Amy Cuthbert

Non-obese diabetic-recombination activating gene-1 (NOD-Rag1 null) interleukin (IL)-2 receptor common gamma chain (IL2r gamma null) null mice: a radioresistant model for human lymphohaematopoietic engraftment

Immunodeficient hosts engrafted with human lymphohaematopoietic cells hold great promise as a preclinical bridge for understanding human haematopoiesis and immunity. We now describe a new immunodeficient radioresistant non‐obese diabetic mice (NOD) stock based on targeted mutations in the recombination activating gene‐1 (Rag1null) and interleukin (IL)‐2 receptor common gamma chain (IL2rγnull), and compare its ability to support lymphohaematopoietic cell engraftment with that achieved in radiosensitive NOD.CB17–Prkdcscid (NOD–Prkdcscid) IL2rγnull mice. We observed that immunodeficient NOD–Rag1null IL2rγnull mice tolerated much higher levels of irradiation conditioning than did NOD–Prkdcscid IL2rγnull mice. High levels of human cord blood stem cell engraftment were observed in both stocks of irradiation‐conditioned adult mice, leading to multi‐lineage haematopoietic cell populations and a complete repertoire of human immune cells, including human T cells. Human peripheral blood mononuclear cells also engrafted at high levels in unconditioned adult mice of each stock. These data document that Rag1null and scid stocks of immunodeficient NOD mice harbouring the IL2rγnull mutation support similar levels of human lymphohaematopoietic cell engraftment. NOD–Rag1null IL2rγnull mice will be an important new model for human lymphohaematopoietic cell engraftment studies that require radioresistant hosts.

Skin Allograft Maintenance in a New Synchimeric Model System of Tolerance

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2k) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2b-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2b allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.

Regulation of skin and islet allograft survival in mice treated with costimulation blockade is mediated by different CD4+ cell subsets and different mechanisms.

Background. Donor-specific transfusion (DST) and a brief course of anti-CD154 monoclonal antibody (mAb) induces permanent islet and prolonged skin allograft survival in mice. Induction of skin allograft survival requires the presence of CD4+ cells and deletion of alloreactive CD8+ cells. The specific roles of CD4+ and CD4+CD25+ cells and the mechanism(s) by which they act are not fully understood. Methods. We used skin and islet allografts, a CD8+ T cell receptor (TCR) transgenic model system, and in vivo depleting antibodies to analyze the role of CD4+ cell subsets in regulating allograft survival in mice treated with DST and anti-CD154 mAb. Results. Deletion of CD4+ or CD25+ cells during costimulation blockade induced rapid rejection of skin but only minimally shortened islet allograft survival. Deletion of CD4+ or CD25+ cells had no effect upon survival of healed-in islet allografts, and CD25+ cell deletion had no effect upon healed-in skin allograft survival. In the TCR transgenic model, DST plus anti-CD154 mAb treatment deleted alloreactive CD8+ T cells, and anti-CD4 mAb treatment prevented that deletion. In contrast, injection of anti-CD25 mAb did not prevent alloreactive CD8+ T cell deletion. Conclusions. These data document that (1) both CD4+CD25+ and CD4+CD25− cells are required for induction of skin allograft survival, (2) CD4+CD25+ T cells are not required for alloreactive CD8+ T cell deletion, and (3) CD4+CD25+ regulatory cells are not critical for islet allograft tolerance. It appears that skin and islet transplantation tolerance are mediated by different CD4+ cell subsets and different mechanisms.

Systemic delivery of triplex-forming PNA and donor DNA by nanoparticles mediates site-specific genome editing of human hematopoietic cells in vivo

In vivo delivery is a major barrier to the use of molecular tools for gene modification. Here we demonstrate site-specific gene editing of human cells in vivo in hematopoietic stem cell-engrafted NOD.Cg-PrkdcscidIL2rγtm1Wjl (abbreviated NOD-scid IL2rγnull) mice, using biodegradable nanoparticles loaded with triplex-forming peptide nucleic acids (PNAs) and single-stranded donor DNA molecules. In vitro screening showed greater efficacy of nanoparticles containing PNAs/DNAs together over PNA-alone or DNA-alone. Intravenous injection of particles containing PNAs/DNAs produced modification of the human CCR5 gene in hematolymphoid cells in the mice, with modification confirmed at the genomic DNA, mRNA and functional levels. Deep sequencing revealed in vivo modification of the CCR5 gene at frequencies of 0.43% in hematopoietic cells in the spleen and 0.05% in the bone marrow: off-target modification in the partially homologous CCR2 gene was two orders of magnitude lower. We also induced specific modification in the β-globin gene using nanoparticles carrying β-globin-targeted PNAs/DNAs, demonstrating this method’s versatility. In vivo testing in an enhanced green fluorescent protein-β-globin reporter mouse showed greater activity of nanoparticles containing PNAs/DNAs together over DNA only. Direct in vivo gene modification, such as we demonstrate here, would allow for gene therapy in systemic diseases or in cells that cannot be manipulated ex vivo.

Alloreactive CD8 T cells rescued from apoptosis during co‐stimulation blockade by Toll‐like receptor stimulation remain susceptible to Fas‐induced cell death

Blockade of co‐stimulatory signals to T cells is extremely effective for the induction of transplantation tolerance in immunologically naive rodents. However, infections and inflammation compromise the efficacy of co‐stimulation blockade regimens for the induction of tolerance, thereby stimulating the rejection of allografts. Previous studies have shown that stimulation of innate immunity abrogates tolerance induction by preventing the deletion of alloreactive CD8+ T cells that normally occurs during co‐stimulation blockade. Although inflammation prevents the deletion of alloreactive T cells during co‐stimulation blockade, it is not known if this resistance to cell death is the result of a mechanism intrinsic to the T cell. Here, we used syngeneic bone marrow chimeric mice that contain a trace population of T‐cell receptor transgenic alloreactive CD8+ T cells to investigate the early apoptotic signature and activation status of alloreactive T cells following exposure to inflammatory signals during co‐stimulation blockade with an antibody specific for CD154. Our findings revealed that the presence of bacterial lipopolysaccharide during co‐stimulation blockade enhanced the early activation of alloreactive CD8+ T cells, as indicated by the up‐regulation of CD25 and CD69, suppressed Fas ligand expression, and prevented apoptotic cell death. However, alloreactive CD8+ T cells from lipopolysaccharide‐treated mice remained sensitive to Fas‐mediated apoptosis in vitro. These findings suggest that alloreactive T cells rescued from deletion during co‐stimulation blockade by inflammation are still sensitive to pro‐apoptotic signals and that stimulating these apoptotic pathways during co‐stimulation blockade may augment the induction of tolerance.