Short-Term In-Vitro Expansion Improves Monitoring and Allows Affordable Generation of Virus-Specific T-Cells against Several Viruses for a Broad Clinical Application

Abstract

Adenoviral infections are a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. Adoptive transfer of donor-derived human adenovirus (HAdV)-specific T-cells represents a promising treatment option. However, the difficulty in identifying and selecting rare HAdV-specific T-cells, and the short time span between patients at high risk for invasive infection and viremia are major limitations. We therefore developed an IL-15-driven 6 to 12 day short-term protocol for in vitro detection of HAdV-specific T cells, as revealed by known MHC class I multimers and a newly identified adenoviral CD8 T-cell epitope derived from the E1A protein for the frequent HLA-type A*02∶01 and IFN-γ. Using this novel and improved diagnostic approach we observed a correlation between adenoviral load and reconstitution of CD8+ and CD4+ HAdV-specific T-cells including central memory cells in HSCT-patients. Adaption of the 12-day protocol to good manufacturing practice conditions resulted in a 2.6-log (mean) expansion of HAdV-specific T-cells displaying high cytolytic activity (4-fold) compared to controls and low or absent alloreactivity. Similar protocols successfully identified and rapidly expanded CMV-, EBV-, and BKV-specific T-cells. Our approach provides a powerful clinical-grade convertible tool for rapid and cost-effective detection and enrichment of multiple virus-specific T-cells that may facilitate broad clinical application.

Adenovirus Infections in Immunocompetent and Immunocompromised Patients

“…Hence, exposure to adenoviruses during childhood and the ensuing generation of cross-reactive cytotoxic T cells are believed to lead to broad HAdV immunity in adults (119)(120)(121). Healthy individuals usually carry HAdV-specific T cells, which can be identified by various methods, such as gamma interferon secretion assays, cytokine flow cytometry, or detection of MHC class I multimers (118,122,123). The absence of HAdVspecific T cells has a negative impact on the course of HAdV infections, and conversely, reconstitution of the HAdV-specific Tcell response correlates with viral clearance (122,124).…”

“…The MHC multimer technology requires knowledge of immunodominant human leukocyte antigen (HLA)-restricted peptide epitopes and facilitates the isolation of antigen-specific CD8 ϩ T cells (MHC class I multimers) or CD4 ϩ T cells (MHC class II multimers) of high purity (251). Short-term in vitro expansion under good manufacturing practice (GMP) conditions can render adoptive T-cell transfer available in less than 2 weeks (123,(252)(253)(254). In addition to a variety of methods based on the isolation of HAdV-specific T cells from the original stem cell donors, several approaches exploiting third-party donors are emerging (141,(255)(256)(257)(258)(259)(260)(261)(262)(263).…”

High-density preculture of PBMCs restores defective sensitivity of circulating CD8 T cells to virus- and tumor-derived antigens

“…22 We therefore investigated whether HD preculture of PBMCs also enhances the responsiveness of human ADV (ADE)-specific CD8 T cells to more readily detectable levels. Indeed, CD8 T cells from 7 tested healthy individuals showed strongly enhanced IFN-g responses to the epitopes HEX 901-910, E1A 19-45, or HEX 37-45 from ADE02, the clinically important strain (Figure 2E-F).…”

“…Functional responses were tested with PepMix CEF standard containing HLAclass I restricted T-cell epitopes from human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and influenza A virus, and PepMix influenza A. PepMix Human Actin served as negative control (JPT Peptide Technologies). Synthetic peptides from HCMV (PP65_HCMV 495-503), human adenovirus (ADV) 2 (HEX_ADE02 901-910, HEX_ADE02 37-45, and E1A_ADE02 [19][20][21][22][23][24][25][26][27], and Wilms tumor 1 protein (WT1) (WT1_HUMAN 126-134 and WT1_HUMAN 356-364) were synthesized as previously described 17 (purity .90%). Clinical grade TAB08 was used at 1 mg/mL.…”

“…22 Indeed, HLA-A0201/pp65 pentamer staining (a procedure unsuitable for routine clinical practice due to the multitude of pentamers required) reveals that the frequency of CD8 T cells expressing the relevant antigen/MHC specificity is unaffected by the preculturing step (supplemental Figure 4). This feature makes HD preculture an ideal and simple tool for immunomonitoring of CD8 T-cell responses to cancer-associated antigens over time ( Figure 5D), and to infectious agents and vaccines.…”