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309 Determining the histocompatibility barriers between universal CAR T (UCART) cells and NK cells
  1. Kimberly Apodaca,
  2. Chong Xu,
  3. Kelsey Stanton,
  4. Beatriz Carreno and
  5. Gerald Linette
  1. University of Pennsylvania, Philadelphia, PA, USA


Background Chimeric antigen receptor (CAR) T cell adoptive therapies have proven efficacy in the treatment of hematological malignancies. However, there are challenges to the current CAR T cell manufacturing process, one being manufacturing failure due to dysfunctional, patient-derived T cells.1 A proposed solution to overcoming this limitation is the development of universal CAR T (UCART) cells engineered from healthy, normal donor-derived T cells. Histocompatibility barriers must be addressed when creating the UCART cell. Ablation of the alpha/beta T cell receptor surface expression will prevent graft-versus-host disease while host-versus-graft responses will be mitigated by ablating the surface expression of the major histocompatibility complex (MHC) class I and II molecules.2,3 These triple knockout (TKO) T cells could serve as universal recipients for development of the UCART cell. Importantly, a consequence of MHC class I ablation is NK cell activation due to the “missing-self” response.4 HLA-E, a non-classical MHC class I molecule, is known to interact with the NK cell inhibitory receptor NKG2A and we propose its overexpression on UCART cells will mitigate NK cell activation.

Methods Because the HLA-E/NKG2A interaction is dependent upon the peptide presented by HLA-E, 10 HLA-E single-chain trimer (SCT) constructs expressing various peptide sequences were created to determine which HLA-E/peptide complex would result in significant NK cell inhibition.5 K562 cells transduced to express these HLA-E/peptide complexes were used as a model for UCART cells in preliminary experiments.

Results An HLA-E SCT presenting an HLA-C leader peptide (HLA-E/C) resulted in significant inhibition of NK cells as determined by flow cytometry-based NK cell degranulation (CD107a) assays. Decreased lysis of K562 HLA-E/C -expressing target cells in 51Cr release assays further validated the inhibitory effect of HLA-E/C complexes on NK cell activation. Finally, HLA-E/C complex expression on TKO T cells lead to protection against NK lytic activity.

Conclusions Altogether these data demonstrate the effectiveness of selected HLA-E/peptide complexes to inhibit NK cell activation due to the “missing-self” response. Modifications such as the one described here may prevent UCART cell clearance due to host recognition (NK cell activation) and may ultimately lead to a more potent, safe, and long-term persistent UCART cell therapy.


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  3. Wang D, Quan Y, Yan Q, Morales J. E, Wetsel RA. Targeted disruption of the ß2-microglobulin gene minimizes the immunogenicity of human embryonic stem cells. Stem Cells Transl Med. 2015; 4:1234–1245.

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  5. Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG, Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. J Exp Med 1998; 187:813–818.

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