Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways

Abstract

Involvement of CD4+ helper T (Th) cells is crucial for CD8+ cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4+ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4+ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4+ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2Kb/OVA257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4+ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4+ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4+ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.

Enhanced suppression of polyclonal CD8+25+ regulatory T cells via exosomal arming of antigen-specific peptide/MHC complexes

“…Naïve CD4 + T and RNA isolation was performed using RNeasy Plus Mini Kit (Qiagen, Toronto, ON, Canada), and cDNA was then synthesized immediately using the RT 2 First Strand Kit (Qiagen). RT-PCR was carried out with RT 2 SYBR Green qPCR Mastermix (Qiagen) using a StepOnePlus Real-time PCR system (Thermo Fisher Scientific) [18]. A panel of primers specific for T cell anergy-associated genes (Ikaros, Grail, Casp3, EGR2, Grg4, and Itch) were synthesized [19].…”

“…A panel of primers specific for T cell anergy-associated genes (Ikaros, Grail, Casp3, EGR2, Grg4, and Itch) were synthesized [19]. Expression of each gene was normalized to b-actin [18].…”

“…Cell lysates (10 mg/lane) were loaded onto 12% acrylamide gels, subjected to SDS-PAGE, and was subsequently transferred onto nitrocellulose membrane (Millipore, Bedford, MA). The membrane was blocked by incubation for 2 h at room temperature with ODYSSEY blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) and immunoblotted with Abs specific for Egr2 and b-actin at 4°C overnight, followed by incubation with goat anti-rabbit IRDye800CW, and scanned using the ODYSSEY instrument, according to manufacturer's instruction [18].…”

Transgenic 4-1BBL-engineered vaccine stimulates potent Gag-specific therapeutic and long-term immunity via increased priming of CD44+CD62Lhigh IL-7R+ CTLs with up- and downregulation of anti- and pro-apoptosis genes

“…8-10 OVA-specific CTLs were then purified from the enriched T-cell population using PE-tetramer staining followed by anti-PE microbeads (Miltenyi Biotec). 25 A highly purified population of OVAspecific CTLs was obtained by passing the anti-PE microbeads-bound cell sample through a MACS column. The expression of a pathway-focused panel of 84 genes related to apoptosis in the above two groups of CTLs was examined using RT 2 Profiler PCR Array Mouse Apoptosis (SuperArray Bioscience, Betheoda, MD).…”

“…The expression of a pathway-focused panel of 84 genes related to apoptosis in the above two groups of CTLs was examined using RT 2 Profiler PCR Array Mouse Apoptosis (SuperArray Bioscience, Betheoda, MD). 25 …”