Jennifer K. Broom, Andrew M. Lew, Hiroaki Azukizawa, Tony J

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Antigen-Specific CD4 Cells Assist CD8 T-Effector Cells in Eliminating Keratinocytes Jennifer K. Broom, Andrew M. Lew, Hiroaki Azukizawa, Tony J. Kenna, Graham R. Leggatt, Ian H. Frazer Journal of Investigative Dermatology Volume 130, Issue 6, Pages 1581-1589 (June 2010) DOI: 10.1038/jid.2010.17 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 hGH graft-primed animals require CD4 T cells for optimal second graft rejection. (a, b) Graft survival time is shown for (a) naive or K14hGH.C57 graft-primed C57Bl/6 animals (n=12) that received a K14hGH.C57 or a C57Bl/6 graft, and for (b) K14hGH.C57 graft-primed animals that received a K14hGH.C57 graft with or without simultaneous depletion of CD4 T cells. Graft survival was compared using the log-rank (Mantel–Cox) test. Multiple mice rejecting grafts on the same day are shown as a single graph point on that day. (c, d) For control antibody-treated (c) and GK1.5 anti-CD4-treated (d) animals, efficacy of CD4 cell depletion was assessed by flow cytometry analysis of peripheral blood, using anti-CD4 and anti-CD3mAb. Percentage of CD4 cells at 7 days is shown for each treatment. Results are combined from two experiments. Journal of Investigative Dermatology 2010 130, 1581-1589DOI: (10.1038/jid.2010.17) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CD40 stimulation abrogates the requirement for CD4 cells to enable rejection of hGH grafts from hGH-primed animals. (a) Graft survival over time for K14hGH.C57 grafts (n=15) and for C57Bl/6 grafts (n=6) placed on hGH graft-primed and CD4-depleted C57Bl/6 recipients treated with agonist CD40 antibody FKG 45. (b) Graft survival over time for K14hGH.C57 grafts placed on graft-primed and CD4-depleted C57Bl/6 recipients, treated either with agonist CD40 antibody (n=15) or with control antibody (n=6). Graft survival differences were assessed for significance using th log-rank (Mantel–Cox) test. Results are from three separate experiments. Journal of Investigative Dermatology 2010 130, 1581-1589DOI: (10.1038/jid.2010.17) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Ovalbumin and SIINFEKL-expressing skin grafts do not require CD4 cells for rejection. (a) Graft survival over time is shown for K5.mOVA grafts placed on naive, or OVA graft-primed and CD4-depleted C57Bl/6-recipient animals (n=12). (b) Graft survival over time is shown for K14 SIINFEKL grafts placed on naive or SIINFEKL graft-primed and CD4-depleted C57Bl/6-recipient animals (n=12). Survival differences were assessed for significance using the log-rank (Mantel–Cox) test. Journal of Investigative Dermatology 2010 130, 1581-1589DOI: (10.1038/jid.2010.17) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Transfer of CD4 T cells alone does not significantly increase graft rejection. (a) Survival over time of OVA grafts placed on immunodeficient Rag1−/- recipients (n=16) reconstituted with 2.5 × 107 OVA-specific helper T cells from OT-II transgenic spleen or on control Rag 1−/- animals receiving no cells (n=8). (b, c) Effective transfer of OVA-specific helper T cells from OTII mice was confirmed by staining of peripheral blood from mice receiving OT-II cells (b) and control mice (c) with anti-CD3 and anti-Vα2 antibodies specific for OT-II cells. Results are from two separate experiments. Journal of Investigative Dermatology 2010 130, 1581-1589DOI: (10.1038/jid.2010.17) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The rate of K5mOVA graft rejection is dependent on CD8 precursor frequency and CD4 T cells. (a) Immunodeficient Rag1−/- recipients of K5.mOVA grafts received increasing numbers of Rag−/- OTI OVA-specific CD8 T cells from spleen as shown, together with sufficient irrelevant TCR transgenic T cells to equalize the number of transferred cells, and graft survival was observed (n≥4 per group). (b) Rag-OT-I OVA-specific CD8 T cells from spleen (106) were administered with (n=4) or without (n=4) OVA-specific T-helper (OT-II) spleen cells (106) to immunodeficient Rag1−/- recipients. After 3 days, K5mOVA skin grafts were placed and followed up to rejection. (c) Rag−/- 2C immunodeficient mice received 102 OVA-specific CD8 OT-1 cells, or 102 OT-1 cells and 106 OVA-specific CD4 OT-2 cells. K5.mOVA skin grafts were placed and followed up to rejection. Graft survival over time is shown. Survival differences were assessed for significance using the log-rank (Mantel–Cox) test. Results are from two separate experiments. Journal of Investigative Dermatology 2010 130, 1581-1589DOI: (10.1038/jid.2010.17) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 CD4 T cells provide help to activated CD8 T cells. (a, b) OT-I CD8 Ova-specific T cells, either activated in vitro (left panels) or not so activated (“naive”; right panels), were assessed for CD8, CD44 (a), or CD62L (b) expression by flow cytometry. (c) OT-II spleen cells were enriched for OVA-specific T-helper cells using anti-CD4 magnetic beads and enrichment assessed with anti-CD4 and -CD3 antibodies by flow cytometry. (d) Mice received 102 activated OVA-specific CD8 OT-I cells (n=7) or 102 activated OT-I and 106 CD4-enriched OVA-specific helper OT-II cells (n=7). K5.mOVA skin grafts were placed and followed up to rejection. Graft survival over time is shown. Survival differences were assessed for significance using log-rank (Mantel–Cox) test. Journal of Investigative Dermatology 2010 130, 1581-1589DOI: (10.1038/jid.2010.17) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions