Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry.

Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acquisition parameters and fluorochrome combination for long-term HSC staining have been specifically designed for concurrent use with DAPI and Ki-67 antibodies. © 2018 by John Wiley & Sons, Inc.

Pre-TCR ligand binding impacts thymocyte development before αßTCR expression.

Adaptive cellular immunity requires accurate self- vs. nonself-discrimination to protect against infections and tumorous transformations while at the same time excluding autoimmunity. This vital capability is programmed in the thymus through selection of αßT-cell receptors (αßTCRs) recognizing peptides bound to MHC molecules (pMHC). Here, we show that the pre-TCR (preTCR), a pTα-ß heterodimer appearing before αßTCR expression, directs a previously unappreciated initial phase of repertoire selection. Contrasting with the ligand-independent model of preTCR function, we reveal through NMR and bioforce-probe analyses that the ß-subunit binds pMHC using Vß complementarity-determining regions as well as an exposed hydrophobic Vß patch characteristic of the preTCR. Force-regulated single bonds akin to those of αßTCRs but with more promiscuous ligand specificity trigger calcium flux. Thus, thymic development involves sequential ß- and then, αß-repertoire tuning, whereby preTCR interactions with self pMHC modulate early thymocyte expansion, with implications for ß-selection, immunodominant peptide recognition, and germ line-encoded MHC interaction.

A conserved E7-derived cytotoxic T lymphocyte epitope expressed on human papillomavirus 16-transformed HLA-A2+ epithelial cancers.

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS(3) Poisson detection mass spectrometry. Only one epitope (E7(11-19)) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7(11-20)), previously used as a vaccine candidate, was neither detected by MS(3) on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.

PlexinD1 glycoprotein controls migration of positively selected thymocytes into the medulla.

Precise intrathymic cell migration is important for thymocyte maturation and organ architecture. The orchestration of thymocyte trafficking, however, is not well understood at a molecular level. Here, we described highly regulated plexinD1 expression on CD4+CD8+ double positive (DP) thymocytes. PlexinD1 expression was further affected by the engagement of T cell receptor complex. Activation of plexinD1 via the ligand, semaphorin 3E, repressed CCL25 chemokine signaling via its receptor CCR9 in CD69+ thymocytes. In the absence of plexinD1, CD69+ thymocytes remained in the cortex, maturing to form ectopic single positive (SP) thymocyte clusters in Plxnd1-deficient fetal liver cell-transplanted mice. As a consequence, the boundary between DP and SP thymocytes at corticomedullary junctions was disrupted and medullary structures formed under the thymic capsule. These results demonstrate the importance of plexinD1 in directing migration of maturing thymocytes via modulation of biological responses to chemokine gradients.

The TCR C beta FG loop regulates alpha beta T cell development.

The TCRbeta chain constant domain contains an unusually elongated, solvent-exposed FG loop. This structural element forms one component of an alphabeta TCR cavity against which CD3epsilongamma may abut to facilitate Ag-specific signaling. Consistent with this notion, in the present study we show that N15alphabeta TCR transfectants expressing a FG loop-deleted chain (betaDeltaFG) stimulate less tyrosine protein phosphorylation than those bearing a wild-type beta-chain (betawt) upon TCR cross-linking. Furthermore, coimmunoprecipitation studies suggest a weakened association between the CD3epsilongamma heterodimer and the beta-chain in TCR complexes containing the betaDeltaFG variant. To further investigate the biologic role of the Cbeta FG loop in development, we competitively reconstituted the thymus of Ly5 congenic or RAG-2-/- mice using bone marrow cells from betawt or betaDeltaFG transgenic C57BL/6 (B6) mice. Both betawt and betaDeltaFG precursor cells generate thymocytes representative of all maturational stages. However, betaDeltaFG-expressing thymocytes dominate during subsequent development, resulting in an excess of betaDeltaFG-expressing peripheral T cells with reduced proliferative and cytokine production abilities upon TCR stimulation. Collectively, our results show that the unique Cbeta FG loop appendage primarily controls alphabeta T cell development through selection processes.

Sca+CD34- murine side population cells are highly enriched for primitive stem cells.

OBJECTIVE: The aim of this study was to characterize murine side population (SP) stem cells and SP cell subpopulations for primitive stem cell capacity. MATERIALS AND METHODS: SP cells, characterized by a specific Hoechst dye efflux pattern, were isolated by flow cytometric analysis and sorting from murine adult whole bone marrow (WBM). Different subpopulations of SP cells were isolated by staining with anti-Sca and anti-CD34 antibodies. Primitive stem cell content of SP cells and SP subsets were determined by cobblestone area-forming cell (CAFC) frequencies. RESULTS: Measurement of CAFC frequencies revealed that SP cells are greatly enriched for both primitive stem cells (day-28-35 CAFC) and somewhat more mature hematopoietic cells (day-14-21 CAFC) compared to WBM. The day-28 and day-35 CAFC enrichments in SP cells vs WBM cells were 1065 and 471, respectively. Analysis of the subpopulations of SP cells revealed that SP(+)Sca(-)CD34(+) cells contained almost exclusively day-7 CAFC and had little day-28-35 CAFC activity. SP(+)Sca(+)CD34(+) cells had high day-7-14 CAFC frequencies, but lower day-35 CAFC frequencies compared to SP(+)Sca(+)CD34(-) cells. SP(+)Sca(+)CD34(-) cells contained very low day-7 CAFC activity, but nearly 2200 times the day-28-35 CAFC activity as normal bone marrow. To evaluate the influence of Hoechst dye efflux capacity, we divided the SP tail into four groups of cells. The SP cells with lowest efflux of Hoechst dye contained the highest progenitor activity (day-7-14 CAFC). The highest day-35 CAFC frequencies, nearly 6000 times those of normal marrow, were seen in the SP cells with the greatest efflux of the Hoechst dye. CONCLUSIONS: Murine SP cells contain both progenitor and primitive populations of hematopoietic stem cells. The most primitive stem cells measured in the in vitro CAFC assay mark for Sca(+) and CD34(-) and have a high ability to efflux Hoechst dye. Isolation of these cells may provide the means to directly study mechanisms of primitive stem cell damage.