Temporal analysis of cytokine gene expression during infiltration of porcine neuronal grafts implanted into the rat brain

Abstract

A large array of evidence supports the involvement of infiltrating T lymphocytes in the rejection process of intracerebral neuronal xenografts. Little is known, however, about the molecular mechanisms that drive the recruitment of this cell type. In the present work, we used real-time RT-PCR methodology to investigate the kinetics of cytokine gene expression during the infiltration of fetal porcine neurons (PNEU) implanted into the striatum of LEW.1A rats. T lymphocyte infiltration was followed by measuring the intracerebral levels of transcripts encoding the beta chain of the T cell receptor. These transcripts remained barely detectable until the fourth week (28 days) postimplantation, when a sudden accumulation occurred. Their kinetics, which support previous immunohistochemical observations, indicate that alphabetaT lymphocyte recruitment occurs rapidly after a delay of several weeks in this experimental model. Infiltration of PNEU grafts by T lymphocytes was accompanied by a concomitant, dramatic augmentation of transcripts coding for monocyte chemotactic protein-1 and RANTES (for regulated on activation, normal T cell expressed and secreted), two chemokines targeting this cell type, among others. Likewise, a sudden accumulation of transcripts of proinflammatory lymphokines [interleukin (IL)-1alpha, tumor necrosis factor-alpha, IL-6] as well as Th1 cytokines (IL-2, interferon-gamma) was also detected. In contrast, IL-4, -10, and -13 mRNA remained barely detectable at the different time points. No significant changes were noticed for IL-12 or transforming growth factor-beta transcripts. These data support the concept that T lymphocyte infiltration of PNEU grafts is actively promoted by a local production of chemokines and proinflammatory lymphokines and is based on a Th1 polarization.

A Regulatory CD4+ T Cell Subset in the BB Rat Model of Autoimmune Diabetes Expresses Neither CD25 Nor Foxp3

“…Transcript levels of Foxp3, CTLA-4, GITR, early growth response 2 (Egr-2), programmed death-1 (PD-1), and the housekeeping gene hypoxanthine phosphoribosyl-transferase (HPRT) were quantified using realtime PCR. Primer sequences were as follows: Foxp3 forward 5Ј-CCCAGG AAAGAC AGCAACCTT-3Ј and reverse 5Ј-CTGCTTGGCAGTGCTTGA GAA-3Ј (26); CTLA-4 forward 5Ј-GGACTGAGGGCTGCTGACAC-3Ј and reverse 5Ј-GGCATGGTTCTGGATCGATG-3Ј (26); GITR forward 5Ј-GCAGACTTTGGACCAACTGTTC-3Ј and reverse 5Ј-AGCGGCTGG GTATTGACCT-3Ј (26); and HPRT forward 5Ј-GCGAAAGTGGAAAA GCCAAGT-3Ј and reverse 5Ј-GCCACATCAACAGGACTCTTGTAG-3Ј (27). Primers for rat Egr-2 (accession no.…”

Gene Editing, Gene Therapy, and Cell Xenotransplantation: Cell Transplantation Across Species

“…Despite 30% of treated patients showing some improvement, very few surviving porcine cells were found in one deceased patient autopsied 7 months post-implantation. Despite immunological privileges of the brain, Tcell-mediated immune rejection of grafted tissue is a major concern in neural cell xenotransplantation [68] and cyclosporine A immunosuppression was not sufficient to protect neural cell xenografts in rats [69,70]. As an alternative to immunosuppression, transgenic expression of human CTLA4 in porcine neurons inhibited human T lymphocytes proliferation in vitro without affecting normal development after transplantation in rats [71].…”

Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation

“…Xenografts, however, are always ultimately rejected. Xenograft rejection is accompanied by a rapid infiltration of the graft itself and surrounding host tissue by T lymphocytes with a Th1 polarization as well as by macrophages/microglial cells (Melchior et al, 2002). This infiltration occurs in a dramatic pro-inflammatory context.…”

“…In the LEW.1A rat, xenogeneic porcine neurons are rejected concomitantly to a sudden infiltration of T cells occurring several weeks after transplantation (Melchior et al, 2002). Despite enabling initial functional recovery, immunosuppressive treatments with calcineurin inhibitors are not sufficient to protect xenogeneic neurons from rejection in the long term (Larsson et al, 2000), possibly because these immunosuppressors inhibit T cell reactivity in the periphery but poorly in the brain, due to the low permeability of the blood-brain barrier (Tsuji et al, 1993).…”