The GATA1 and GATA2 transcription factors play a central role in early cell fate decisions in hematopoietic progenitor cells. Although the switch from GATA2 to GATA1 occupancy at GATA-binding sites in erythroblast-specific genes has been extensively studied, the underlying molecular mechanisms controlling this switch are not fully elucidated. An antisense promoter in the 5' region of the GATA2 gene produces a long non-coding RNA that has been shown to affect GATA2 transcription and erythroblast differentiation. The recent identification of an antisense promoter in the first intron of the GATA1 gene indicates that similar promoter competition mechanisms operate in these genes, potentially controlling GATA1/GATA2 levels in a probabilistic manner. In the current study we perform a comprehensive evaluation of GATA1 and GATA2 transcripts in human CD34 progenitors either freshly isolated or differentiated in vitro and the human leukemia cell lines K562 and HL60. The ratio of competing sense and antisense transcripts varied significantly with differentiation status, suggesting that promoter competition controls cell fate decisions. Treatment of HL60 cells with differentiating agents resulted in significant changes in the ratio of GATA2 sense to antisense promoter activity, in agreement with previous results showing that sense and antisense transcription are associated with distinct cellular phenotypes.

Previous studies of the murine Ly49 and human KIR gene clusters implicated competing sense and antisense promoters in the control of variegated gene expression. In the current study, an examination of transcription factor genes defines an abundance of convergent and divergent sense/antisense promoter pairs, suggesting that competing promoters may control cell fate determination. Differentiation of CD34+ hematopoietic progenitors in vitro shows that cells with GATA1 antisense transcription have enhanced GATA2 transcription and a mast cell phenotype, whereas cells with GATA2 antisense transcription have increased GATA1 transcripts and an erythroblast phenotype. Detailed analyses of the AHR and RORC genes demonstrate the ability of competing promoters to act as binary switches and the association of antisense transcription with an immature/progenitor cell phenotype. These data indicate that alternative cell fates generated by promoter competition in lineage-determining transcription factors contribute to the programming of cell differentiation.

The human KIR genes encode a family of class I MHC receptors that are expressed on subsets of NK cells. The expression of KIR proteins is controlled by a stochastic process, and competition between sense and antisense promoter elements has been suggested to program the variegated expression of these genes. Previous studies have demonstrated distinct roles of distal, intermediate, and proximal sense promoter/enhancer elements in gene activation and expression. Conversely, proximal and intronic antisense promoter transcripts have been associated with gene silencing at different stages of NK cell development. In the current study, we examine the effect of intermediate promoter deletion on KIR2DL1 expression in the YTS cell line. Homozygous deletion of the KIR2DL1 intermediate element did not affect proximal promoter activity but resulted in increased detection of upstream transcripts. No significant changes in alternative mRNA splicing or expression levels of KIR2DL1 protein were observed. However, intermediate element deletion was associated with a reduced frequency of gene activation by 5-azacytidine. Taken together, these results indicate that the intermediate element is not an enhancer required for KIR expression; however, it is required for the efficient activation of the gene.

Previous studies of the murine Ly49 and human KIR gene clusters have revealed a role for bidirectional promoters in the control of variegated gene expression. Whether or not competing promoters control other instances of cell fate determination remains an outstanding question. Although divergent transcripts within 300 bp are found in ~6% of human genes, an analysis of human transcription factor (TF) genes (1640) revealed that 33% possess stable divergent transcripts with a transcription start site (TSS) less than 300 bp upstream, with many separated by less than 30 bp, indicating an enrichment for potential binary switches in TFs. We have performed a detailed examination of putative bidirectional promoter switches in three lineage-determining TF genes: AHR, GATA3 , and RORγT . These genes also contain additional pairs of opposing promoters that would prevent simultaneous transcription of sense and antisense, and thus may represent simple on/off switches rather than probabilistic switches. In situ RNA hybridization of human tissues revealed mutually exclusive expression of sense versus antisense transcription, indicating switching between stable sense or antisense transcriptional states. Single-cell RNAseq confirmed the separate sense/antisense states, and revealed the identity of cells with active switch elements. Differential gene expression analysis of cells with antisense switch transcripts revealed an enrichment for genes found in immature/stem cells and a lack of genes associated with terminally differentiated cells. Taken together, these data indicate that there is a digital component to the differentiation program mediated by binary promoter switches in lineage-determining transcription factors.

A complex system regulating HLA-C expression in NK cells, driven by an NK-specific promoter that produces alternatively spliced variants of the 5 '-UTR has been recently identified. Exon content of the NK-specific 5 '-UTR varies strikingly across HLA-C alleles, with some exons being allele specific. In order to investigate the possibility that allelic variation in the 5 '-UTR modulates HLA-C expression levels, cDNAs containing several distinct classes of 5 '-UTR were compared. Subtle changes in 5 '-UTR content had a significant effect on the expression of HLA-C*03 and HLA-C*12 cDNA clones, suggesting that alternative splicing can fine-tune the level of protein expression. The HLA-C*06 allele was found to be highly expressed in relation to the other alleles studied. However, its increased expression was primarily associated with differences in the peptide-binding groove. Although the impact of allele-specific alternative splicing of NK-Pro transcripts on protein levels can be modest when compared with the effect of changes in peptide-loading, alternative splicing may represent an additional regulatory mechanism to fine-tune HLA-C levels within NK cells in distinct tissue environments or at different stages of maturation in order to achieve optimal levels of missing-self recognition.

We analysed data from 80 patients who tested positive for SARS-CoV-2 RNA who had previously been HLA typed to support transplantation. Data were combined from two adjacent centres in Manchester and Leeds to achieve a sufficient number for early analysis. HLA frequencies observed were compared against two control populations: first, against published frequencies in a UK deceased donor population (n = 10,000) representing the target population of the virus, and second, using a cohort of individuals from the combined transplant waiting lists of both centres (n = 308), representing a comparator group of unaffected individuals of the same demographic. We report a significant HLA association with HLA- DQB1*06 (53% vs. 36%; p < .012; OR 1.96; 95% CI 1.94-3.22) and infection. A bias towards an increased representation of HLA-A*26, HLA-DRB1*15, HLA-DRB1*10 and DRB1*11 was also noted but these were either only significant using the UK donor controls, or did not remain significant after correction for multiple tests. Likewise, HLA-A*02, HLA-B*44 and HLA-C*05 may exert a protective effect, but these associations did not remain significant after correction for multiple tests. This is relevant information for the clinical management of patients in the setting of the current SARS-CoV-2 pandemic and potentially in risk-assessing staff interactions with infected patients.

Human rhinoviruses cause the common cold and exacerbate chronic respiratory diseases. Although infection elicits neutralizing antibodies, these do not persist or cross-protect across multiple rhinovirus strains. To analyze rhinovirus-specific B cell responses in humans, we developed techniques using intact RV-A16 and RV-A39 for high-throughput high-dimensional single-cell analysis, with parallel assessment of antibody isotypes in an experimental infection model. Our approach identified T-bet+ B cells binding both viruses that account for similar to 5% of CXCR5- memory B cells. These B cells infiltrate nasal tissue and expand in the blood after infection. Their rapid secretion of heterotypic immunoglobulin G (IgG) in vitro, but not IgA, matches the nasal antibody profile post-infection. By contrast, CXCR5+ memory B cells binding a single virus are clonally distinct, absent in nasal tissue, and secrete homotypic IgG and IgA, mirroring the systemic response. Temporal and spatial functions of dichotomous memory B cells might explain the ability to resolve infection while rendering the host susceptible to re-infection.

Background: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. Objective: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. Methods: Circulating virus-specific T(H)1 cells and allergenspecific T(H)2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. Results: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1(+) T(H)1 cells, but not allergenspecific T(H)2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral T(H)1 response in asthmatic subjects versus controls, with synchronized allergen-specific T(H)2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, T(H)2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-gamma, TNF-alpha) correlated with both egress of circulating virus-specific T(H)1 cells and worse symptoms. Conclusions: Rhinovirus induces robust T(H)1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.

Despite the recognition of various endotypes in allergic disease, surprisingly little is known regarding the complexity of T helper (Th) cells that respond to house dust mite (HDM), especially given the IgE dominance of HDM in patients with asthma and atopic dermatitis (AD). Thus, we rigorously analyzed dust mite-reactive Th cells using a novel 22-marker immunophenotyping panel for spectral flow cytometry in patients with different diseases. Peripheral blood mononuclear cells isolated from allergic adults with asthma and/or AD (ImmunoCAP class ≥3 for HDM and Der p 1) were labeled with cell trace dye (CellTraceTM Violet), and cultured for 7 days with HDM or Der p 1. Cells were then stained for surface (CD3, CD4, CD27, CRTH2, CD45RO, CD45RA, CD25) and intracellular (IL-4, IL-5, IL-9, IL-10, IL-13, IL-22, TNF-α, IFN-γ, IL-17A) markers. Dust mite-reactive cells (cell trace dye low) were analyzed by spectral flow cytometry. As described previously, Th2 signatures were enriched within CD27-CRTH2+ cells and Th1/Th17 signatures dominated CD27+/-CRTH2- subsets. However, HDM- and Der p 1-reactive T cells encompassed a broad spectrum of cytokine profiles (Th1, Th2, Th17, Th1/Th17, Th9, Th22), and generally lacked CRTH2 expression. The extent of heterogeneity and dominance of specific Th types varied among patients with the same or different disease. Moreover, addition of IL-33 and endotoxin augmented Der p 1-specific Th1 responses. Multi-dimensional analysis by spectral flow cytometry provides a valuable tool to classify dust mite-specific Th cells and assess their mechanisms of induction in the context of discrete clinical diseases.

There are several aspects of HLA‐C gene expression that distinguish it from the HLA‐A and HLA‐B genes. First, HLA‐C is expressed by extravillous trophoblasts, whereas HLA‐A and HLA‐B are not. Second, its cell‐surface expression is much lower, which has been linked to changes in transcription and efficiency of peptide loading and export. Third, HLA‐C possesses a NK cell‐specific promoter and a complex alternative splicing system that regulates expression during NK cell development. In this study, we investigate the contribution of the HLA‐C core promoter to trophoblast‐specific expression. Analysis of transcription start sites showed the presence of a trophoblast‐associated start site and additional upstream TATA and CCAAT‐box elements in the HLA‐C promoter, suggesting the presence of an overlapping trophoblast‐specific promoter. A comparison of in vitro promoter activity showed that the HLA‐C promoter was more active in trophoblast cell lines than either the HLA‐A or HLA‐B promoters. Enhanced trophoblast activity was mapped to the central enhanceosome region of the promoter, and mutational analysis identified changes in the RFX‐binding region that generated a trophoblast‐specific enhancer.

Background. Little is known about T cells that respond to human rhinovirus in vivo, due to timing of infection, viral diversity, and complex T-cell specificities. We tracked circulating CD4(+) T cells with identical epitope specificities that responded to intranasal challenge with rhinovirus (RV)-A39, and we assessed T-cell signatures in the nose. Methods. Cells were monitored using a mixture of 2 capsid-specific major histocompatibility complex II tetramers over a 7-week period, before and after RV-A39 challenge, in 16 human leukocyte antigen-DR4(+) subjects who participated in a trial of Bifidobacterium lactis (Bl-04) supplementation. Results. Pre-existing tetramer+ T cells were linked to delayed viral shedding, enriched for activated CCR5(+) Th1 effectors, and included a minor interleukin-21(+) T follicular helper cell subset. After RV challenge, expansion and activation of virus-specific CCR5(+) Th1 effectors was restricted to subjects who had a rise in neutralizing antibodies, and tetramer-negative CCR5(+) effector memory types were comodulated. In the nose, CXCR3-CCR5(+) T cells present during acute infection were activated effector memory type, whereas CXCR3(+) cells were central memory type, and cognate chemokine ligands were elevated over baseline. Probiotic had no T-cell effects. Conclusions. We conclude that virus-specific CCR5(+) effector memory CD4(+) T cells primed by previous exposure to related viruses contribute to the control of rhinovirus.

The HLA-C gene appears to have evolved in higher primates to serve as a dominant source of ligands for the KIR2D family of inhibitory MHC class I receptors. The expression of NK cell-intrinsic MHC class I has been shown to regulate the murine Ly49 family of MHC class I receptors due to the interaction of these receptors with NK cell MHC in cis. However, cis interactions have not been demonstrated for the human KIR and HLA proteins. We report the discovery of an elaborate NK cell-specific system regulating HLA-C expression, indicating an important role for HLA-C in the development and function of NK cells. A large array of alternative transcripts with differences in intron/exon content are generated from an upstream NK-specific HLA-C promoter, and exon content varies between HLA-C alleles due to SNPs in splice donor/acceptor sites. Skipping of the first coding exon of HLA-C generates a subset of untranslatable mRNAs, and the proportion of untranslatable HLA-C mRNA decreases as NK cells mature, correlating with increased protein expression by mature NK cells. Polymorphism in a key Ets-binding site of the NK promoter has generated HLA-C alleles that lack significant promoter activity, resulting in reduced HLA-C expression and increased functional activity. The NK-intrinsic regulation of HLA-C thus represents a novel mechanism controlling the lytic activity of NK cells during development.

Rhinovirus (RV) induces acute episodes of asthma in allergic individuals. The T-cell mechanisms underlying the interplay between RV and allergy are unclear. Here, for the first time, we simultaneously monitor RV- and allergen-specific CD4+ T cells in asthmatics following experimental RV challenge using peptide/MHCII tetramers. HLA-diverse allergic asthmatics (n=9) and healthy control subjects (n=10) were inoculated intranasally with RV-A16. PBMC were collected before inoculation, and at days 4, 7, and 21 post-inoculation. Antigen-specific T cells were identified by dual staining with RV and allergen MHCII tetramers selected based on IgE profile. Molecular signatures were analyzed by multi-color flow cytometry and mass cytometry. Both RV- and allergen-specific memory T cells were readily detectable prior to RV infection in asthmatics, whereas allergen-specific T cells were rare in controls. RV-specific Th1 cells (CXCR3+CCR4neg) and allergen-specific Th2 cells (CXCR3negCCR4+) expanded following infection in asthmatics. RV-specific cells became activated and increased expression of the respiratory-homing marker CCR5 regardless of disease status; however, RV-specific responses were augmented in asthmatics. Analysis of lineage-specifying transcription factors in T cells by mass cytometry revealed a major population of memory CD4+ T cells that co-expressed T-bet and CCR5. These cells were rapidly sequestered from the periphery following inoculation, consistent with homing to the respiratory tract. Parallel monitoring of circulating RV- and allergen-specific T cells during experimental infection reveals an enhanced RV-specific Th1 response in allergic asthmatics, concomitant with a minor allergen-specific Th2 response. Our findings are consistent with dysregulated Th1 immunity to rhinovirus in allergic asthma.

Rhinovirus (RV) is a major cause of common cold and an important trigger of asthma exacerbations. T-cell responses to RV are poorly understood and no preventive strategies exist. We sought to determine whether circulating RV-specific CD4+ T-cell responses were modulated in subjects who received probiotic supplementation. Our substudy included sixteen HLA-DRB1*0401+ subjects who tested seronegative for RV-39, and who were enrolled in a double-blind placebo-controlled trial of Bifidobacterium lactis Bl-04 supplementation. After 4 weeks of supplementation, subjects received intranasal RV-39. PBMCs isolated on days -28 (pre-supplementation), 0 (pre-inoculation), 5 and 21 (post-inoculation) were stained with HLA-DRB1*0401 tetramers presenting two RV-39 capsid peptides, and analyzed by flow cytometry. RV-specific CD4+ T-cell frequencies and phenotypes were comparable among probiotic (n=11) and placebo (n=5) groups. Among infected subjects (n=12), RV-specific CD4+ T-cells were expanded during the effector phase (day 5) and displayed an activated effector memory phenotype. This expansion was absent in 2 infected subjects who lacked neutralizing antibodies, and in 4 non-infected subjects. Pre-existing RV-specific memory cells recognized epitopes conserved across RV strains, comprised a mixture of T helper (CXCR5neg) and T follicular helper (CXCR5+) populations, and were predominantly IFN-γ+IL-4neg, with minor IL-17+ and IL-21+ populations. Moreover, they were cross-reactive with a corresponding epitope of RV-16. High pre-existing RV-specific T-cell frequencies were linked to reduced viral infection (p<0.05); however this effect was attenuated after accounting for probiotic supplementation (p=0.11). Circulating RV-specific CD4+ T cells display attributes that might be harnessed for the treatment or prevention of common cold.

Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A/K251A/F162Y showed an ∼100-fold reduced capacity to bind IgE, while preserving the native molecular fold, as proven by x-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and the CD4(+) T-cell phenotype in peripheral blood mononuclear cell cultures. Although T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T-cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.

Inhibitory natural killer (NK) cell receptors specific for major histocompatibility complex class I (MHC-I) molecules include Ly49 receptors in mice and killer immunoglobulin-like receptors (KIR) in humans. The “licensing” or “arming” models imply that engagement of these receptors to self MHC-I molecules during NK cell development educates NK cells to be more responsive to cancer and viral infection. We recently reported that hematopoietic stem cell transplantation (HSCT) induced rapid and preferential expansion of functionally competent Ly49G+, but not other Ly49 family, NK cells independent of NK cell licensing via Ly49–MHC-I interactions. We now extend these studies to evaluate expression of the two Ly49G receptor isoforms Ly49GB6 and Ly49GBALB, using mice with different MHC-I haplotypes that express one or both of the isoforms. NK cells from CB6F1 (H-2bxd) hybrid mice express two different alleles for Ly49G receptor, Ly49GB6 and Ly49GBALB. We found that CB6F1 mice had more Ly49GB6+ NK cells than Ly49BALB+ NK cells, and that only Ly49GB6+ NK cells increased in relative numbers and in Ly49G mean fluorescence intensity values after HSCT similar to the B6 parental strain. We further observed that Ly49G+ NK cells in BALB/c (H-2d) and BALB.B (H-2b) mice, which have the same background genes, recover slowly after HSCT, in contrast to Ly49G+ NK cells in B6 (H-2b) recipients. The difference in expression of Ly49GB6 relative to Ly49GBALB was linked to differences in the activity of the Pro1 promoter between the two alleles. Thus, we conclude that the Ly49GB6 receptor dominates Ly49G expression on NK cells after HSCT in strains in which that allele is expressed. The data suggest that Ly49 allelic polymorphism within a particular Ly49 family member can differentially affect NK cell recovery after HSCT depending on the background genes of the recipient, not on the MHC-I haplotype.

The variegated expression of the human KIR family of class I MHC receptors provides an interesting model system for the study of stochastic activation of gene transcription. Previous studies have linked distal KIR promoter transcription to the initiation of KIR expression from the proximal promoter. In order to identify novel genetic alterations associated with decreased KIR expression, a group of 182 donors was characterized for KIR gene content, KIR transcripts, and FACS analysis of KIR surface expression. An individual was discovered that possessed a single copy of the KIR2DL1 gene but had a low level of gene expression by either FACS or Q-PCR. Complete sequencing of the KIR2DL1 gene confirmed the presence of an intact coding region. Analysis of the three known promoter regions revealed a cluster of 3 single nucleotide polymorphisms (SNPs) in the distal promoter region approximately 1 kb upstream from the start of KIR2DL1 translation. These SNPs are also found in the distal promoter region of the non-transcribed KIR2DL5*002 allele as well as the KIR3DP1 pseudogene. One of these SNPs creates a binding site for the inhibitory ZEB1 transcription factor that overlaps a c-Myc binding site previously implicated in the activation of KIR genes by distal transcription. We therefore propose that the generation of this novel ZEB1-binding site antagonizes the Myc-associated activation of the KIR2DL1 gene by the distal promoter.

This chapter describes the techniques our lab has used to find the multiple promoters present in individual KIR genes. Our previous studies in the murine Ly49 gene family led us to expect the presence of distal promoters, antisense transcripts, and bi-directional promoters in the KIR gene cluster. We present here all of the techniques used to systematically determine if a gene possesses these types of control elements.

Background: Thymic stromal lymphopoietin (TSLP) is expressed at sites of allergic inflammation, including eczematous skin. This cytokine has been reported to exert its T(H)2-inducing properties through dendritic cells. Expression of TSLP receptor on the surface of activated T(H)2 cells could amplify T(H)2 responses at inflamed sites through the direct actions of TSLP.Objective: To test rigorously whether T(H)2 cells induced by "proallergic" factors express TSLP receptor and characterize these cells using an experimental platform that combines flow cytometry with microscopic capabilities.Methods: CD4(+) T cells isolated from patients with atopic dermatitis or normal healthy controls were cocultured with autologous dendritic cells in the presence of T(H)2-promoting stimuli (TSLP +/- allergen and staphylococcal enterotoxin B +/- TSLP). Surface expression of TSLP receptor was analyzed by image-based flow cytometry, and responsiveness of purified T cells to TSLP was assessed by phosphorylation of signal transducer and activator of transcription-5 and cytokine secretion.Results: T(H)2-promoting stimuli induced a robust population of activated T(H)2 cells (CD25(+)IL-4(+)). Regardless of the nature of the stimulus, flow cytometry imaging confirmed that T cells expressing TSLP receptor were rare, constituting a minor fraction of the IL-4(+) T cell pool; however, TSLP responsiveness was nonetheless detectable. Analysis of cell size and nuclear morphology revealed preferential expression of TSLP receptor on IL-4-expressing cells undergoing mitosis. Analysis of lesional skin in atopic dermatitis supported the view that rare IL-4(+) T cells expressing TSLP receptor are present at inflamed sites.Conclusion: In a "proallergic" milieu, TSLP receptor is preferentially expressed on rare actively dividing T(H)2 cells. The direct action of TSLP on T cells could amplify T(H)2 responses at

The KIR genes are in a silent methylated state in NK progenitors prior to their selective activation by developing NK cells. We have recently identified an additional antisense transcript originating in the second KIR intron. The KIR2DL1 antisense transcript is composed of three exons that contain: (1) a segment from intron 2; (2) exon 2 plus flanking intron sequences; (3) exon 1 plus the bidirectional promoter region. The composition of the spliced antisense KIR transcript (KIRaS) suggests that it functions to silence KIR gene expression. KIRaS was detected in human embryonic stem (ES) cells, an embryonal carcinoma (EC) cell line (Ntera2), and the HEK293 cell line, but not NK, T, or B cell lines. In vitro promoter analysis revealed that KIRaS promoter activity is only detected in the cell lines where transcript is present. Promoter activity is controlled by the MZF‐1 transcription factor that is expressed in totipotent hematopoieticcells and myeloid progenitors. KIR gene activation appears to be under the control of four distinct promoter units: distal forward; proximal forward or reverse; distal antisense. The potential interplay of the multiple KIR promoters and the timing of their expression during development will be discussed.Funded by NCI Contract N01‐CO‐12400