The experimental design is two-fold. One is to direct differentiation of ES cells to the DC lineage but identify the timepoint at which DC lineage-committed ES cells, which we call ES-DC precursors, or ES-DCp, emerge. These are not to be confused with immature ES-DC, which derive from them. The second part is to implant these ES-DCp in allogeneic mice and test the hypothesis that accepted precursors differentiate and colonize lymphoid organs, potentially affecting host immunity to donor alloantigens. These cells should phenotypically be more like ES cells than DCs. To monitor their appearance, we check for transcription factors that guide their commitment, and not necessarily for surface markers. We wanted to establish proof of concept that these precursors can differentiate in vivo in an allogeneic host. We were not sure if commitment was reversible, but more importantly, if they were sustainable in the absence of cytokines. Emphasize “in the absence of cytokines” in the second question.
The mice were given either 0.5 mg or 0.25 of antibody. We homogenized lymph nodes, thymus, and spleen and checked for donor Class I and Class II by rt-PCR. Detection of donor Class I was improved with combined treatment of anti-CD4 and anti-CD8. Except for this signal, Class II signal was not strong in thymus, most likely because the tissue was not treated with collagenase prior to homogenization. Nevertheless, detection of Class II in thymus of the double Ab-treated mouse was promising, and this needed to be confirmed by IHC.
Luis P. Fernández December 5, 2009 Image from Ken Shortman and Yong-Jun Liu, Nature Reviews Immunology 2, 151-161 (2002). “ Embryonic stem cell-based therapy for transplantation tolerance"
Relevant Financial Relationship Disclosure Statement Luis P. Fernández, PhD Transplantation Biology Research Center/ Massachusetts General Hospital I have no financial relationships to disclose within the past 12 months relevant to my presentation AND My presentation does not include discussion of off-label or investigational use
Goals <ul><li>Circumvent DC immunogenicity by introducing allogeneic DC in the form of poorly immunogenic progenitors </li></ul><ul><li>Characterize the differentiation potential of these progenitors in vitro and in vivo. </li></ul><ul><li>Assess the spectrum of tolerogenicity induced by ES cell-derived DC. </li></ul>
Experimental Design ES cells (ESC) ES-DC precursors (ES-DCp) Immature ES-DCs C57BL/6 Balb/c Immunomodulation In vitro In vivo Hypothesis <ul><li>Similar to ESC, ES-DCp are weakly immunogenic and will be accepted by mildly conditioned allogeneic hosts. </li></ul><ul><li>ES-DCp will continue to differentiate in vivo and colonize lymphoid organs. </li></ul><ul><li>It is predicted that allogeneic DCs will "redefine self" and downmodulate T cell responses to allogeneic antigens. </li></ul>Mild conditioning
H&E CD11c , I-Ab Ig isotype 400x 100x kd cap 100x kd cap 100x kd cap cap kd P15 ESC Implanted ES-DCp BM-DC MEF spleen PU.1 CX3CR1 CD11b CD11c Oct3/4 -Actin water GM-CSF IL-3 ES-DCp Follow up on weeks 1, 2, 3, 6 ES-DCp in immunosuppressed mice: test of sustained commitment 1 mg anti-CD4 & -CD8 ESC
K b I-A b actin LN Thy Spn (anti-CD4) LN Thy Spn (anti-CD4 &-CD8) LN Thy Spn (anti-CD4) LN Thy (anti-CD8) 0.5 mg 0.25 mg Spn ES-DCp ESC Thymus, week 3 CD11c (red) I-Ab (green) I-Ab actin I-Ab -actin Kb ESC ES-DCp BM-DC Water Responder mice injected with: ES-DCp ESC % of ESC-treated BALB/c response to B6 p= 0.001 25 50 75 100
Summary <ul><li>Immunocompetent hosts: rejection of derivatives of ES-DCp and other cells does not permit further study </li></ul><ul><li>Immunocompromised host: ES-DCp differentiate in situ in a narrow window before teratomas take over </li></ul><ul><li>Modulation of immunosuppression to allow ES-DCp differentiation and T cell recovery </li></ul><ul><li>Thymic migration of ES-DCp derivatives and potential to downmodulate alloimmune response. </li></ul><ul><li>The need to purify ES-DCp </li></ul>
PU.1 promoter Transfect C57BL/6 ES cells ES-DCp Balb/c LIF withdrawal Expand G418-resistant colonies RFP+ B220- SSEA1- cells Strategy to enrich ES-DCp for implantation (pDsRed1-1 ™ , Clontech) Three-week culture GMCSF+IL3 Non-adherent plate RFP Neo R <ul><li>Eliminate teratogenic cells/purify ES-DCp. </li></ul><ul><li>In the ES-DCp-treated animal: </li></ul><ul><ul><li>Detect donor-derived cells in lymphoid organs by IHC. </li></ul></ul><ul><ul><ul><li>Determine phenotype of donor DCs in lymphoid organs and establish correlates between phenotype and tolerance induction. </li></ul></ul></ul><ul><ul><ul><li>Assess host immunity to donor antigens over a longer time period. </li></ul></ul></ul><ul><ul><li>Examine signs of regulation (T regs) </li></ul></ul><ul><li>Test the impact of ES-DCp therapy on transplantation tolerance. </li></ul>
ACKNOWLEDGEMENTS TBRC/Massachusetts General Hospital Christian Leguern Paulo Martins Sharon Germana Kaela Goldstein Yuanto Wang Guotong Man Akira Shimizu Childrens Hospital Boston George Q. Daley