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.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

571 ANV419 is a novel CD122-selective IL-2/anti-IL-2 antibody fusion protein with potent CD8 T cell and NK cell stimulatory function in vitro and in vivo

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A605-A605
Author(s):  
Christoph Huber ◽  
Andreas Katopodis ◽  
Barbara Branetti ◽  
Jean-Michel Rondeau ◽  
Simone Popp ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Binding Site ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
High Dose

BackgroundANV419 is a uniquely engineered IL-2 fusion to an antibody selectively blocking the IL-2 receptor alpha (CD25) binding site. It signals selectively through the CD122/CD132 dimeric IL-2 receptor and stimulates the proliferation of CD8 T cells and NK cells while avoiding the proliferation of immunosuppressive regulatory T cells (Treg). Therefore, ANV419 has the potential to substantially separate targeted T-cell and NK cell proliferation and anti-tumor responses from the dose limiting toxicities of recombinant IL-2 (aldesleukin). ANV419 has antibody like stability and behavior and is currently in late preclinical development for tumor immunotherapy.MethodsThe crystal structure of ANV419 has been solved and its binding affinity to CD25 and CD122 has been determined. In vitro and in vivo studies, including pharmacodynamics and toxicity, have been performed in rodents and non-human primates. The ability of ANV419 to inhibit tumor growth has been studied in mouse syngeneic models.ResultsStructural analysis demonstrates that the CD25 binding site of IL-2 is completely blocked in ANV419 while the CD122/CD132 sites are available for binding. As a result, ANV419 lacks CD25 binding activity but retains IL-2 receptor beta (CD122) affinity comparable to native IL-2. In human peripheral blood monocyte cultures, ANV419 induces STAT5 phosphorylation with high selectivity for CD8 and NK cells but not Treg. Concordantly, it stimulates the proliferation of purified human CD8 T cells and NK cells but not CTLL-2 cells. A single injection of ANV419 in mice results in strong induction of the proliferation marker Ki67 specifically in CD8 T cells and NK cells but not Tregs and a selective increase of the respective cell numbers in the spleen and peripheral blood of animals. Single agent anti-tumor activity was observed in checkpoint sensitive (H22) and resistant (Renca, B16F10) syngeneic mouse tumor models. Combination of ANV419 with trastuzumab in the gastric cancer N87 xenograft model in BALB/c nude mice led to significant tumor reduction relative to trastuzumab monotherapy. In non-human primates, ANV419 is well tolerated and induces expression of Ki67 and sustained expansion in CD8 T cells and NK cells with no signs of vascular leak syndrome observed with high dose aldesleukin in patients.ConclusionsThe pre-clinical data suggest that ANV419 possesses a unique structure and is potent in expanding CD8 T-cells and NK cells with a marked safety window in non-human primates. This data warrants further translational development of ANV419 as an immune therapeutic in oncology.

scholarly journals T cell-mediated eradication of murine metastatic melanoma induced by targeted interleukin 2 therapy.

1996 ◽  
Vol 183 (5) ◽  
pp. 2361-2366 ◽  
Author(s):  
J C Becker ◽  
J D Pancook ◽  
S D Gillies ◽  
K Furukawa ◽  
R A Reisfeld
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Tumor Model ◽  
Melanoma Metastases ◽  
Tumor Eradication

Induction of a T-cell mediated antitumor response is the ultimate goal for tumor immunotherapy. We demonstrate here that antibody-targeted IL2 therapy is effective against established pulmonary and hepatic melanoma metastases in a syngeneic murine tumor model. The effector mechanisms involved in this tumor eradication are not dependent on NK cells, since the therapeutic effect of antibody-IL2 fusion protein was not altered in NK cell-deficient mice. In contrast, T cells are essential for the observed antitumor effect, since therapy with antibody IL2 fusion proteins is unable to induce tumor eradication in T cell-deficient SCID mice. In vivo depletion studies characterized the essential effector cell population further as CD8 + T cells. Such CD8 + T cells, isolated from tumor bearing mice after antibody-directed IL2 therapy, exerted a MHC class I-restricted cytotoxicity against the same tumor in vitro. These data demonstrate the ability of antibody-targeted IL2 delivery to induce a T cell-dependent host immune response that is capable of eradicating established melanoma metastases in clinically relevant organs.

scholarly journals Neoleukin-2 enhances anti-tumour immunity downstream of peptide vaccination targeted by an anti-MHC class II VHH

Open Biology ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 190235 ◽  
Author(s):  
Stephanie J. Crowley ◽  
Patrick T. Bruck ◽  
Md Aladdin Bhuiyan ◽  
Amelia Mitchell-Gears ◽  
Michael J. Walsh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Class Ii ◽  
Tumour Immunity ◽  
Tumour Rejection

Cancer-specific mutations can lead to peptides of unique sequence presented on MHC class I to CD8 T cells. These neoantigens can be potent tumour-rejection antigens, appear to be the driving force behind responsiveness to anti-CTLA-4 and anti-PD1/L1-based therapies and have been used to develop personalized vaccines. The platform for delivering neoantigen-based vaccines has varied, and further optimization of both platform and adjuvant will be necessary to achieve scalable vaccine products that are therapeutically effective at a reasonable cost. Here, we developed a platform for testing potential CD8 T cell tumour vaccine candidates. We used a high-affinity alpaca-derived VHH against MHC class II to deliver peptides to professional antigen-presenting cells. We show in vitro and in vivo that peptides derived from the model antigen ovalbumin are better able to activate naive ovalbumin-specific CD8 T cells when conjugated to an MHC class II-specific VHH when compared with an irrelevant control VHH. We then used the VHH-peptide platform to evaluate a panel of candidate neoantigens in vivo in a mouse model of pancreatic cancer. None of the candidate neoantigens tested led to protection from tumour challenge; however, we were able to show vaccine-induced CD8 T cell responses to a melanoma self-antigen that was augmented by combination therapy with the synthetic cytokine mimetic Neo2/15.

Double Loading of Dendritic Cell MHC Class I and MHC Class II with an AML Antigen Repertoire Enhances Primary and Secondary T-Cell Responses In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2529-2529
Author(s):  
William K. Decker ◽  
Dongxia Xing ◽  
Sufang Li ◽  
Simon N. Robinson ◽  
Hong Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Cd8 T Cells ◽  
Class Ii ◽  
T Cell Response ◽  
Class I ◽  
Cross Presentation ◽  
Ifn Γ

Abstract Despite improvements in therapy for acute myelogenous leukemia (AML), a significant percentage of patients still relapse and succumb to their disease. Dendritic cell immunotherapy offers the promise of potentially effective supportive therapy for a variety of neoplastic conditions; and the use of DCs loaded with tumor antigens is now recognized as an important investigational therapy. Though a variety of methods have been used to load DC vaccines, the loading of the MHC class II compartment with tumor lysate has predominated. The priming of a class II-mediated (CD4) T-cell response may be crucial to the success of DC immunotherapy as such a response is likely required for the development of memory CD8+ T-cells. DC cross-presentation is credited with the ability of lysate-loaded DCs to prime both CD4 and CD8 T-cell responses, enabling the generation of CD8+ CTLs without the loading of the MHC class I compartment (i.e. the cytoplasm). Recently, however, several reports have raised doubts as to the efficiency of cross-presentation as a mechanism for CTL priming in vivo. To examine this issue, we have loaded human DCs with both AML tumor lysate and mRNA. This technique allows the full repertoire of class I antigens to be presented without dependence upon cross-presentation; and, moreover, provides a full complement of class II antigens necessary for CD4 T-cell priming and the generation of memory responses. Methods: CD14+ precursors were isolated from normal donor PBPCs by magnetic separation. Immature DCs were then generated by culturing precursors for six days in GM-CSF and IL-4. Lysate was produced by three successive freeze/thaw cycles of blasts. mRNA was extracted from blasts using Trizol and oligo-dT separation. Immature DCs were pulsed for three hours with AML lysate and subsequently electroporated with AML mRNA. Loaded DCs were matured for 48 hours with IL-1β, TNF-α, IL-6, and PGE2 and then used to prime autologous T-cells. Short-term responses were assayed on day 5 of the 1st stimulation. Memory responses were assayed on day 10 of a tertiary stimulation. Results: Doubly-loaded DCs can prime a superior T-cell response in vitro in comparison to that of singly-loaded DCs, demonstrating a 30–70% increase in IFN-γ ELISpots over lysate-loaded DCs (p<0.001) and a 3–4 fold increase in ELISpots in comparison to mRNA loaded DCs (p<0.001). These results were verified by flow cytometry which showed 35% of CD8+ T-cells primed by doubly-loaded DCs were CD69+/IFN-γ+ vs. 14% of CD8+ T-cells primed by lysate-loaded DCs (p<0.001). This enhancement may be based upon both an upregulation of CD83 surface expression (p<0.0019) of doubly-loaded DCs and/or the upregulation of B7.1/B7.2 that accompanies elevated CD40L signaling. Memory responses were also greatly improved, with a 126% increase in total ELISpots (double loaded DCs versus lysate loaded DCs; p<0.03) and a 187% increase in total IFN-γ secretion (p<0.03). Unloaded (p<0.01) and mRNA (p<0.007) loaded DCs exhibited a virtual inability to generate memory T-cells in vitro, suggesting that the perpetuation of the memory response is reliant upon T-cell help. Conclusion: DCs doubly-loaded with lysate and mRNA are more efficient in the generation of primary and secondary immune responses than are singly-loaded DCs. The clinical administration of such doubly-loaded DCs may offer an important therapeutic option to patients with AML.

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.

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.

HLAs, TCRs, and KIRs, a Triumvirate of Human Cell-Mediated Immunity

2020 ◽  
Vol 89 (1) ◽  
pp. 717-739 ◽  
Author(s):  
Zakia Djaoud ◽  
Peter Parham
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
T Cell Receptors ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Antigenic Specificity ◽  
Class I Mhc ◽  
Cell Receptors

In all human cells, human leukocyte antigen (HLA) class I glycoproteins assemble with a peptide and take it to the cell surface for surveillance by lymphocytes. These include natural killer (NK) cells and γδ T cells of innate immunity and αβ T cells of adaptive immunity. In healthy cells, the presented peptides derive from human proteins, to which lymphocytes are tolerant. In pathogen-infected cells, HLA class I expression is perturbed. Reduced HLA class I expression is detected by KIR and CD94:NKG2A receptors of NK cells. Almost any change in peptide presentation can be detected by αβ CD8+ T cells. In responding to extracellular pathogens, HLA class II glycoproteins, expressed by specialized antigen-presenting cells, present peptides to αβ CD4+ T cells. In comparison to the families of major histocompatibility complex (MHC) class I, MHC class II and αβ T cell receptors, the antigenic specificity of the γδ T cell receptors is incompletely understood.

scholarly journals In vitro education of human natural killer cells by KIR3DL1

2019 ◽  
Vol 2 (6) ◽  
pp. e201900434
Author(s):  
Jason Pugh ◽  
Neda Nemat-Gorgani ◽  
Zakia Djaoud ◽  
Lisbeth A Guethlein ◽  
Paul J Norman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Surface Expression ◽  
Class I ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Basic Protocol

During development, NK cells are “educated” to respond aggressively to cells with low surface expression of HLA class I, a hallmark of malignant and infected cells. The mechanism of education involves interactions between inhibitory killer immunoglobulin–like receptors (KIRs) and specific HLA epitopes, but the details of this process are unknown. Because of the genetic diversity of HLA class I genes, most people have NK cells that are incompletely educated, representing an untapped source of human immunity. We demonstrate how mature peripheral KIR3DL1+ human NK cells can be educated in vitro. To accomplish this, we trained NK cells expressing the inhibitory KIR3DL1 receptor by co-culturing them with target cells that expressed its ligand, Bw4+HLA-B. After this training, KIR3DL1+ NK cells increased their inflammatory and lytic responses toward target cells lacking Bw4+HLA-B, as though they had been educated in vivo. By varying the conditions of this basic protocol, we provide mechanistic and translational insights into the process NK cell education.

Identification and Characterization of HLA Class I-Restricted MYD88 L265P-Derived Peptides As Tumor-Specific Targets for Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2750-2750
Author(s):  
Annika Nelde ◽  
Juliane S Stickel ◽  
Daniel Johannes Kowalewski ◽  
Oliver Olaf Wolz ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Hla Class I ◽  
Class I ◽  
Specific Cell ◽  
Specific Lysis ◽  
Effector Cells

Abstract Non-Hodgkin lymphomas (NHL) are frequent malignancies with considerable mortality. A recurrent somatic and oncogenic driver mutation in the Toll-like receptor adaptor gene MYD88, Leu265Pro (L265P) has been identified in up to 90% of certain NHL subtypes. Genetic alterations affecting a protein-coding region have the potential to generate mutation-derived peptides that are presented by HLA class I proteins and might be recognized by cytotoxic T cells. Because MYD88L265P is a widely occurring and tumor-specific mutation, we investigated the potential of MYD88L265P -containing peptides for CD8+ T cell mediated immunotherapy as a new therapeutic approach for MYD88L265P+ NHL. Based on in silico prediction we identified potential HLA ligands encompassing the MYD88L265P mutation for several HLA class I allotypes. Functional characterization of the candidate HLA class I MYD88L265P-derived HLA class I ligands with regard to induction of T cell responses identified a set of immunogenic peptides for HLA-B*07 and -B*15. In one MYD88L265P-mutated NHL patient, memory T cell responses targeting three different MYD88L265P-derived HLA class I ligands were detected by IFN-γ ELISPOT. Efficient T cell priming was demonstrated in vitro using naïve T cells of healthy volunteers (HVs). In detail, three HLA-B*07 peptides (P1-3B*07) and one HLA-B*15-restricted peptide (P4B*15) were analyzed using artificial antigen-presenting cell-based (aAPC) in vitro priming experiments in three to six HVs, respectively. For all tested peptides proliferation of peptide-specific CD8+ T cells could be detected after in vitro priming. For the HLA-B*07-restricted ligands, peptide-specific CD8+ T cells could be induced in 6/6 (P1B*15), 1/3 (P2B*07) and 3/4 (P3B*07) HVs, respectively, with a maximum frequency of 14.1% peptide-specific CD8+ T cells. For the HLA-B*15-restricted ligand (P4B*15), peptide-specific CD8+ T cells could be induced in 2/3 HVs with a maximum frequency of 9.5% tetramer-positive CD8+ T cells. The functionality and specificity of peptide-specific CD8+ T cells after aAPC-based in vitro priming was validated by intracellular cytokine staining for IFN-γ and TNF-α as well as for the expression of the degranulation marker CD107a. In 3/3 HVs primed with P1B*07 (RPIPIKYKAM) as well as in 1/2 HVs primed with P4B*15 (HQKRPIPIKY), we detected specific and functional CD8+ T cell populations after stimulation with the mutated peptides, but not after stimulation with the corresponding wild type peptides (P1WT: RLIPIKYKAM, P4WT: HQKRLIPIKY). Furthermore, the peptide-specific cytotoxic activity of specific CD8+ T cells was demonstrated in a VITAL assay. The polyclonal P1B*07- and P4B*15-specific CD8+ T cells (0.12% and 0.76% peptide-specific T cells, respectively) lysed autologous peripheral blood mononuclear cells loaded with the mutated peptides, but not cells presenting the wild type peptides. P4B*15-specific CD8+ T cells showed 17.9% (±1.2%) MYD88L265P-peptide-specific cell killing at an E/T ratio of 1:1 compared to 2.6% (±1.2%) of non-specific cell lysis of unspecific effector cells against the same targets in three independent replicates, respectively. The specific lysis showed an E/T ratio-dependent manner as the specific lysis decreases with reducing E/T ratios. P1B*07-specific CD8+ T cells specifically killed 11.4% (±1.7%) of MYD88L265P loaded targets at an E/T ratio of 0.7:1 in comparison to 2.1% unspecific lysis of unspecific effector cells. In this study, we identified and characterized MYD88L265P mutation-derived HLA class I ligands for T cell mediated immunotherapy. The strong immunogenicity of the HLA-B*07 and HLA-B*15-restricted mutation-derived peptides as well as the functionality and specificity of peptide-specific CD8+ T cells, demonstrated by cytotoxicity assays, underline the potential of the MYD88L265P mutation as tumor-specific target. These data highlight the potential of MYD88L265P mutation-specific immunotherapy as a novel broadly applicable and tumor-specific treatment approach for patients with MYD88L265P+ NHL. Disclosures Langerak: InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam).

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