We developed a highly contiguous chromosome-level reference genome for North American bison to provide a platform to evaluate the conservation, ecological, evolutionary, and population genomics of this species. Generated from a F1 hybrid between a North American bison dam and a domestic cattle bull, completeness and contiguity exceeds that of other published bison genome assemblies. To demonstrate the utility for genome-wide variant frequency estimation, we compiled a genomic variant database consisting of three true albino bison and 44 wild-type pelage color bison. Through the examination of genomic variants fixed in the albino cohort and absent in the controls, we identified a nonsynonymous single nucleotide polymorphism (SNP) mutation on chromosome 29 in exon 3 of the tyrosinase gene (c.1114C > T). A TaqMan SNP Genotyping Assay was developed to genotype this SNP in a total of 283 animals across 29 herds. This assay confirmed the absence of homozygous variants in all animals except 7 true albino bison included in this study. In addition, the only heterozygous animals identified were 2 wild-type pelage color dams of albino offspring. Therefore, we propose that this new high-quality bison genome assembly and incipient variant database provides a highly robust and informative resource for genomics investigations for this iconic North American species.
687 Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation. We previously showed that recipient antigen-presenting cells (APCs) are required for CD8-dependent GVHD, while donor APCs promote GVHD in a MHC matched (C3H.SW (right arrow) B6; both H-2b) model (Shlomchik et al, Science 1999; Matte et al., Nat Med 2004). However, how donor APCs promote maximal GVHD was not addressed. The LTFNYRNL peptide from H60 is a dominant minor histocompatibility antigen (miHA) presented by H-2Kb. To study cross-presentation of H60, we crossed B6 mice congenic for H60 (CH60; hematopoietically restricted) or transgenic for H60 driven by an actin promoter (actH60; H60 is ubiquitously expressed) with B6 Kb-/- mice. These mice express H60 but cannot directly present it to donor CD8 cells as they do not express Kb. CH60C*Kb-/-and actH60*Kb-/- were irradiated and reconstituted with C3H.SW (H60-) bone marrow,106(6 superscript) CD8 T cells and 2*105( 5 superscript) CD4 (to promote the CD8 response to H60). Using H60-MHC tetramers, we detected H60-specific CD8 T cell expansion as early as day 8, with a peak at day14, demonstrating cross-priming by donor C3H.SW APCs. Intracellular IFN-γ staining and an in vivo CTL assay showed that these cross-primed CD8 T cells had effector functions. Surprisingly, accumulation of H60-tetramer+ cells was greater when it was exclusively cross-presented. SIINFEKL, a peptide derived from ovalbumin (OVA), is also presented by Kb. Therefore to confirm our findings we used B6 mice transgenic for ovalbumin crossed to Kb-/- mice (ova*Kb-/-) as recipients in the same model. SIINFEKL-tetramer+ T cells expansion was also observed in ova*Kb-/- recipients, demonstrating cross-priming. The source of miHA did not affect the cross-priming as similar SIINFEKL-reactive T cell expansion occurred in retransplanted (right arrow)ova*Kb-/-, ova*Kb-/-(right arrow) Kb-/- bone marrowγKb-/- chimeras. Cross-priming of SIINFEKL-reactive CD8 cells even occurred when BALB/c mice transgenic for OVA (BALB/c-ova; (H-2d)) were transplanted with B6 BM and a mix of B6 CD4 and CD8 cells. SIINFEKL-reactive cells produced IFN-γ and killed SIINFEKL-pulsed B6 cells in vivo. Because of the availability of knockout/transgenic mice backcrossed to B6, we used this system to explore mechanisms of cross-presentation. Donor CD11c+ dendritic cells (DCs) were required as cross-priming was abrogated when BALB/c-OVA mice were transplanted with BM from mice constitutively lacking CD11c+ DCs (Birnberg et al, Immunity 2008). CD4 help has been reported to be important for cross-priming. Surprisingly, however, cross-priming by donor APCs was unaffected when BALB/c-OVA mice were transplanted with B6 MHCII-/- BM but was greatly reduced in recipients of B6 CD40-/- BM. Thus, while CD40L activation of cross-priming DCs is important, CD4 cells which are likely the source of the CD40L need not actually make T cell receptor:MHC contacts with the cross-presenting DC. CD40L-conditioning of donor APCs is not required to cross-prime memory cells, as sort-purified memory CD8 cells from SIINFEKL-vaccinated mice expanded robustly in actOVA*Kb-/- but not Kb-/- mice. Cross-priming also occurred in recipients of IFNAR1-/- BM, indicating that Type I IFN activation of donor APCs is not required as has been reported in nontransplant settings. Taken together, our data demonstrate that cross-presentation by donor DCs occurs in MHC-matched and -mismatched transplants, and this cross-presentation likely explains the reduced GVHD we observed in recipients of MHCI- donor bone marrow. That T cells can be cross-primed to nonhematopoietic antigens provides a basis for persistent GVHD and for the generation of CD8 responses against antigens not initially targeted. We also found transplantation to be a permissive environment for cross-priming in that CD4 help could be delivered in trans, type I IFN APC activation was not required and memory cells could be activated without CD4 help. These data provide further rationale for targeting donor DCs and pathways required for cross-presentation to prevent and treat GVHD. Disclosures: No relevant conflicts of interest to declare.
689 In MHC-mismatched allogeneic bone marrow/stem cell transplantation (alloBMT), T cells that at least in-part recognize the allogeneic MHC are likely the dominant mediators of acute GVHD. This would explain why far fewer T cells induce acute GVHD in MHC-mismatched as opposed to MHC-matched alloBMT. However, despite this difference, in human recipients of haploidentical allografts, the organ distribution and histologic appearance of GVHD, especially chronic GVHD, are similar. Moreover, late-onset GVHD in MHC-mismatched alloBMT occurs when recipient antigen presenting cells (APCs) have been replaced by donor APCs. We therefore investigated how indirect presentation of host minor histocompatibility antigens (miHAs) by donor APCs contributes to GVHD. To test this, we employed the MHC/miHA mismatched B6 (H-2b) (right arrow) BALB/c (H-2d) strain pairing. We compared GVHD in irradiated BALB/c mice that were reconstituted with B6 CD4 cells and either B6 BM or B6 MHCII-/- BM. We reasoned that if BALB/c miHAs presented by donor B6 APCs were important, GVHD would be reduced in recipients of MHCII-/- BM. As measured by weight loss and survival, this was indeed the case. However, it was possible that receiving MHCII-/- donor BM reduced GVHD for reasons unrelated to the presentation of BALB/c miHAs. To address this we compared GVHD in irradiated B6.C (H-2d) recipients of B6 CD4 cells and either B6 or B6 MHCII-/- BM. This strain pairing shares the same MHC mismatch as does the B6(right arrow)BALB/c model, but lacks miHAs except those encoded by the congenic H-2d region. In contrast to the prior experiments, B6.C recipients of MHCII-/- or wild type BM developed similar GVHD. To further define indirect-presentation of miHAs in MHC/miHA-mismatched alloBMT, we used B6 Marilyn CD4+ T cell receptor transgenic T cells that recognize an IAb-restricted peptide derived from the male DBY protein. Male or female BALB/c mice were irradiated and reconstituted with female B6 BM and female polyclonal B6 CD4 cells along with graded numbers of Marilyn T cells. At day 7 and 14 post transplant Marilyn cells expanded at least 1000-fold greater in male than in female recipients. Marilyn T cell expansion was similar when male antigen was restricted to hematopoietic or nonhematopoietic cells. There was little expansion of Marilyn cells in male BALB/c mice if donor BM was MHCII-/-, confirming that expansion of Marilyn T cells depended on donor APCs presenting DBY. Indirectly primed Marilyn cells were functional as measured by IFN-γ production after restimulation by the DBY peptide in vitro. As we saw reduced GVHD in the B6 (right arrow) BALB/c model in recipients of MHCII-/- BM, we reasoned we should be able to detect polyclonal CD4 cells responding to host miHA presented by donor APCs. That was the case as we found an increase in DBY-reactive polyclonal CD4 cells by ELISPOT in male recipients as compared to female recipients at day 7 (1.5 fold for IFN-γ and TNF-αa). This difference was lost when donor BM was MHCII-/-. In summary, indirect presentation of host miHAs by donor APCs in MHC-mismatched alloBMT is surprisingly efficient and alloreactive CD4 cells that target host miHAs presented by donor APCs contribute to GVHD in MHC/miHA-disparate alloBMT. The generation of T cells that target host miHAs presented by donor APCs allows GVHD to persist even when host APCs that prime T cells that recognize the allogeneic MHC have been eradicated. This provides a mechanistic explanation for why late onset GVHD, including chronic GVHD, in MHC-mismatched alloBMT resembles that in MHC-matched alloBMT as both may target host miHAs indirectly presented by donor-derived APCs. These data also provide further rationale for targeting donor-derived APCs as a method for preventing and treating GVHD. Disclosures: No relevant conflicts of interest to declare.
The skin is an essential barrier that protects organisms from the external environment. Despite recent advances in our understanding of skin homeostasis, the ways by which diverse cell types interact with each other to sustain this process is not fully understood. Challenges in addressing this question have been the inability to simultaneously follow behaviors of different cell types and to define their functional interactions in a live mammal. Skin epidermis is an ideal system because of its accessibility and well-characterized epithelial and coexisting epidermal immune cells. Epithelial cells in the skin are intermixed with different types of immune cells such as dendritic epidermal T-cells (DETCs) and Langerhans cells (LCs). By using our intravital imaging platform, I have established novel genetic and cell biological approaches to define and manipulate epithelial-immune interactions in an intact epidermis in live mice. In addition to immune surveillance, our data shows that both immune populations can perceive and respond to the changes of their neighbors. Furthermore, epidermal immune cells retain spatial organization within their own population while neighboring epithelial cells continuously divide and differentiate. Lastly, skin epithelial cells act as regional checkpoints for the organization and number of epidermal immune populations, but not vice versa, during epidermal homeostasis. This study reveals new principles of immune organization within the epidermis, and elucidates dynamic epithelial-immune interactions that are in place to maintain homeostasis of the epidermis.
Magnesium regulates permeability barrier homeostasis in human epidermal equivalent cultures J Meyer, D Crumrine, TM Mauro and PM Elias Dermatology, VA Med Ctr/UCSF, San Francisco, California, United States While magnesium (Mg) deficiency was shown to cause diffuse dermatitis in laboratory animals over 80 years ago, the function of this cation in the skin remains unclear. The recent discovery of an ichthyosis with a permeability barrier abnormality caused by loss-of-function mutations in the Mg transporter, ichthyin, suggests that Mg is important for permeability barrier homeostasis. In the current study, we explored the role of Mg in barrier function in human epidermal equivalent (HEE) keratinocyte cultures. The Mg content in HEEs correlated with [Mg] in the culture media, independent of changes in calcium concentrations, and was reduced by more than 50% under low Mg conditions. Permeability barrier function, as assessed by transepithelial electrical resistance, was reduced by more than 65% when Mg in the media was reduced from 1 mM (physiologic) to 25 mM, but was preserved at higher
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.