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In contrast to previous notions of the help-independency of memory CD8 T cells during secondary expansion, here we show that CD4 help is indispensable for the re-expansion of once-helped memory CD8 T cells, using a hematopoietic cellspecific dominant minor histocompatibility (H) antigen, H60, as a model antigen. H60-specific memory CD8 T cells generated during a helped primary response vigorously expanded only when rechallenged under helped conditions. The help requirement for an optimal secondary response was confirmed by a reduction in peak size by CD4 depletion, and was reproduced after skin transplantation. Helpless conditions or noncognate separate help during the secondary response resulted in a significant reduction in the peak size and different response kinetics. Providing CD4 help again during a tertiary challenge restored robust memory expansion; however, the repeated deprivation of help further reduced clonal expansion. Adoptively transferred memory CD8 T cells did not proliferate in CD40L ؊/؊ hosts. In the CD40 ؊/؊ hosts, marginal memory expansion was detected after priming with male H60 cells but was completely abolished by priming with peptideloaded CD40 ؊/؊ cells, suggesting the essential role of CD40 and CD40L in memory responses. These results provide insight into the control of minor H antigenspecific CD8 T-cell responses, to maximize the graft-versus-leukemia response. IntroductionMinor histocompatibility (H) antigens are naturally processed peptide fragments derived from proteins with polymorphisms that generate differential major histocompatibility complex (MHC)-presenting epitopes. 1 They are recognized as foreign during allotransplantation between MHC-matched subjects, resulting in the induction of specific CD4 and CD8 T-cell responses that can lead to life-threatening graft-versus-host disease (GVHD); however, they have also been found to exert a graft-versus-leukemia (GVL) effect. 2 Therefore, characterization of the immune response to minor H antigens with specific expression on hematopoietic lineage cells is necessary to apply the minor H antigen-specific T-cell response most productively toward eradicating malignant leukemic cells while suppressing GVHD.Allo-reactive CD8 T cells are major effectors of rejection after allogeneic transplantation. The CD8 T-cell response to antigen exposure generally involves an initial expansion phase followed by a contraction phase and a memory phase. [3][4][5] During this process, help conferred by CD4 T cells is essential for generating the CD8 T-cell response to noninflammatory Th-dependent antigens, such as minor H antigens, although the timing and mechanism of delivery of this help to CD8 T cells is controversial. CD40 and CD40L are believed to be major mediators of CD4 help. Signaling through CD40 via an interaction with CD40L expressed on activated CD4 T cells activates antigen-presenting cells (APCs), which subsequently increases their antigen presentation and costimulatory capacity, priming CD8 T cells directly. [6][7][8][9] In contrast to n...

The zebra finch has been used as a valuable vocal learning animal model for human spoken language. It is representative of vocal learning songbirds specifically, which comprise half of all bird species, and of Neoaves broadly, which comprise 95% of all bird species. Although transgenesis in the zebra finch has been accomplished, it is with a very low efficiency of germ-line transmission and far from the efficiency with a more genetically tractable but vocal nonlearning species, the chicken (a Galloanseriformes). To improve germ-line transmission in the zebra finch, we identified and characterized its primordial germ cells (PGCs) and compared them with chicken. We found striking differences between the 2 species, including that zebra finch PGCs were more numerous, more widely distributed in early embryos before colonization into the gonads, had slower timing of colonization, and had a different developmental gene-expression program. We improved conditions for isolating and culturing zebra finch PGCs in vitro and were able to transfect them with gene-expression vectors and incorporate them into the gonads of host embryos. Our findings demonstrate important differences in the PGCs of the zebra finch and advance the first stage of creating PGC-mediated germ-line transgenics of a vocal learning species.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated protein 9 (Cas9) have facilitated the production of genome‐edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9‐mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome–targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off‐target effects. The genome‐modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome‐editing method could be used to develop many chicken models and should significantly expand the application of genome‐modified avians.—Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development. FASEB J. 33, 8519–8529 (2019). http://www.fasebj.org

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BackgroundThe persistence of latently Human immunodeficiency virus-1 (HIV-1) infected cellular reservoirs in resting CD4+ T cells is a major obstacle to HIV-1 eradication. The detailed mechanism of HIV-1 latency remains unclear. We investigated histones and their post-translational modification associated with HIV-1 latency in novel HIV-1 latently infected cell lines established previously, NCHA cells.MethodsTo examine histones and their modification linked with HIV-1 latency, the expression profiles for core histone proteins and histone deacetylases (HDACs) in NCHA cells were characterized by RT-PCR, ELISA, and western blot. The levels of histone acetylation and methylation at histone H3 Lys9 (H3K9) and Lys27 (H3K27) in HIV-1 latently infected cells were analyzed by western blot and chromatin immunoprecipitation-sequencing (ChIP-seq).ResultsThe expression levels for four core histone proteins (H2A, H2B, H3 and H4) and HDACs (HDAC1-8) in NCHA cells were not significantly different from those in their parental cells. Histone H3K9 and H3K27 acetylations in NCHA cells showed no difference in parental and NCHA cells, whereas the levels of di- and tri-methylation were increased in NCHA cells. The expression of EED which is a component of polycomb repressive complex 2 (PRC2), and BMI1 and RING2 which are constituents of PRC1, were upregulated in NCHA cells. In addition, more ubiquitylation at histone H2A was detected in NCHA cells.ConclusionsOur results suggest that tri-methylation of histone H3K27 and H2A ubiquitylation via polycomb group protein may play a crucial role in epigenetic silencing accounting for HIV-1 latency in NCHA cells.

Primordial germ cells (PGCs) are undifferentiated gametes with heterogeneity, an evolutionarily conserved characteristic across various organisms. Although dynamic selection at the level of early germ cell populations is an important biological feature linked to fertility, the heterogeneity of PGCs in avian species has not been characterized. In this study, we sought to evaluate PGC heterogeneity in zebra finch using a single-cell RNA sequencing (scRNA-seq) approach. Using scRNA-seq of embryonic gonadal cells from male and female zebra finches at Hamburger and Hamilton (HH) stage 28, we annotated nine cell types from 20 cell clusters. We found that PGCs previously considered a single population can be separated into three subtypes showing differences in apoptosis, proliferation, and other biological processes. The three PGC subtypes were specifically enriched for genes showing expression patterns related to germness or pluripotency, suggesting functional differences in PGCs according to the three subtypes. Additionally, we discovered a novel biomarker, SMC1B, for gonadal PGCs in zebra finch. The results provide the first evidence of substantial heterogeneity in PGCs previously considered a single population in birds. This discovery expands our understanding of PGCs to avian species, and provides a basis for further research.