽  
Xianguo Kong ◽  
Shaji Abraham ◽  
Lin Ma ◽  
Leonard C. Edelstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Cell Lines ◽  
Binding Sites ◽  
Transgene Expression ◽  
Cell Types ◽  
Jurkat Cells ◽  
Cell Type ◽  
Hematological Disorders

Abstract Abstract 3296 Genetic modification of hematopoietic stem cells (HSCs) has the potential to benefit acquired and congenital hematological disorders. Despite the use of so-called “tissue-specific” promoters to drive expression of the desired transgene, off-target (and consequent deleterious) effects have been observed. MicroRNAs (miRNAs) are important regulators of gene expression. They associate with Argonaute proteins and most typically target 3'UTRs, where complementary base-pairing results in repressed gene expression via RNA decay and translation inhibition. Most miRNAs are ubiquitously expressed, and although some are claimed to be “tissue specific,” such claims have generally not been rigorously validated. The long-term goal of this work is identifying “cell preferential” miRNA expression that could be exploited in expression vectors to minimize off-target transgene expression in HSCs. Initially, total RNA was extracted with Trizol from the megakaryocyte and T-lymphocyte cell lines, Meg-01 and Jurkat, and miRNAs were profiled by Nanostring technology (Nanostring Technologies, Denver, CO). MiR-495 was determined to be highly expressed in Meg-01 and very low in Jurkat cells. A luciferase reporter construct was generated with four canonical binding sites for miR-495 in the 3'UTR and transfected into both cell lines. Compared to control vector without miR-495 binding sites, luciferase expression showed a 50% reduction in Meg-01 cells, but no knock down in Jurkat cells. These experiments indicated that different levels of endogenous miRNA levels can regulate transgene expression through a novel design in the 3'UTR. We next turned our attention to human hematopoietic cells. We reasoned that the long-term goal of minimal off-target transgene expression in HSCs would require knowledge of miRNAs that had little or no detectable expression (“selectively reduced [SR]”) in one cell type and were highly expressed in other cell types. In this manner, the transgene expression would be dampened only in the non-target cells. As a surrogate for bone marrow progenitors and as proof of principle, we used primary cells in normal human peripheral blood. T-cells, B-cells, platelets and granulocytes were purified by density centrifugation followed by immunoselection from five healthy human donors. Flow cytometry using membrane specific markers demonstrate >97% purity of each specific cell preparation. Total RNA was extracted and miRNAs were profiled as above. First, we identified 277 miRNAs that were differentially expressed between any pair of cell types (p-value<0.05 by ANOVA). Second, we performed ranked pair-wise comparisons across all cell types to determine SR miRNAs. This analysis revealed 5 platelet SR-miRNAs, 6 B-cell SR-miRNAs, 2 T-cell SR-miRNAs and 4 granulocyte SR-miRNAs. Lastly, we considered which of these 17 SR-miRNAs would be the best single SR-miRNA within and across cell types. SR-miRNAs were normalized to let-7b, a miRNA we determined to be equivalently expressed across all cell types, and hence, an ideal normalizer. Lineage-specific SR-miRNAs were selected based on extremely low expression in only one cell type and highest fold change of expression compared to the other cell types. The best SR-miRNAs were miR-29b (SR in platelets), miR-125a-5p (SR in B-cells) and miR-146a (SR in granulocytes). The SR expression levels of these 3 miRNAs were validated by qRT-PCR. Our analysis identified no good SR-miRNAs in T-cells. On-going experiments are testing the selective effects of the SR miRNAs in lentiviral vector infection of cord blood CD34+ cells differentiated along specific lineages. In summary, we have demonstrated in hematopoietic cell lines that SR endogenous miRNAs can regulate the expression of transgenes via tandem arrangement of their target sites in the 3'UTR. Additionally, we have identified miRNAs that are specifically expressed at a very low level in one blood cell type and at high levels in other cell types. These miRNAs could potentially be utilized as new biological tools in gene therapy for hematological disorders to restrict transgene expression and avoid the negative consequences of off-target expression. Disclosures: No relevant conflicts of interest to declare.

Production of colony-stimulating factors by murine T cells in limiting dilution and long-term cultures

Nature ◽  
1982 ◽  
Vol 298 (5869) ◽  
pp. 79-82 ◽  
Author(s):  
Fritz G. Staber ◽  
Lothar Hültner ◽  
Fabrizio Marcucci ◽  
Peter H. Krammer
Keyword(s):  
T Cells ◽  
Colony Stimulating Factors ◽  
Limiting Dilution

Targeting of glioblastoma with activated T cells armed with anti-CD3xanti-HER2/neu (HER2Bi) and anti-CD3xanti-EGFR (EGFRBi) bispecific antibodies

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 3038-3038
Author(s):  
I. M. Zitron ◽  
O. Norkina ◽  
Z. Al-Kadhimi ◽  
G. R. Barger ◽  
L. G. Lum ◽  
...  
Keyword(s):  
T Cells ◽  
Malignant Glioma ◽  
Cell Lines ◽  
Malignant Gliomas ◽  
Interleukin 2 ◽  
Bispecific Antibodies ◽  
Cell Populations ◽  
Activated T Cells ◽  
Primary Glioblastoma

3038 Background: Malignant gliomas are the most common primary brain tumors in adults. The prognosis for patients with glioblastoma remains poor despite aggressive multimodality treatment including surgery and chemoradiotherapy. The receptor tyrosine kinases EGFR, mutant EGFR (EGFRvIII), and HER2/neu are expressed on the majority of glioblastomas and are potential targets for activated T cells (ATCs) armed with bispecific antibodies (BiAbs). Methods: ATCs were generated from human peripheral blood mononuclear cells (PBMC) by culture for 14 days with monoclonal anti-CD3 and interleukin-2 and armed with HER2Bi and/or EGFRBi. HER2Bi- and/or EGFRBi-armed ATCs were examined for in vitro cytotoxicity (MTT and 51Cr release assays) against long-term malignant glioma lines (U87MG, U118MG, and U251MG) as well as primary glioblastoma lines derived from surgical specimens. Expression of EGFR and HER2/neu were evaluated by FACS. Anti-CD133 coated magnetic microbeads were used to separate CD133-positive and CD133-negative cell populations. Results: EGFRBi-armed ATCs killed up to 85% of U87, U118, and U251 targets. HER2Bi-armed ATCs exhibited comparable cytotoxicity against U118 and U251, but did not kill HER2-negative glioma U87. Cytotoxicity exhibited by either HER2Bi- or EGFRBi-armed ATCs against four primary glioblastoma cell lines was 50–80%. We found that both CD133-negative and CD133-positive cell populations were susceptible to killing by armed ATCs. When we armed ATCs simultaneously with HER2Bi and EGFRBi, killing by doubly armed ATCs was equal to or greater than that by EGFRBi-armed ATCs against the tested cell lines. Conclusions: BiAbs efficiently target ATCs to kill EGFR and/or HER2/neu expressing glioblastomas. Long-term malignant glioma cell lines and primary lines derived from surgical specimens are equally susceptible. Both CD133-negative and CD133-positive (the putative glioma stem cells) are killed. ATCs armed with BiAbs represent a potentially valuable adjuvant to current treatment. No significant financial relationships to disclose.

scholarly journals Influences of antigen processing on the expression of the T cell repertoire. Evidence for MHC-specific hindering structures on the products of processing.

1988 ◽  
Vol 168 (1) ◽  
pp. 357-373 ◽  
Author(s):  
S J Brett ◽  
K B Cease ◽  
J A Berzofsky
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Processing ◽  
Sperm Whale ◽  
T Cell Response ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Similar Dose ◽  
Sperm Whale Myoglobin

Two lines of evidence in the current study indicate that antigen processing is a major factor, in addition to MHC binding and T cell repertoire, that determines Ir gene responsiveness and epitope immunodominance. First, immunization with synthetic peptides of myoglobin sequences revealed new reactivities that had not appeared after priming with native myoglobin. For example, B10.S mice (H-2S) immune to equine myoglobin predominantly responded to peptide 102-118, whereas there was little, if any, response to this peptide in B10.BR (H-2k) mice immunized with native equine myoglobin. However, after immunization with the 102-118 peptide, both strains responded to the peptide. After in vitro restimulation, B10.BR T cells responded as well as B10.S T cells. Similarly, some individual 102-118-specific T cell clones from mice of both haplotypes showed similar dose responses and fine specificity patterns. Thus, low responsiveness to this site is due neither to a hole in the repertoire nor to a failure to bind to the appropriate MHC molecule. An alternative explanation was suggested by the observation that, whereas B10.S T cells from peptide 102-118-immune mice responded almost as well to whole myoglobin as to the peptide, the B10.BR T cells from peptide immune mice, while responding well to peptide, were poorly stimulated by whole myoglobin. Thus, the product of natural processing of equine myoglobin probably has hindering structures in the regions flanking the core epitope 102-118 that interfere with presentation by I-Ak but not I-AS. The second line of evidence that processing of native myoglobin may influence the apparent specificity of the T cell response was obtained using the I-Ad-restricted sperm whale myoglobin 102-118-specific clone 9.27. This clone discriminated readily between whole sperm whale myoglobin and equine myoglobin, but it did not distinguish between peptides corresponding to 102-118 of the sperm whale and equine sequences. This distinction between equine peptide and native equine myoglobin could be overcome by artificial "processing" of equine myoglobin with cyanogen bromide. In both sets of experiments, F1 APCs that present the same epitope well to T cells of another haplotype failed to overcome the defect, which was therefore not due to the availability of different processed cleavage fragments in APC of different haplotypes, as would be expected if there were MHC-linked processing. Thus, the differential responses to peptides versus native molecule for both I-Ad- and I-Ak-restricted clones appeared to depend on the restricting molecule used.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals A New Promising Third Generation CAR-CD30 T-Cell Therapy for CD30+ Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2069-2069
Author(s):  
Biagio De Angelis ◽  
Marika Guercio ◽  
Domenico Orlando ◽  
Stefano Di Cecca ◽  
Matilde Sinibaldi ◽  
...  
Keyword(s):  
T Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Short Term ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T

Prognosis of a significant proportion of patients with chemotherapy-refractory or multiply-relapsed CD30+ Non-Hodgkin's Lymphoma (NHL) or Hodgkin lymphoma (HL) still remain poor. Targeting CD30 with monoclonal antibodies in HL and anaplastic large cell lymphoma was shown to induce remarkable clinical activity; however, occurrence of adverse events (mainly neuropathy) may result into treatment discontinuation in many patients. Immunotherapeutic approaches targeting CD30 by chimeric antigen receptor (CAR) has been demonstrated to be of value in two independent clinical trials, although clinical benefit was sub-optimal. We designed a new CAR construct characterized by an anti-CD30 single-chain variable-fragment cassette (AC10), linked to CD3ζ by the signaling domains of two costimulatory molecules, namely either CD28.4-1BB or CD28.OX40. The inducible Caspase-9 (iCasp9) safety switch was included in both constructs with the goal of promptly controlling undue toxicity. As a selectable marker, we added in frame the CD34 antigen. The in vitro anti-tumor efficacy was evaluated by using either the NHL cell line: Karpas299, or the HL cell lines: L428, in both short-term cytotoxic assay (51Cr release assays) and long-term co-cultures for 6 days. Supernatant from co-culture experiments was analyzed by Elisa. We assessed the antitumor effect of CAR.CD30 T cells in a in vivo NSG mouse model engrafted i.v. with lymphoma FF-luciferase cell lines Karpas299 or L428, and monitored tumor growth by IVIS Imaging system. For tumor re-challenging, mice of the NHL model surviving until day +140, were i.v. infused with 0.2x106 Karpas299 cells, and subsequently followed for additional 110 days. Persistence of CAR.CD30 T cells was evaluated, together with a deep characterization of memory profile of T cells. Independently from the costimulatory domains CD28.OX40 or CD28.4-1BB, the generated retroviral vectors showed similar transduction efficiency of T cells (86.5±5.1% and 79.3±5.3%, respectively). Nevertheless, CD28.OX40 costimulatory domains was associated with more stable expression of the CAR over time, during extensive in vitro culture (84.72±5.30% vs 63.98±11.51% CD28.4-1BB CAR T cells at 30 days after transduction; p=0.002). For both CAR constructs, we did not observe any significant difference in the suicide gene iCasp9 activity, both in vitro and in vivo. In short-term cytotoxic assay, both CAR.CD30 T cells significantly and specifically lysed CD30+ NHL and HL tumor cell lines. In long-term co-culture, CD28.OX40 showed a superior anti-lymphoma in vitro activity as compared to CD28.41BB T cells, when challenged at very high tumor/effector ratio (8:1) (for Karpas 299; p=0.03). Moreover, the antigen stimulation was associated to higher levels of Th1 cytokine production, with CD28.OX40 T cells secreting a significantly higher amount of IFNγ, IL2 and TNFα as compared to CD28.41BB T cells (p= 0.040; p=0.008; p=0.02; respectively). Bioluminescence in HL (L428) tumor-bearing mice, treated with NT T cells, rapidly increased up to 5 log in less than 50 days and mice either died or were sacrificed due to morbidity. The best outcome was observed in mice treated with CD28.OX40, as three out of five mice were still alive at the experimental end-point of day+165, as compared with mice treated with CD28.4-1BB (60% vs 0%, p=0.0021). In NHL (Karpas 299) mouse models, CD28.OX40 had an extensive anti-tumor control superior to that of CD28.41BB T cells, leading to a significant reduction of tumor bioluminescence at day 45 (3.32x10 vs 2.29x10, p=0.04). The median survival of mice treated with NT and CD28.4-1BB CAR T cells was 45.5 and 58 days respectively, but undetermined for mice treated with CD28.OX40 CAR T cells (p=0.0002). After 140 days, cured mice were re-challenged with Karpas 299; mice were followed for other 100 days. Bioluminescence analysis showed rapid progression of the tumor in the control mice cohort, as well as in CD28.4-1BB treated mice. In contrast, in CD28.OX40 treated mice, at day+240 days, 4 out of 6 mice were tumor-free, resulting into a statistically significant survival benefit (p=0.0014). Only in mice treated with 28.OX40 T cells, we observed a long-lasting persistence of circulating CAR-T cells up to day +221. In summary, we have developed a novel CAR.CD30 construct displaying features that make it a particularly suitable candidate for a clinical trial in patients suffering from CD30+ tumors. Disclosures Merli: Novartis: Honoraria; Sobi: Consultancy; Amgen: Honoraria; Bellicum: Consultancy. Locatelli:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BluebirdBio: Consultancy; Miltenyi: Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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