Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell “mass cytometry” to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.

The engagement of programmed death 1 (PD-1) to its ligands, PD-L1 and PD-L2, inhibits proliferation and cytokine production mediated by antibodies to CD3 (refs. 5,6,7). Blocking the PD-1-PD-L1 pathway in mice chronically infected with lymphocytic choriomeningitis virus restores the capacity of exhausted CD8(+) T cells to undergo proliferation, cytokine production and cytotoxic activity and, consequently, results in reduced viral load. During chronic HIV infection, HIV-specific CD8(+) T cells are functionally impaired, showing a reduced capacity to produce cytokines and effector molecules as well as an impaired capacity to proliferate. Here, we found that PD-1 was upregulated on HIV-specific CD8(+) T cells; PD-1 expression levels were significantly correlated both with viral load and with the reduced capacity for cytokine production and proliferation of HIV-specific CD8(+) T cells. Notably, cytomegalovirus (CMV)-specific CD8(+) T cells from the same donors did not upregulate PD-1 and maintained the production of high levels of cytokines. Blocking PD-1 engagement to its ligand (PD-L1) enhanced the capacity of HIV-specific CD8(+) T cells to survive and proliferate and led to an increased production of cytokines and cytotoxic molecules in response to cognate antigen. The accumulation of HIV-specific dysfunctional CD8(+) T cells in the infected host could prevent the renewal of a functionally competent HIV-specific CD8(+) repertoire.

Correlates of immune-mediated protection to most viral and cancer vaccines are still unknown. This impedes the development of novel vaccines to incurable diseases such as HIV and cancer. In this study, we have used functional genomics and polychromatic flow cytometry to define the signature of the immune response to the yellow fever (YF) vaccine 17D (YF17D) in a cohort of 40 volunteers followed for up to 1 yr after vaccination. We show that immunization with YF17D leads to an integrated immune response that includes several effector arms of innate immunity, including complement, the inflammasome, and interferons, as well as adaptive immunity as shown by an early T cell response followed by a brisk and variable B cell response. Development of these responses is preceded, as demonstrated in three independent vaccination trials and in a novel in vitro system of primary immune responses (modular immune in vitro construct [MIMIC] system), by the coordinated up-regulation of transcripts for specific transcription factors, including STAT1, IRF7, and ETS2, which are upstream of the different effector arms of the immune response. These results clearly show that the immune response to a strong vaccine is preceded by coordinated induction of master transcription factors that lead to the development of a broad, polyfunctional, and persistent immune response that integrates all effector cells of the immune system.

Su ibity to systec lupus erythematosus has been unequivocally e d to be an inherited trai, but the exact genes and how they confer susceptibility n largely unknown. In this study of (NZB x NZW)F2 inte mice, we used linkage analysis ofmarkers covering >90% ofthe automal genome and idenified eit ptl ld chomosomes 17,(4)(5)(6)(7)18,1,11, pectively) ated with antichromatin autoantibody production, gm nphris, and/or it. Only one locus, the major his patibt complx, was linked to all three traits. Two other loci were ed with both glomerulonephitis and mortalt, whereas the remaining loci were linked to one of the above traits. Two adial loci (Sbwl and -2) that conibut to p m were also iden- Among the murine lupus strains, the (NZB x NZW)F1 (BWF1) hybrid has clinical features most closely resembling human SLE with markedly accelerated disease compared with parental strains and a striking female predilection (2). Conventional genetic studies indicate that each of the parental strains contributes at least one or two genes (3, 4), one of which is linked to the MHC locus, with heterozygosity (H-2dz) conferring maximal susceptibility (5,6).The recent identification of thousands of polymorphic dinucleotide repeats (microsatellites) that can be used to create dense linkage maps between inbred strains (7, 8) has made it feasible to systematically search the entire mouse genome for susceptibility gene loci. We have used this approach to map the genes predisposing to early disease in the BWF1 hybrid and report the identification of several loci predisposing to mortality, glomerulonephritis (GN), antichromatin antibody production, and splenomegaly.MATERIALS AND METHODS Mice. NZB/BlScr, NZW/LacScr, BWF1, and (NZB x NZW)F2 (BWF2) intercross mice were bred and maintained in our animal colony. Female mice from 6 mo of age were examined daily for disease, bled monthly for sera, and sacrificed at either 1 yr of age or earlier if moribund.Phenotyping of Mice. Autopsies and histologic examinations were done as described (9). Severity of GN was graded from 0 to 4+ (9), and mice were considered to have severe GN ifthey were 4+ at 12 mo or .3+ ifmice eitherdied earlier or had anasarca. Survival comparisons and cumulative antichromatin antibody levels were analyzed with the generalized Wilcoxon test. Comparisons of spleen size were done with the Mann-Whitney U test for small sample sizes and the Student's t test for larger samples. ELISA for chromatin was done as described (10). For linkage analysis, BWF2 mice with antichromatin antibody levels of OD > 0.5 by 11 mo were considered positive.Cbromosoma Markers and Genotyping of Mice. Chromosomal markers consisted of simple-sequence-length polymorphisms (SSLPs) identified by PCR (refs. 7,8

Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Lack of effector T cell (Teff) function and gain of suppressive activity by Treg are dependent on the transcriptional program induced by Foxp3. Here we report repression of SATB1, a genome organizer regulating chromatin structure and gene expression, as crucial for Treg phenotype and function. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly through induction of microRNAs that bound the SATB1 3′UTR. Release of SATB1 from Foxp3 control in Treg caused loss of suppressive function, establishment of transcriptional Teff programs and induction of Teff cytokines. These data support that inhibition of SATB1-mediated modulation of global chromatin remodelling is pivotal for maintaining Treg functionality.