Engineered Antigen- Presenting Cells Expressing Human HLA Class-II DR Alleles Support Efficient Enrichment and Expansion of Antigen Specific Th1 CD4[+] T-Cells Which Are Functionally Augmented by IL-15Rα/IL-15 Complexes
Authors: Hasan Aisha N. , Ricafort Rosanna J. , Selvakumar Annamalai , Doubrovina Ekaterina , Riviere Isabelle , Sadelain Michel , O'Reilly Richard J. 
Published: October 2019

Abstract
Abstract 1905
Previous studies have affirmed the therapeutic efficacy of adoptively transferred antigen specific CD8+ and CD4+ T-cells (TC) against viral infections and tumors. A major challenge in optimizing this approach is to develop strategies to permit generation of CD4+ and long lived CD8+ TC of defined antigen specificity.
We previously described a panel of NIH 3T3 based artifical antigen presenting cells (AAPC) for the immediate generation of HLA class-I restricted CMVpp65 specific CD8+ TC. We now describe a panel of NIH 3T3 based AAPC, each transduced to express a shared HLA DRA 0101 alpha chain and one of the following β chains of the human HLA class-II alleles DRB1 0301, 0701, 1501, 0401 and 1101. At least one of these alleles is inherited by 61% and 59% of caucasians and blacks respectively. These AAPCs were also transduced to co-express the human TC costimulatory molecules B7.1, ICAM-1 and LFA-3. Sensitization of TC from seropositive donors in the presence of IL-2 with AAPCs sharing one of these alleles, either loaded with a CMVpp65 peptide pool or transduced to express the CMV pp65 protein, resulted in 33–71 fold expansion of CMVpp65 specific CD4+ TC that exhibited a Th1 cytokine profile, producing TNF-α and IFNγ in response to the same CMVpp65 epitopes. These TC were also cytotoxic against peptide loaded HLA class-II sharing targets. Epitope mapping demonstrated that the HLA DRB1 0301 restricted TC responded to a CMVpp65 epitope known to be presented by this allele QEFFWDANDIY (aa 509–527) and to an unreported epitope DVEEDLTMTRN (aa 245–263). The DRB1 0701 restricted CD4+ TC responded to 4 different epitopes. Two of these also included nonamer peptide sequences previously reported to be presented by HLA class-I alleles; Q IFLEV QAIRE and PQYSEH PTFTS presented by HLA B44, and a third AGILARNLVPM, contained an epitope presented by HLA B0801. The fourth epitope, KYQEFFWDANDIY is known to be also presented by HLA DRB1 0301. The DRB1 1501 restricted CD4+ TC were also responsive to a known class-II epitope LLQTGIHVRVS (aa 37–55) as well as a new epitope LVSQYTPDSTP (aa 53–71). CD4+ TC from 3 donors also responded to CMVpp65 when sensitized with autologous DCs loaded with CMVpp65 peptide pool, and each recognized the same epitopes as TC sensitized with the class-II AAPCs.
Supplementation of TC cultures with soluble IL-15Rα/ IL-15 complexes markedly augmented the proportion of IFNg+ CD4+ TC, while increasing concentrations of IL-2 resulted in generation of Th2 type CD4+ TC generating IL-4, IL-5 and IL-2 in response to re-stimulation with CMVpp65 peptides. This system can therefore be harnessed by cytokine modulation to selectively generate CD4+ TC with a Th1, or Th2 cytokine profile.
The fact that the class-II AAPC transduced to express the full sequence of CMVpp65 are able to process and present antigenic epitopes on the surface of the expressed HLA class-II allele in the absence of the human invariant chain and HLA-DM suggests that the mouse 3T3 cells contain sufficiently homologous proteins to permit the transfer of processed peptides to human Class-II alleles for presentation. Alternatively, invariant chain independent pathways could permit delivery of certain immunogenic epitopes to the expressed class II HLA alleles. The repertoire of epitopes presented by the Class-II AAPCs with or without the invariant chain is currently under study.
Our results suggest that the panel of AAPCs expressing these HLA DRB1 alleles provides a novel and rapid approach for the generation of Th1 CD4+ virus-specific TC of desired HLA class-II restriction for adoptive therapy of CMV disease to foster lasting immunity with co-infused CMVpp65-specific CD8+ TC. Class-II AAPCs used with different concentrations or types of cytokines may also be useful to generate other functional subsets of CD4+ TC to promote tolerance or enhance tumor- specific immunity.

Disclosures:
No relevant conflicts of interest to declare.