A Series of Human Cell-Based Artificial APC Expands Long-Lived, Th1-Biased, Viral Antigen-Specific CD4+ T Cells with a Central/Effector Memory Phenotpype Restricted by Common HLA-DR Alleles
Authors: Butler Marcus O. , Ansén Sascha , Tanaka Makito , Imataki Osamu , Berezovskaya Alla , Mooney Mary M. , Metzler Genita , Milstein Matthew I. , Nadler Lee M. , Hirano Naoto 
Published: October 2019

Abstract
Abstract 354
Adoptive cell therapy utilizes unique mechanisms of action to prevent the development of infections in immunocompromised patients and treat chemotherapy resistant malignancies. In adoptive cell therapy, the major effector cells appear to be CD8+ T cells, since they are armed with antigen-specific effector functions, i.e. cytotoxicity and cytokine secretion. However, the roles of antigen-specific CD4+ T cells in T cell immunity are also critical. In immunocompromised patients adoptively transferred with CMV-specific CD8+ T cells, long-term in vivo persistence was achieved only when CMV-specific CD4+ T cells were also present in vivo. Recently, adoptive transfer of a NY-ESO-1 specific CD4+ T cell clone was reported to induce a complete response in a patient with metastatic melanoma. These results suggest that adoptive cell therapy for infectious diseases and cancer can be improved by infusing both antigen-specific CD4+ helper T cells as well as CD8+ CTL. Unfortunately, however, few versatile systems are available for producing large numbers of antigen-specific human CD4+ T cells for the purpose of adoptive therapy.
K562 is a human erythroleukemic cell line, which lacks the expression of HLA class I and II, invariant chain (Ii), and HLA-DM, but does express adhesion molecules such as ICAM-1 and LFA-3. Given this unique immunologic phenotype, K562 has served as a useful tool in clinical cancer immunotherapy trials. Previously, we reported the generation of a K562-based artificial APC (aAPC), which expresses HLA-A2, CD80, and CD83. aAPC/A2 can uniquely support the priming and prolonged expansion of large numbers of antigen-specific CD8+ CTL which display a central/effector memory phenotype, possess potent effector function, and can be maintained in vitro without any feeder cells or cloning. aAPC/A2 is equipped with constitutive proteasome and inducible immunoproteasome machinery and can naturally process and present CD8+ T cell peptides via transduced A2 molecules. We have successfully generated clinical grade aAPC/A2 under cGMP conditions and conducted a clinical trial where patient with advanced melanoma are infused with large numbers of MART1-specific CTL generated ex vivo using aAPC/A2, IL-2 and IL-15.
Based on our experience with aAPC/A2 and CD8+ T cells, we have generated a series of novel aAPC (aAPC/DR1, DR3, DR4, DR7, DR10, DR11, DR13, and DR15) to stimulate HLA-DR-restricted antigen-specific CD4+ T cells. K562 has been engineered to express HLA-DRα and β chains as a single HLA allele in conjunction with Ii, HLA-DMα and β chains, CD80 and CD83. CD83 delivers a CD80-dependent T cell stimulatory signal that allows T cells to be long-lived. Following the transduction of Ii, CLIP (class II invariant chain-associated peptide) appeared on the cell surface of aAPC. Furthermore, CLIP expression on aAPC was almost completely abrogated by the introduction of HLA-DM. This result is in accordance with previous studies showing that HLA-DM catalyzes the removal of CLIP from DR thus enabling exogenous peptides to bind to empty DR molecules in late endosomes. In addition to its endogenous pinocytic activity, aAPC was made capable of Fcγ receptor-mediated endocytosis by transduction of CD64. Comparison of naïve aAPC and CD64-transduced aAPC confirmed that Fcγ receptor-mediated endocytosis is more efficient than pinocytosis to take up soluble protein and process and present DR-restricted peptides to CD4+ T cells. Using these standardized and renewable aAPC, we determined novel viral protein-derived DR-restricted CD4+ T cell epitopes and expanded large numbers of viral antigen-specific CD4+ T cells without growing bystander Foxp3+ regulatory T cells. Without any feeder cells or cloning, expansion of CD4+ T cells using aAPC and low dose IL-2 and IL-15 was sustainable up to 150 days. Immunophenotypic analysis using HLA-DR tetramers and specific mAbs revealed that expanded CD4+ T cells were CD45RA−, CD45RO+, CD62L+-, demonstrating a central/effector memory phenotype. Furthermore, intracellular cytokine analysis showed that expanded DR-restricted viral-specific CD4+ T cells secreted IL-2 and IFN-γ but much less IL-4, displaying a Th1-biased phenotype. Taken all together, these results suggest that K562-based aAPC may serve as a translatable platform to generate both antigen-specific CD4+ helper T cells and CD8+ CTL.

Disclosures:
No relevant conflicts of interest to declare.