Co-Administration Of Gene-Modified CD4+ T Cells Targeting HLA Class I-Restricted WT1 Epitope Diversely Enhances The Antitumor Effect Mediated By Redirected T-Cell Based Adoptive Immunotherapy Against Human Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 144-144
Author(s):  
Hiroshi Fujiwara ◽  
Fumihiro Ochi ◽  
Toshiki Ochi ◽  
Hiroaki Asai ◽  
Yukihiro Miyazaki ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells

Abstract Purpose In the context of redirected T-cell based antitumor adoptive immunotherapy, the therapeutic roles played by co-infused CD4+ T cells genetically redirected to the predefined HLA class I-restricted epitope which had been originally recognized by effector CD8+ T cells has not yet been fully discussed. In this study, using an HLA class I-restricted WT1 -specific T-cell receptor (TCR) gene transfer, we in detail examined antileukemia functionality mediated by these gene-modified CD4+ T cells co-infused with similarly gene-modified effector CD8+ T cells as the redirected T cell-based adoptive immunotherapy. Methods Using our unique retroviral vector expressing HLA-A*2402-restricted and WT1235-243-specific TCR a/b genes and shRNAs for endogenous TCRs (WT1-siTCR vector), we genetically modified both CD4+ and CD8+ T cells from the same healthy donor or leukemia patients (termed WT1-siTCR/CD4 and WT1-siTCR/CD8, respectively). First, target-responsive cellular outputs mediated by WT1-siTCR/CD4 was thoroughly examined using flowcytometry, ELISA, 51Cr-release assay, CFSE dilution assay and bioluminescence assay. Next we similarly assessed impacts of WT1-siTCR/CD4 on the antileukemia functionality mediated by concurrentWT1-siTCR/CD8 both in vitro and in vivo. Eventually, we assessed the in vivo therapeutic efficacy of combined administration of WT1-siTCR/CD8 with WT1-siTCR/CD4 using a xenografted mouse model. Results The transcription factor profile demonstrated that WT1-siTCR/CD4 turned a terminal effector, but not regulatory phenotype. Activated WT1-siTCR/CD4 expressed cell-surface CD40L. Target-responsive cytokine production profile of WT1-siTCR/CD4 represented the Th1 helper function in the context of HLA-A*2402. HLA class II molecules expressed by leukemia cells facilitated the recognition of leukemia cells by WT1-siTCR/CD4 in the context of HLA-A*2402. WT1-siTCR/CD4 displayed the delayed cytocidal activity determined by 51Cr release assay. WT1-siTCR/CD4 could produce IFN-g in response to freshly isolated leukemia cells. WT1-siTCR/CD4 displayed the leukemia trafficking activity in vivo. WT1-siTCR/CD4 represented the potential to migrate into bone marrow via CXCR4/CXCL12 axis both in vitro and in vivo. Concurrent WT1-siTCR/CD4 augmented IFN-g production and cytotoxic degranulation mediated by WT1-siTCR/CD8 in response to the cognate epitope via humoral factors. Consequently, the cytocidal activity against autologous leukemia cells mediated by WT1-siTCR/CD8 was augmented in the presence of WT1-siTCR/CD4, both of them generated from normal lymphocytes of the same patient with leukemia in a complete remission. Upon the target recognition, activated WT1-siTCR/CD4 recruited WT1-siTCR/CD8 via CCL3/4-CCR5 axis. Proliferative response and differentiation into central memory T-cell subset mediated by WT1-siTCR/CD8 in response to the cognate epitope and leukemia cells were enhanced in the presence of autologousWT1-siTCR/CD4, but not gene-modified CD4+ T cells (NGM-CD4). CD127 expression on activated WT1-siTCR/CD8 also increased in parallel to this differentiation. Co-infused WT1-siTCR/CD4 augmented the tumor trafficking and persistence of WT1-siTCR/CD8 in vivo, resulting in the greater suppression of leukemia cells in a xenografted mouse model. Finally, in the therapeutic mouse model, co-infusion of WT1-siTCR/CD8 with of WT1-siTCR/CD4 significantly suppressed the growth of inoculated leukemia cells compared to that in mice received co-infusion of WT1-siTCR/CD8 with NGM-CD4 (Fig.1). Correlation between the therapeutic efficacy and survival of infused gene-modified T cells was also observed. Conclusion In results, the combined infusion of WT1-siTCR/CD8 with WT1-siTCR/CD4, but not NGM-CD4 obviously demonstrates the enhanced antileukemia efficacy via diverse mechanisms. Now we have just started a clinical trial using gene-modified T cells with WT1-siTCR vector for the treatment of patients with refractory acute myeloid leukemia and myeloid dysplastic syndrome. Because redirected T cells employed in this trial encompassed both WT1-siTCR/CD4 and WT1-siTCR/CD8, we are planning to clinically verify the significance of WT1-siTCR/CD4 in the redirected T cell-based antileukemia adoptive immunotherapy. (Fig.1) Disclosures: No relevant conflicts of interest to declare.

Redirected CD4+ T Cells towards HLA Class I-Restricted WT1 Epitope Successfully Display Multifactorial Helper Function for the Enhancement of Antileukemia Efficacy Mediated by Redirected T-Cell Based Immunocelltherapy.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3153-3153
Author(s):  
Yukihiro Miyazaki ◽  
Hiroshi Fujiwara ◽  
Toshiki Ochi ◽  
Sachiko Okamoto ◽  
Hiroaki Asai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Proliferation And Differentiation ◽  
Ifn Γ

Abstract Abstract 3153 Purpose: In antitumor adoptive immunotherapy, the utility of tumoricidal CD8+ T cells are mainly highlighted, while in tumor immunity, the importance of tumor-reactive CD4+ T cells is also well documented. However, because the number of well-characterized tumor-associated epitopes recognized by CD4+ T cells still remains small, application of tumor-reactive CD4+ T cells is limited. In order to circumvent this drawback, redirection of CD4+ T cells to well-characterized HLA class I-restricted CD8+ T-cell epitope seems promising. In this study, using an HLA class I-restricted and WT1-specific T-cell receptor (TCR) gene transfer, we, in detail, examined helper functions mediated by those gene-modified CD4+T cells in redirected T cell-based antileukemia adoptive immunotherapy. Methods: HLA-A*2402-restricted and WT1235–243-specific TCR α/β genes were inserted into our unique retroviral vector encoding shRNAs for endogenous TCRs (WT1-siTCR vector), and was employed for gene-modification both of CD4+ and CD8+ T cells to express WT1-specific TCR. (1) WT1 epitope-responsive cytokine production mediated by WT1-siTCR-transduced CD4+ T cells (WT1-siTCR/CD4) was measured using bead-based immunoassay and ELISA assay. (2) WT1 epitope-ligation induced co-stimulatory molecules by WT1-siTCR/CD4 was assessed using flow cytometry. (3) Impacts on WT1 epitope and leukemia-specific responses; cytocidal activity, proliferation and differentiation into memory T-cell phenotype, mediated by WT1-siTCR-transduced CD8+ T cells (WT1-siTCR/CD8) provided by concurrent WT1-siTCR/CD4 were assessed using 51Cr-release assay, CD107a/intracellular IFN-γ assay, CFSE dilution assay and flow cytometry. (4) WT1 epitope-ligation triggered chemokine production mediated by WT1-siTCR/CD4 was assessed using real-time PCR, then chemotaxis mediated by WT1-siTCR/CD8 in response to those chemokines was assessed using a transwell experiment. (5) In vivo tumor trafficking mediated by WT1-siTCR/CD4 was assessed using bioluminescence imaging assay. (6) Finally, WT1-siTCR/CD4-caused in vivo augmentation of antileukemia functionality mediated by WT1-siTCR/CD8 was assessed similarly using a xenografted mouse model. Results: WT1-siTCR/CD4 showed a terminal effector phenotype; positive for transcription factor T-bet, but negative for Bcl-6 or Foxp3. Upon recognition of WT1 epitope, WT1-siTCR/CD4 produced Th1, but not Th2 cytokines in the context of HLA-A*2402, which simultaneously required HLA class II molecules on target cells. WT1 epitope-ligation enhanced WT1-siTCR/CD4 to express cell-surface OX40. In the presence of WT1-siTCR/CD4, but not non-gene-modified CD4, effector functions mediated by WT1-siTCR/CD8 in response to WT1 epitope and leukemia cells, including cytocidal activity based on CD107a expression and IFN-γ production was enhanced. Such augmentation was mediated by humoral factors produced by WT1 epitope-ligated WT1-siTCR/CD4. Additionally, proliferation and differentiation into memory phenotype, notably CD45RA- CD62L+ central memory phenotype, mediated by WT1-siTCR/CD8 in response to both WT1 epitope and leukemia cells were also augmented, accompanied with increased expression of intracellular Bcl-2 and cell-surface IL-7R. Next, CCL3/4 produced by activated WT1-siTCR/CD4 triggered chemotaxis of WT1-siTCR/CD8 which express the corresponding receptor, CCR5. Using bioluminescence imaging, intravenously infused WT1-siTCR/CD4 successfully migrated towards leukemia cells inoculated in a NOG mouse. Finally, co-infused WT1-siTCR/CD4 successfully augmented immediate accumulation towards leukemia cells and antileukemia reactivity mediated by WT1-siTCR/CD8 in a xenografted mouse model. Conclusion: Using GMP grade WT1-siTCR vector, redirected CD4+ T cells to HLA class I-restricted WT1 epitope successfully recognized leukemia cells and augmented in vivo antileukemia functionality mediated by similarly redirected CD8+ T cells, encompassing tumor trafficking, cytocidal activity, proliferation and differentiation into memory cells. The latter seem to support the longevity of transferred antileukemia efficacy. Taking together, coinfusion of redirected CD4+ T cells to HLA class I-restricted WT1 epitope seems feasible and advantageous for the successful WT1-targeting redirected T cell-based immunotherapy against human leukemia. Disclosures: No relevant conflicts of interest to declare.

Extracellular Domains of CD8a and β Subunits Are Required and Sufficient for HLA Class I Restricted Helper Activity of TCR-Engineered CD4+ T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3574-3574
Author(s):  
Marleen M van Loenen ◽  
Renate S. Hagedoorn ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
Mirjam H.M. Heemskerk
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Extracellular Domain ◽  
Class I ◽  
Α Subunit ◽  
Extracellular Domains ◽  
Helper Activity

Abstract Abstract 3574 Poster Board III-511 Adoptive transfer of T cell receptor (TCR)-transferred T cells may be an attractive strategy to treat patients with hematological malignancies relapsing after allogeneic stem cell transplantation. Transfer of HLA class I restricted TCRs into CD8+ T cells demonstrated redirected antigen specificity. However, for persistence of anti-leukemic responses in vivo, CD4+ T cells may be important. Therefore, redirecting specificity of CD4+ T cells with well defined HLA class I restricted TCRs might be an attractive strategy for providing help. HLA class I restricted TCRs mostly are CD8-dependent, so for optimal HLA class I restricted reactivity, it was demonstrated that co-expression of the CD8-coreceptor is necessary. The CD8 molecule is expressed on the T cell surface as an αα or an αβ dimer. The α subunit of the CD8 coreceptor binds to the non-polymorphic residues in the α3 domain of the HLA class I molecules thereby enhancing the avidity of the TCR/MHC complex, and the cytoplasmatic tail of the α subunit directly associates with the protein tyrosine kinase Lck (p56lck), promoting signal transduction after T cell activation. The β subunit of the CD8 coreceptor is able to strengthen the avidity of the CD8/MHC/TCR interaction via its extracellular domain, and the intracellular domain enhances the association with the intracellular molecules p56Lck and LAT. Previously, it was reported that for optimal HLA class I restricted specific reactivity with respect to proliferation, cytokine production and cytotoxicity, co-expression of the CD8αβ; coreceptor was needed whereas co-expression of the CD8αα coreceptor marginally increased HLA class I restricted functional activity. Since the regulation of the introduced TCR as well as the CD8 coreceptor in redirected CD4+ T cells will be mediated by retroviral LTRs, we prefer to co-transfer a signaling deficient CD8 coreceptor, thereby minimizing the risk of overstimulation of the redirected T cells. In this study, we investigated whether co-transfer of a signaling deficient CD8 coreceptor would still result in optimal HLA class I restricted functionality of HLA class I restricted TCR engineered CD4+ T cells. For this purpose, we constructed retroviral constructs encoding either wild type CD8α or CD8β subunits, CD8α subunits in which the LCK binding domain was mutated, CD8 subunits composed of the CD8α extracellular domain coupled to the intracellular CD8β signalling domain, and intracellular truncated CD8α or CD8β subunits. pp65-KYQ specific CD4+ T cells were isolated using the IFNγ capture assay and transduced with HLA class I restricted TCRs. Subsequently, TCR transduced virus specific CD4+ T cells were sorted based on marker gene expression, and transduced with the different CD8α and CD8β combinations. In agreement with previous studies we demonstrate that for optimal helper activity of the HLA class I restricted TCR transferred CD4+ T cells coexpression of the CD8αβ coreceptor was required. T cells produced IFNγ, TNFα and IL-2, upregulated CD40L and proliferated upon antigen specific stimulation of the HLA class I restricted TCR. Truncation of the intracellular domains of the CD8α and CD8β subunits did not change the functionality of the HLA class I restricted TCR transferred CD4+ T cells. Whereas in the thymus both the intracellular and extracellular domains of CD8β contribute independently to positive selection and development of CD8+ T cells, our results demonstrate that for optimal HLA class I restricted functionality of TCR modified virus specific CD4+ T cells only the extracellular domains of the CD8a and β subunits are required and sufficient. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 4473 Immune control of plasma cell disorders – in-depth analysis of Sox2 immunity in MGUS

2020 ◽  
Vol 4 (s1) ◽  
pp. 136-136
Author(s):  
Sandra Patricia Susanibar Adaniya ◽  
Casey L. Cummins ◽  
Miren L. Baroja ◽  
Beatriz Carreno ◽  
Gerald P. Linette ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Hla Class I ◽  
Cross Sectional Study ◽  
Class I ◽  
Cell Reactivity ◽  
Cross Sectional ◽  
Depth Analysis

OBJECTIVES/GOALS: We aim to identify and characterize anti-Sox2-specific CD8+ T cell responses in stable MUGS patients expressing HLA class I alleles-A*02:01 and /or -B*07:02. METHODS/STUDY POPULATION: Cross sectional study of patients with stable MGUS defined as stable serum paraprotein for ≥ 12 months from the MM Research Clinic at the Abramson Cancer Institute. Sox2 T cell reactivity will be assessed by IFN-γ ELISPOT assays. Rested PBMC will be pulsed with candidate Sox2-derived peptides predicted to display high affinity to HLA class I alleles and known to be processed and presented as determined by “targeted MS/MS” (mass spectrometry). The presence of anti-Sox2-specific CD8+ T cells will be confirmed in peptide/HLA multimer assays using flow cytometry. Anti-Sox2-specific CD8+ T cells will be characterized for HLA restriction and TCR αβ composition. RESULTS/ANTICIPATED RESULTS: Our work is still in progress. From Aug to Dec 2019, 22 MGUS subjects have been analyzed, 11 of which were found to have the HLA of interest. Positive Sox-2 reactivity by ELISpot was found in 3 subjects. DISCUSSION/SIGNIFICANCE OF IMPACT: Anti-Sox2 immune responses may maintain MGUS in a clinical indolent state by eliminating Sox2-expressing clonogenic MM cells. A detailed characterization of anti-Sox2 T cells followed by in-vivo assessment of their anti-myeloma activity could provide the foundation for a Sox2 based immunotherapy approach in MM.

scholarly journals A Novel Strategy for Off-the-Shelf T Cell Therapy Which Evades Allogeneic T Cell and NK Cell Rejection

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1711-1711
Author(s):  
Yong Zhang ◽  
Surbhi Goel ◽  
Aaron Prodeus ◽  
Utsav Jetley ◽  
Yiyang Tan ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Class Ii ◽  
Class I

Abstract Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of "off-the-shelf" T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a "missing-self" signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed. Methods. Here, we developed an immunologically stealth "off-the-shelf" T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Additionally, we utilize potent TCR-α and -β constant chain (TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo. Results. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. With a relatively small bank of donors, we can provide an "off-the-shelf" CAR or TCR-T cell solution for a large proportion of the population. Conclusions. We have successfully developed a differentiated "off-the-shelf" approach, which is expected to be safe and cost-effective. It is designed to provide long-term persistence without the need for an immune suppressive regimen. This promising strategy is being applied to our T cell immuno-oncology and autoimmune research candidates. Disclosures Zhang: Intellia Therapeutics: Current Employment. Goel: Intellia Therapeutics: Current Employment. Prodeus: Intellia Therapeutics: Current Employment. Jetley: Intellia Therapeutics: Current Employment. Tan: Intellia Therapeutics: Current Employment. Averill: Intellia Therapeutics: Current Employment. Ranade: Intellia Therapeutics: Current Employment. Balwani: Intellia Therapeutics: Current Employment. Dutta: Intellia Therapeutics: Current Employment. Sharma: Intellia Therapeutics: Current Employment. Venkatesan: Intellia Therapeutics: Current Employment. Liu: Intellia Therapeutics: Current Employment. Roy: Intellia Therapeutics: Current Employment. O′Connell: Intellia Therapeutics: Current Employment. Arredouani: Intellia Therapeutics: Current Employment. Keenan: Intellia Therapeutics: Current Employment. Lescarbeau: Intellia Therapeutics: Current Employment. Schultes: Intellia Therapeutics: Current Employment.

Control Of Cytomegalovirus Reactivation Post Stem Cell Transplantation Is Mediated By Simultaneous Reconstitution Of CD4+ and CD8+ CMV-Specific T Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4530-4530
Author(s):  
Mohammad Raeiszadeh ◽  
Annette Pachnio ◽  
Charles Craddock ◽  
Paul Moss ◽  
Frederick Chen
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Hla Class I ◽  
Viral Reactivation ◽  
Class I ◽  
Effector Memory ◽  
Cell Immunity ◽  
Cmv Reactivation

Haematopoietic stem cell transplant (HSCT) patients commonly suffer from Cytomegalovirus(CMV) reactivation due to the eradication and delayed reconstitution of CMV-specific T cell immunity resulting from T cell depletion and conditioning chemotherapy. Our knowledge of T cell immunity and in particular of antigen specific CD8+ T cell responses has advanced rapidly following the introduction of HLA- class I tetramers which enables the direct enumeration and characterisation of CMV-specific CD8+ T cells. In order to study the broader CMV specific T cells reconstitution post HSCT, we used a novel HLA-class II tetramer to monitor the reconstitution of CD4+ T cells specific to a CMV-derived peptide restricted to HLA-DRB1*0701, in parallel with different HLA class I tetramers identifying CMV specific CD8+ T cells. We analysed longitudinally the immune reconstitution of a cohort of thirteen HLA-DRB1*0701 –matched HSCT patients treated for haematological malignancies and who were at high risk of CMV reactivation where both donors and recipients were CMV seropositive. Twelve received reduced intensity conditioning and T-cell depletion with in vivo Alemtuzumab, and one had non-T cell depleted myeloablative conditioning. Twelve out of 13 longitudinally studied HSCT patients experienced CMV reactivation which included multiple episodes of viremia in 8 out of 12 .The viremia resolved in less than a month of onset in 8 patients where rapid expansion of CMV specific CD4+ and CD8+ T cells was observed in response to onset of viral reactivation. In four patients, late reconstitution of CMV-specific CD4 and CD8 T cells was observed between three and six months post HSCT; these patients suffered from prolonged and multiple episodes of CMV reactivation. In reconstituting patients, there was a considerable increase in the number of CMV-specific CD4+ and CD8+ T cells, from undetectable levels before viral reactivation up to 50x10^3 cells/ml and 370x10^3 cells/ml, respectively. CMV-specific CD4+ and CD8+ T cells expanded in parallel and statistically significant correlation between these cells were observed((p=0.06)). The patient treated with myeloablative conditioning chemotherapy retained considerable numbers of (CMV-specific CD4 cells (28.7x10^3 cells/ml) at four weeks post HSCT and did not have CMV reactivation. Phenotypic analysis showed that CMV specific CD4+ T cells were predominantly effector memory cells (CCR7-CD45RA-) whilst CD8+ T cells were predominantly effector memory/CD45RA+ cells. The majority of CMV specific T cells expressed CD57 molecule and we documented a strong correlation between expansion of specific CD4+ T cells and generation of CD4+CD57+ cells post HSCT. Both CD4 and CD8 T cells specific to CMV appear to be required for the control of viremia. The use of HLA class I and class II tetramers in combination with antibodies against surface markers such as CD57 provides a broader picture of the global T cell immune response to CMV and may inform on clinical outcome and treatment guidance. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single Cell RNA Sequencing Identifies Transcriptional Programs That Enhance Anti-Tumor Function of Transgenic CD4+ T Cells Redirected with TCR and CD8αβ

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 250-250
Author(s):  
Jan A Rath ◽  
Gagan Bajwa ◽  
Benoit Carreres ◽  
Isabelle Gruber ◽  
Elisabeth Hoyer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Leukemia Cells ◽  
Advisory Committees ◽  
Equity Ownership

Introduction:Transgenic co-expression of a major histocompatibility complex class I restricted tumor associated antigen specific TCR and CD8αβ (TCR8) has been previously proposed as a strategy to redirect CD4+ T cells to tumors. However, it is unknown whether forced TCR8 expression induces more fundamental transcriptional consequences in both CD4+ and CD8+ T cells, and whether T cell lineage origin affects this outcome. Here we deeply interrogate the effects of transgenic TCR and TCR8 in human CD4+ and CD8+ T cells upon leukemia challenge by single cell RNA sequencing (scRNAseq) and investigate T cell function in vitro and in vivo. We identify profound changes of gene expression that have significant functional consequences. Methods:A previously characterized HLA-A*02:01 restricted survivin-specific TCR was used (Arber et al, JCI, 2015 Jan;125(1):157-68) and a new polycistronic vector with this TCR and CD8αβ was generated. CD4+ and CD8+ T cells were isolated and scRNAseq (25'474 cells in total) was performed on (1) freshly isolated cells, (2) retrovirally transduced (TCR or TCR8) expanded cells, and (3) TCR+CD8+, TCR8+CD8+ and TCR8+CD4+ T cells co-cultured with BV173 leukemia cells (HLA-A*02:01+survivin+). scRNAseq results were cross-validated in independent experiments with FACS analysis of selected markers, in vitro stress-killing assays, analysis of cytokine production, and assessment of anti-tumor function in vivo in xenograft mice. Results:CD4+ T cells only killed BV173 leukemia cells when redirected with TCR8 but not with TCR alone (p=0.0004, n=7), while killing by TCR+CD8+ and TCR8+CD8+ T was comparable (p=NS). To explore some of the possible underlying mechanisms, we used dimensionality reduction and unsupervised clustering of the scRNASeq data and identified 19 distinct cell clusters. CD4+ and CD8+ lineage origin clearly separated the samples, but separation by transgene type only became apparent upon co-culture. Analyzing differentially expressed genes, we found that co-cultured samples contained clusters with high expression of cytotoxic markers but with significant differences between CD4+ and CD8+ lineages (e.g. transcription of GZMB in CD4+ T cells and GNLY, NKG7, GZMK in CD8+ T cells). Next, we analyzed which genes were upregulated from the expanded to co-cultured states. Co-cultured TCR8+CD4+ T cells had more upregulated genes with a broader diversity compared to TCR+CD8+ or TCR8+CD8+ T cells. Among these upregulated pathways were cytotoxicity, co-stimulation, oxidative phosphorylation, NFkB regulation, cell growth and transcription factors. TCR8+CD4+ T cells also retained a less differentiated phenotype (e.g. high IL7R, SELL, CCR7, CXCR4) with preservation of replicative potential. Furthermore, co-cultured TCR8+CD4+ T cells expressed more co-stimulatory and less activation/ exhaustion markers. In addition, co-cultured TCR8+CD4+ T cells heavily relied on oxidative phosphorylation and had higher mitochondrial activity compared to co-cultured TCR+ or TCR8+ CD8+ T cells. In stress co-cultures with multiple rounds of tumor challenge, TCR8+CD4+ T cells outperformed TCR+CD8+ T cells (number of killings TCR8+CD4+ vs TCR+CD8+: 3.3±0.5 vs 1.3±1.1, p=0.01, n=7), but were comparable to TCR8+CD8+ T cells (TCR8+CD4+ vs TCR8+CD8+: 3.3±0.5 vs 2±1.4,p=NS, n=7). TCR8+CD4+ T cells expanded significantly better than TCR+CD8+ T cells (p=0.002) and TCR8+CD8+ T cells (p=0.015) and produced TH1 type cytokines. In the xenograft mouse model, we observed significant BV173 leukemia control in mice treated with TCR+CD8+ T cells compared to controls (NT), and further enhancement in mice treated with TCR8+CD8+ T cells (NT vs TCR: p=0.0002, NT vs TCR8: p<0.0001, TCR vs TCR8: p=0.01, n=5). TCR8+CD4+ T cells also significantly delayed leukemia progression compared to TCR+CD4+ or NT T cells (p=0.001, n=5). Conclusion:Transgenic TCR8 expression has previously been proposed as a strategy to enhance TCR-pMHC recognition. Here we identify profound transcriptional changes involving multiple pathways that are important for sustained anti-tumor function upon adoptive T cell transfer in vivo, such as cytotoxicity, co-stimulation, cell cycle and metabolism. Our results point towards previously unrecognized mechanisms by which TCR8 transgenes mediate their beneficial effect in both CD4+ and CD8+ T cells. Disclosures Brenner: T Scan: Membership on an entity's Board of Directors or advisory committees; Marker Therapeutics: Equity Ownership; Allovir: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Equity Ownership; Memgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees. Arber:Cell Medica: Patents & Royalties.

scholarly journals Selective anergy of V beta 8+,CD4+ T cells in Staphylococcus enterotoxin B-primed mice.

1990 ◽  
Vol 172 (4) ◽  
pp. 1065-1070 ◽  
Author(s):  
Y Kawabe ◽  
A Ochi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Anergy ◽  
Specific Cytotoxicity ◽  
Enterotoxin B ◽  
Staphylococcus Enterotoxin B

The cellular basis of the in vitro and in vivo T cell responses to Staphylococcus enterotoxin B (SEB) has been investigated. The proliferation and cytotoxicity of V beta 8.1,2+,CD4+ and CD8+ T cells were observed in in vitro response to SEB. In primary cytotoxicity assays, CD4+ T cells from control spleens were more active than their CD8+ counterparts, however, in cells derived from SEB-primed mice, CD8+ T cells were dominant in SEB-specific cytotoxicity. In vivo priming with SEB abrogated the response of V beta 8.1,2+,CD4+ T cells despite the fact that these cells exist in significant number. This SEB-specific anergy occurred only in V beta 8.1,2+,CD4+ T cells but not in CD8+ T cells. These findings indicate that the requirement for the induction of antigen-specific anergy is different between CD4+ and CD8+ T cells in post-thymic tolerance, and the existence of coanergic signals for the induction of T cell anergy is suggested.

scholarly journals Efficient Targeting of Protein Antigen to the Dendritic Cell Receptor DEC-205 in the Steady State Leads to Antigen Presentation on Major Histocompatibility Complex Class I Products and Peripheral CD8+ T Cell Tolerance

2002 ◽  
Vol 196 (12) ◽  
pp. 1627-1638 ◽  
Author(s):  
Laura Bonifaz ◽  
David Bonnyay ◽  
Karsten Mahnke ◽  
Miguel Rivera ◽  
Michel C. Nussenzweig ◽  
...  
Keyword(s):  
T Cells ◽  
Major Histocompatibility Complex ◽  
Steady State ◽  
T Cell ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Class I ◽  
Histocompatibility Complex ◽  
Dc Maturation

To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal αDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c− cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When αDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4–48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of αDEC-205:OVA to DCs in the steady state initially induced 4–7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with αDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic αCD40 antibody produced large amounts of interleukin 2 and interferon γ, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.

Generation of Cytomegalovirus-Specific T Lymphocytes Using Protein-Spanning Pools of pp65-Derived Over-Lapping Pentadecapeptides for Adoptive Immunotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2236-2236
Author(s):  
Guenther Koehne ◽  
Deepa Trivedi ◽  
Roxanne Y. Williams ◽  
Richard J. O’Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Alleles ◽  
Hla Genotypes ◽  
T Cell Lines

Abstract Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We utilized a pool of 138 synthetic overlapping pentadecapeptides over-spanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T-cells and in analyses of T-cell lines from 5 separate HLA-A*0201+ individuals demonstrate that this pp65-derived pentadecapeptide-pool selectively induced T-cells specifically reactive against sub-pools of pentadecapeptides which contained the HLA-A*0201 binding epitope NLVPMVATV. The specificity of these T-cells for this immunodominant nonapeptide was confirmed by MHC-tetramer staining and intracellular interferon-γ production, demonstrating that 38 – 60% of the CD8+ cell population were specific for this A*2-restricted peptide after 3 weeks of culture. These T cells also killed both nonapeptide-pulsed and CMV-infected target cells. In subsequent experiments using auotlogous monocyte-derived DC’s pulsed with the pentadecapeptide pool for the stimulation of CMV-specific T-cell lines in individuals other than HLA-A*2, the generated T cells selectively recognized 1–3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide loaded targets sharing single HLA class I or II alleles permitted the identification the restricting HLA alleles. Those T-cell lines from HLA-A*2 neg. donors contained high frequencies of CD4 and/or CD8 T-cells selectively reactive against peptides presented by other HLA alleles including known epitopes such as aa 341–350QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as aa 267–275HERNGFTVL (HLA-B*4001 and B* 4002). In some donors, the peptide-specific IFN-g+ T-cells generated have been predominantly CD4+ T-cells. Like the peptide-specific CD8+ T-cells, we could determine both epitope and HLA-class II restricting element, e.g. aa513–523 FFWDANDIYRI (HLA-DRB1* 1301). These CD4+ T-cells also consistently exhibited cytotoxic activity against infected targets as well as peptide-loaded cells expressing the restricting HLA class II allele. Thus, synthetic overlapping pentadecapeptides spanning the sequence of the immunodominant protein of CMV-pp65, when loaded on DCs can consistently stimulate the in vitro generation of CD8+ and CD4+ T-cell lines from seropositive donors of diverse HLA genotypes. These cell lines are selectively enriched for T-cells specific for a limited number of immunodominant epitopes each presented by a single HLA class I or class II allele. This approach fosters expansion and selection of HLA-restricted CMV-pp65-reactive T-cell lines of high specificity which also lyse CMV-infected targets and may have advantages for generating virus-specific T-cells for adoptive immunotherapy.

Separation of Gvhd from GVL Responses By Modulation of TCR Signaling

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3822-3822
Author(s):  
Mobin Karimi ◽  
Martha Jordon ◽  
Taku Kambayashi
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Tumor Growth ◽  
Cd8 T Cells ◽  
Leukemia Cells ◽  
Cell Phenotype ◽  
Tcr Signaling ◽  
Allogeneic Hsct

Abstract In allogeneic hematopoietic stem cell transplantation (HSCT), devising new strategies to separate GVHD and GVL responses is of critical importance. However, this is a difficult task, as GVHD and GVL rely on the same recognition of allogeneic MHC by donor-derived T cells. CD8+ T cells are key effector cells that mediate both GVHD and GVL. In mouse models of allogeneic HSCT, the infusion of donor-derived CD8+ T cells eliminates tumor growth but also causes severe GVHD. The activation of CD8+ T cells can be potentially manipulated by perturbing the signaling pathways downstream of the T cell receptor (TCR). TCR signaling depends on the formation of a proximal multimolecular complex, which is nucleated by adaptor proteins such as SLP-76. The phosphorylation of the Y145 residue of SLP-76 is critical for activation of the downstream enzyme PLCg1. As such, a YàF mutation at Y145 of SLP-76 (Y145F) causes decreased TCR-mediated signaling and attenuated T cell function. Here, we investigated how the SLP-76 Y145F mutation in CD8+T cells may impact GVHD and GVL responses in a mouse model of allogeneic HSCT. We employed a major MHC-mismatch mouse model of GVHD involving the transplantation of C57BL/6 (B6)-derived bone marrow (BM) into lethally irradiated Balb/c mice (B6àBalb/c). BM-transplanted mice were also injected with FACS-sorted CD8+ T cells either B6 wildtype (WT) mice or Y145F mice. Recipients of Y145F CD8+ T cells showed significantly (p<0.001) less weight loss, lower clinical score, and improved survival compared to mice injected with WT CD8+ T cells. Next, to determine whether the Y145F CD8+ T cells could mediate GVL effects, BM-transplanted Balb/c mice were additionally challenged intravenously with 1 x 105 luciferase-positive A20 leukemia cells. As expected, BM-transplanted Balb/c mice succumbed from A20 tumor growth, whereas mice injected with WT CD8+ T cells cleared the tumor but developed GVHD. Surprisingly, mice receiving Y145F CD8+ T cells eradicated the leukemic cells but did not develop GVHD. These data suggest that the Y145F mutation in CD8+T cells may be able to separate GVHD from GVL effects. In addition to defective TCR signaling observed in peripheral T cells of Y145F mice, a majority of Y145F KI CD8+ T cells adopt a memory-like CD44hi phenotype through exposure to high levels of IL-4 produced in the thymus of these mice. To test whether the CD44hi CD8+ T cell phenotype was necessary and/or sufficient for the separation of GVHD and GVL effects, BM-transplanted Balb/c mice were injected with FACS-sorted CD44hi or CD44lo CD8+ T cells from WT or Y145F KI mice and challenged with A20 leukemia cells. While BM-transplanted mice receiving CD44hi CD8+ T cells from Y145F mice displayed intact GVL responses without causing GVHD, mice injected with CD44lo CD8+ T cells from Y145F mice displayed impaired ability to clear the tumor cells. Moreover, recipients of CD44hi or CD44lo CD8+ T cells from WT mice cleared the tumor but exhibited severe GVHD. These findings were corroborated with data obtained with an inducible system, whereby CD8+ T cells are affected by the Y145F mutation only after full maturation and thus do not display a CD44hi phenotype (Y145F conditional knock-in mice). Bone marrow-transplanted recipients receiving Y145F conditional knock-in CD8+ T cells developed GVHD and exhibited an attenuated GVL response, suggesting that the Y145F mutation needed to be present during T cell development. Together, these data suggest that either the Y145F mutation or CD44hi phenotype alone in CD8+T cells is insufficient to separate GVHD from GVT. Our data demonstrate that perturbation of the TCR signaling pathway downstream of Y145 of SLP-76 in CD8+ T cells results in separation of GVHD from GVL effects. Experiments to mechanistically test how the Y145F signaling mutation synergizes with the CD44hi phenotype of CD8+ T cells to allow for the separation of the GVHD and GVL effects are currently underway. Our novel and unexpected finding could lead to a novel therapeutic strategy for treatment of acute GVHD after allogeneic HSCT. Disclosures No relevant conflicts of interest to declare.

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