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Key Points Typical features of GATA2 deficiency were associated with a de novo tandem duplication of GATA2 and increased expression of GATA2-AS1.The duplication contained a maternally inherited deletion copy number polymorphism esv2725896/nsv513733., A 3-year-old girl of nonconsanguineous healthy parents presented with cervical and mediastinal lymphadenopathy due to Mycobacterium fortuitum infection. Routine blood analysis showed normal hemoglobin, neutrophils, and platelets but profound mononuclear cell deficiency (monocytes < 0.1 × 109/L; B cells 78/μL; NK cells 48/μL). A 548 902-bp region containing GATA2 was sequenced by targeted capture and deep sequencing. This revealed a de novo 187-kb duplication of the entire GATA2 locus, containing a maternally inherited copy number variation deletion of 25 kb (GRCh37: esv2725896 and nsv513733). Many GATA2-associated phenotypes have been attributed to amino acid substitution, frameshift/deletion, loss of intronic enhancer function, or aberrant splicing. Gene deletion has been described, but other structural variation has not been reported in the germline configuration. In this case, duplication of the GATA2 locus was paradoxically associated with skewed diminished expression of GATA2 messenger RNA and loss of GATA2 protein. Chimeric RNA fusion transcripts were not detected. A possible mechanism involves increased transcription of the anti-sense long noncoding RNA GATA2-AS1 (RP11-472.220), which was increased several fold. This case further highlights that evaluation of the allele count is essential in any case of suspected GATA2-related syndrome.

Publisher's Note: There is a Blood Commentary on this article in this issue.

Heterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FceRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3-CD56bright, canonical, or adaptive CD3-CD56dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.

Summary Dendritic cell (DC)-mediated cross-presentation of exogenous antigens acquired in the periphery is critical for the initiation of CD8+ T cell responses. Several DC subsets are described in human tissues but migratory cross-presenting DCs have not been isolated, despite their potential importance in immunity to pathogens, vaccines, and tumors and tolerance to self. Here, we identified a CD141hi DC present in human interstitial dermis, liver, and lung that was distinct from the majority of CD1c+ and CD14+ tissue DCs and superior at cross-presenting soluble antigens. Cutaneous CD141hi DCs were closely related to blood CD141+ DCs, and migratory counterparts were found among skin-draining lymph node DCs. Comparative transcriptomic analysis with mouse showed tissue DC subsets to be conserved between species and permitted close alignment of human and mouse DC subsets. These studies inform the rational design of targeted immunotherapies and facilitate translation of mouse functional DC biology to the human setting., Graphical Abstract Highlights ► Human tissues contain CD1c+ DCs, CD14+ DCs, and a CD141hi cross-presenting DC subset ► CD141hi DCs migrate to draining lymph nodes and probably arise from blood CD141+ DCs ► Human tissue CD141hi DCs are homologous to mouse CD103+ or CD8+ DCs ► Human tissue CD1c+ DCs are homologous to mouse CD4+ DCs

Human immunodeficiency syndrome with loss of DCs, monocytes, and T reg cells; preservation of Langerhans cells; associated loss of BM multilymphoid progenitors; and overproduction of Flt3 ligand., Congenital or acquired cellular deficiencies in humans have the potential to reveal much about normal hematopoiesis and immune function. We show that a recently described syndrome of monocytopenia, B and NK lymphoid deficiency additionally includes the near absence of dendritic cells. Four subjects showed severe depletion of the peripheral blood HLA-DR+ lineage− compartment, with virtually no CD123+ or CD11c+ dendritic cells (DCs) and very few CD14+ or CD16+ monocytes. The only remaining HLA-DR+ lineage− cells were circulating CD34+ progenitor cells. Dermal CD14+ and CD1a+ DC were also absent, consistent with their dependence on blood-derived precursors. In contrast, epidermal Langerhans cells and tissue macrophages were largely preserved. Combined loss of peripheral DCs, monocytes, and B and NK lymphocytes was mirrored in the bone marrow by complete absence of multilymphoid progenitors and depletion of granulocyte-macrophage progenitors. Depletion of the HLA-DR+ peripheral blood compartment was associated with elevated serum fms-like tyrosine kinase ligand and reduced circulating CD4+CD25hiFoxP3+ T cells, supporting a role for DC in T reg cell homeostasis.

We investigated whether the repulsive SLIT/ROBO pathway is expressed in the endometrium and is negatively regulated during implantation. We also examined whether deficient expression in the Fallopian tube (FT) may predispose to ectopic pregnancy (EP). Endometrium (n = 21) and FT (n = 17) were collected across the menstrual cycle from fertile women with regular cycles. Decidualized endometrium (n = 6) was obtained from women undergoing termination, and FT (n = 6) was obtained from women with EP. SLIT/ROBO expression was quantified by reverse transcription-PCR and protein localized by immunohistochemistry. The regulation of SLIT/ROBO expression in vitro, by sex steroids and hCG, was assessed in endometrial (hTERT-EEpC) epithelial cells, and the effects of Chlamydia trachomatis infection and smoking were studied in oviductal (OE-E6/E7) epithelial cells. Endometrial SLIT3 was highest in the mid-secretory phase (P = 0.0003) and SLIT1,2 and ROBO1 showed a similar trend. ROBO2 was highest in proliferative phase (P = 0.027) and ROBO3,4 showed a similar trend. SLIT2,3 and ROBO1, 4 were lower in decidua compared with mid-secretory endometrium (P, link_to_OA_fulltext

The human corpus luteum (CL) undergoes luteolysis, associated with marked tissue and vascular remodeling, unless conception occurs and the gland is rescued by human chorionic gonadotropin (hCG). In Drosophila the Slit gene product, a secreted glycoprotein, acts as a ligand for the roundabout (robo) transmembrane receptor. Together they influence the guidance and migration of neuronal and nonneuronal cells. In vertebrates three Slit (Slit1, Slit2, Slit3) and four Robo (Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Robo) genes have been identified. ROBO1, SLIT2, and SLIT3 are also inactivated in human cancers and may regulate apoptosis and metastasis. Because processes such as apoptosis and tissue remodeling occur during the regression of the CL, the aim of this study was to investigate the expression, regulation, and effects of the SLIT and ROBO genes in human luteal cells. Immunohistochemistry and RT-PCR revealed that SLIT2, SLIT3, ROBO1, and ROBO2 are expressed in luteal steroidogenic cells and fibroblast-like cells of the human CL. Furthermore, using real-time quantitative PCR, expression of SLIT2, SLIT3, and ROBO2 was maximal in the late-luteal phase and significantly reduced after luteal rescue in vivo with exogenous hCG (P < 0.05). Additionally, hCG significantly inhibited SLIT2, SLIT3, and ROBO2 expression in cultured luteinized granulosa cells (P < 0.05). Blocking SLIT-ROBO activity increased migration and significantly decreased levels of apoptosis in primary cultures of luteal cells (P < 0.05). Overall, these results suggest the SLIT/ROBO pathway could play an important role in luteolysis in women.

In Drosophila, the Slit gene product, a secreted glycoprotein, acts as a midline repellent to guide axonal development during embryogenesis. Three human Slit gene orthologues have been characterised and recently we reported frequent promoter region hypermethylation and transcriptional silencing of SLIT2 in lung, breast, colorectal and glioma cell lines and primary tumours. Furthermore, re-expression of SLIT2 inhibited the growth of cancer cell lines so that SLIT2 appears to function as a novel tumour suppressor gene (TSG). We analysed the expression of SLIT3 (5q35-34) and SLIT1 (1q23.3-q24) genes in 20 normal human tissues. Similar to SLIT2 expression profile, SLIT3 is expressed strongly in many tissues, while SLIT1 expression is neuronal specific. We analysed the 5' CpG island of SLIT3 and SLIT1 genes in tumour cell lines and primary tumours for hypermethylation. SLIT3 was found to be methylated in 12 out of 29 (41%) of breast, one out of 15 (6.7%) lung, two out of six (33%) colorectal and in two out of (29%) glioma tumour cell lines. In tumour cell lines, silenced SLIT3 associated with hypermethylation and was re-expressed after treatment with 5-aza-2'-deoxycytidine. In primary tumours, SLIT3 was methylated in 16% of primary breast tumours, 35% of gliomas and 38% of colorectal tumours. Direct sequencing of bisulphite-modified DNA from methylated tumour cell lines and primary tumours demonstrated that majority of the CpG sites analysed were heavily methylated. Thus, both SLIT2 and SLIT3 are frequently methylated in gliomas and colorectal cancers, but the frequency of SLIT3 methylation in lung and breast cancer is significantly less than that for SLIT2. We also demonstrated SLIT1 promoter region hypermethylation in glioma tumour lines (five out of six; 83%), the methylation frequency in glioma tumours was much lower (two out of 20; 10%). Hence, evidence is accumulating for the involvement of members of the guidance cues molecules and their receptors in tumour development.

The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P

The human syndrome of dendritic cell, monocyte, B and natural killer lymphoid deficiency presents as a sporadic or autosomal dominant trait causing susceptibility to mycobacterial and other infections, predisposition to myelodysplasia and leukemia, and, in some cases, pulmonary alveolar proteinosis. Seeking a genetic cause, we sequenced the exomes of 4 unrelated persons, 3 with sporadic disease, looking for novel, heterozygous, and probably deleterious variants. A number of genes harbored novel variants in person, but only one gene, GATA2, was mutated in all 4 persons. Each person harbored a different mutation, but all were predicted to be highly deleterious and to cause loss or mutation of the C-terminal zinc finger domain. Because GATA2 is the only common mutated gene in 4 unrelated persons, it is highly probable to be the cause of dendritic cell, monocyte, B, and natural killer lymphoid deficiency. This disorder therefore constitutes a new genetic form of heritable immunodeficiency and leukemic transformation.

The secreted SLIT glycoproteins and their Roundabout (ROBO) receptors were originally identified as important axon guidance molecules. They function as a repulsive cue with an evolutionarily conserved role in preventing axons from migrating to inappropriate locations during the assembly of the nervous system. In addition the SLIT-ROBO interaction is involved in the regulation of cell migration, cell death and angiogenesis and, as such, has a pivotal role during the development of other tissues such as the lung, kidney, liver and breast. The cellular functions that the SLIT/ROBO pathway controls during tissue morphogenesis are processes that are dysregulated during cancer development. Therefore inactivation of certainSLITsandROBOsis associated with advanced tumour formation and progression in disparate tissues. Recent research has indicated that the SLIT/ROBO pathway could also have important functions in the reproductive system. The fetal ovary expresses most members of theSLITandROBOfamilies. TheSLITsandROBOsalso appear to be regulated by steroid hormones and regulate physiological cell functions in adult reproductive tissues such as the ovary and endometrium. Furthermore severalSLITsandROBOsare aberrantly expressed during the development of ovarian, endometrial, cervical and prostate cancer. This review will examine the roles this pathway could have in the development, physiology and pathology of the reproductive system and highlight areas for future research that could further dissect the influence of the SLIT/ROBO pathway in reproduction.

In humans and domestic mammals, pivotal processes in ovary development, including primordial follicle assembly, occur prenatally. These events are essential for determining fertility in adult life; however, they remain poorly understood at the mechanistic level. In mammals, the SLITs (SLIT1, SLIT2 and SLIT3) and their ROBO (ROBO1, ROBO2, ROBO3/RIG-1 and ROBO4/MAGIC ROBO) receptors regulate neural, leukocyte, vascular smooth muscle cell and endothelial cell migration. In addition, the SLIT/ROBO pathway has functional roles in embryonic development and in the adult ovary by inhibiting cell migration and promoting apoptosis. We therefore characterised follicle formation and investigated the expression and localisation of the ROBO/SLIT pathway in the ovine fetal ovary. Using RT-PCR, we identifiedSLIT2,SLIT3,ROBO1,ROBO2andROBO4in sheep ovaries harvested across gestation. The real-time quantitative PCR results implied thatROBO2expression andROBO4expression were elevated during the early stages of follicle formation and stayed abundant during primordial follicle maturation (PP

Recently a mouse model of T/natural killer acute lymphoblastic leukemia was used to assess global promoter methylation across the mouse genome using the restriction landmark genomic scanning technique. One of the methylated mouse genes identified in this way was Slit2. There are three mammalian SLIT genes (SLIT1, SLIT2, SLIT3), that belong to a highly conserved family of axon guidance molecules. We have previously demonstrated that SLIT2 is frequently inactivated in lung, breast, colorectal and glioma tumors by hypermethylation of a CpG island in its promoter region, whilst inactivating somatic mutations are rare. Furthermore, we demonstrated that SLIT2 acts as a tumor suppressor gene in breast and colorectal cancer cells. In this report we determined the methylation status of the SLIT2 gene in leukemias (CLL and ALL). SLIT2 was methylated in all ten leukemia cell lines analyzed (eight completely and two partially methylated). SLIT2 expression was restored after treating ALL lines with 5-aza-2dC. In primary ALL and CLL samples, SLIT2 was also frequently methylated, 58% (30/52) B-ALL; 83% (10/12) T-ALL and in 80% (24/30) CLL. Whilst DNA from peripheral blood and bone marrow from healthy control samples showed no SLIT2 methylation. Methylation results in leukemia cell lines and ALL and CLL primary samples were confirmed by direct sequencing of bisulfite modified DNA. Our results demonstrate that methylation of the SLIT2 5' CpG island is conserved between mice and humans, and therefore is likely to be of functional importance.

The human LH receptor (LHR) plays a key role in luteal function and the establishment of pregnancy through its interaction with the gonadotropins LH and human chorionic gonadotropin. We previously identified four splice variants of the LHR in human luteinized granulosa cells (LGCs) and corpora lutea (CL). Real-time quantitative PCR revealed that expression of the full-length LHR (LHRa) and the most truncated form (LHRd) changed significantly in CL harvested at different stages of the ovarian cycle (P < 0.01, ANOVA). LHRa expression was reduced in the late luteal CL (P < 0.05). Conversely, an increase in LHRd expression was observed in the late luteal CL (P < 0.01). Chronic manipulation of human chorionic gonadotropin in LGC primary cultures supported the in vivo findings. LHRd encodes a protein lacking the transmembrane and carboxyl terminal domains. COS-7 cells expressing LHRd were unable to produce cAMP in response to LH stimulation. COS-7 cells coexpressing LHRd and LHRa also failed to generate cAMP in response to LH, suggesting that this truncated form has a negative effect on the signaling of LHRa. Immunofluorescence staining of LGC and COS-7 cells implied that there is a reduction in cell surface expression of LHRa when LHRd is present. Overall, these results imply expression of LHR splice variants is regulated in the human CL. Furthermore, during functional luteolysis a truncated variant could modulate the cell surface expression and activity of full-length LHR.

RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras-association domain. RASSF2 resides at 20p13, a region frequently lost in human cancers. In this report we investigated methylation status of the RASSF2 promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2 was frequently methylated in breast tumour cell lines (65%, 13/20) and in primary breast tumours (38%, 15/40). RASSF2 expression could be switched back on in methylated breast tumour cell lines after treatment with 5'-aza-2'deoxycytidine. RASSF2 was also frequently methylated in NSCLC tumours (44%, (22/50). The small number of corresponding normal breast and lung tissue DNA samples analysed were unmethylated. We also did not detect RASSF2 methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2 in these ovarian tumours. We identified a highly conserved putative bipartite nuclear localization signal (NLS) and demonstrated that endogenous RASSF2 localized to the nucleus. Mutation of the putative NLS abolished the nuclear localization. RASSF2 suppressed breast tumour cell growth in vitro and in vivo, while the ability of NLS-mutant RASSF2 to suppress growth was much diminished. Hence we demonstrate that RASSF2 has a functional NLS that is important for its tumour suppressor gene function. Our data from this and a previous report indicate that RASSF2 is frequently methylated in colorectal, breast and NSCLC tumours. We have identified RASSF2 as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles.

Summary Dendritic cells (DCs), monocytes, and macrophages are leukocytes with critical roles in immunity and tolerance. The DC network is evolutionarily conserved; the homologs of human tissue CD141hiXCR1+CLEC9A+ DCs and CD1c+ DCs are murine CD103+ DCs and CD64−CD11b+ DCs. In addition, human tissues also contain CD14+ cells, currently designated as DCs, with an as-yet unknown murine counterpart. Here we have demonstrated that human dermal CD14+ cells are a tissue-resident population of monocyte-derived macrophages with a short half-life of, Graphical Abstract, Highlights • Human dermal CD14+ cells are a transient population of macrophages • Dermal CD14+ cells are derived from circulating blood monocytes • Human CD14+ cells are homologous to murine CD11b+CD64+ monocyte-derived macrophages • Human and mouse mononuclear phagocyte network organization is conserved, It is unclear whether human dermal CD14+ cells are bona fide dendritic cells. Haniffa and colleagues demonstrate that dermal CD14+ cells are monocyte-derived macrophages and complete the human and mouse tissue dendritic cell network alignment.

During the development of the nervous system, guidance cues are required to correctly direct the developing axons. These cues are highly conserved in evolution and may have diverse functions and receptors. The Slit proteins are members of these cues and along with their roundabout (robo) receptors, they act as repulsive cues for robo-expressing axons preventing them from crossing or re-crossing the midline. There is increasing evidence that the slit-robo interactions are not limited to axon guidance. The repulsive effect on axons due to slit-robo binding is mirrored in the immune system as well as in breast tumour cells; Slit proteins act as inhibitors of cell migration and invasion. Our group recently demonstrated that both SLIT and ROBO genes are inactivated in human cancers by promoter region CpG island hypermethylation with the subsequent silencing of gene expression. Restoring expression after treatment with a demethylating agent, provided further evidence that promoter hypermethylation was responsible for silencing SLIT-ROBO genes in several human cancers. Whilst Robo1 homozygous mutant mice die at birth due to incomplete lung development, the heterozygous mice show increased predisposition to tumour development concurrent with the inactivation of the remaining wild type Robo1 allele by promoter region CpG island hypermethylation. SEMA3B, another axon guidance molecule, was recently demonstrated to be epigenetically inactivated in human cancers and suppressed tumour growth. Hence, evidence is accumulating for the role of axon guidance molecules in human cancer development. Unlike mutational inactivation, epigenetic inactivation is a reversible event. This presents new and exciting opportunities for clinical management of cancer. In addition, promoter hypermethylation of genes is increasingly being developed as molecular biomarkers for non-invasive screens for early detection of cancer. Furthermore, the SLIT(s) gene products are secretary proteins, which may also lead to the development of novel therapeutic approaches. This chapter summarises the literature on SLIT-ROBO gene families in relation to human diseases.

Abstract 2045 The novel syndrome of Dendritic Cell, Monocyte, B and NK lymphocyte (DCML) deficiency has recently been characterised and found to be caused by mutations in the transcription factor GATA-2 (Dickinson et al. Blood 2011, Hsu et al. Blood 2011). In this report we describe a series of 10 patients and show that there is a high risk of death unless curative haematopoietic stem cell transplantation (HSCT) is performed. Our case series includes 10 patients; 4 sporadic cases and 6 cases from 2 pedigrees showing autosomal dominant inheritance. 7 patients died; of the 3 survivors, 2 were transplanted and show marked resolution of their disease with a follow up of 36 and 11 months, respectively. Causes of death in the patients who died include disseminated mycobacterium avium infection (2) H1N1 influenza (1) candidiasis (1) pulmonary alveolar proteinosis (2) and CMV pneumonitis (1). At least 8 patients had chronic HPV infection and 2 patients had autoimmune phenomena including inflammatory arthritis and erythema nodosum. One patient also developed lymphoma. All patients had monocytopenia and decreased B and NK cells with normal T cells at presentation. Haemoglobin, platelets and neutrophil counts were within normal ranges or mildly reduced. In 4 patients, near absolute dendritic cell (DC) deficiency and elevated Flt-3 ligand was also confirmed. BM aspirate showed dysplastic megakaryocytes and increased fibrosis in some instances. Macrophages were present in dermis, bone marrow and lung, epidermal Langerhans cells were largely preserved and plasma cells were found in inflammatory lesions by immunohistochemistry. GATA-2 mutation was confirmed by Sanger sequencing in all cases. Of the 2 patients treated with transplantation, the first patient presented aged 12 with disseminated BCG sepsis 6 months following BCG vaccination. Skin biopsy showed acid fast bacilli but no well-formed granulomata, and ongoing sepsis failed to respond to antitubercular therapy including addition of IFN gamma. The second patient presented to gynaecology services at the age of 20 with vulval intraepithelial neoplasia stage 3 (VIN3), and then to respiratory physicians a year later with breathlessness, productive cough, weight loss and night sweats; pulmonary alveolar proteinosis was diagnosed on biopsy. She continued to deteriorate despite whole lung lavage, and developed respiratory failure requiring 35% oxygen and nocturnal non-invasive ventilation (NIV). The patient's family history included two generations affected by haematological malignancy or respiratory illness in early adulthood, inherited in a fashion suggesting an autosomal dominant trait. Both patients underwent reduced intensity allogeneic haematopoietic stem cell transplantation with PBMC from unrelated adult donors (patient 1 – 10/12 HLA match; patient 2 – 9/12); transplant conditioning was with Fludarabine 150mg/m2, Melphalan 140mg/m2 and Alemtuzumab 60mg (patient 1), and Fludarabine 150mg/m2, Busulphan 6.4mg/kg, Alemtuzumab 60mg (patient 2). GVHD prophylaxis was with Ciclosporin and Mycophenolate Mofetil. Both transplants were uneventful. 32 months post-transplantation, patient 1 is well and off all medication. The monocyte count, lymphocyte subsets and immunoglobulins are normal and there were good responses to tetanus and HIB vaccinations. Chimerism shows 100% donor myeloid and B cells and 85% donor T cells. 9 months post-transplantation patient 2 has improved significantly and no longer requires oxygen or NIV. Lung function tests and radiology have significantly improved. Monocytes are in the normal range and chimerism shows 100% donor CD15 and 85% donor CD3. Follow-up continues with gynaecology for VIN3 associated with HPV16/18 infection. Neither patient has developed GVHD. Here we show that DCML deficiency caused by GATA-2 mutation incurs a high risk of mortality due to infection or respiratory failure unless treated by HSCT. Rapid correction of both immunodeficiency and pulmonary alveolar proteinosis is possible, even in the context of severe infection or respiratory failure, using reduced intensity conditioning. The absence of GVHD in both these cases, despite significant HLA-mismatching, may reflect the absence of recipient DC at transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 2546 Alemtuzumab-containing reduced intensity transplantation regimens frequently induce a state of partial T cell chimerism in the blood of the recipient. It has been widely shown that partial T cell chimerism is associated with freedom from graft versus host disease (GVHD) and that the occurrence of GVHD is often associated with rapidly rising donor T cell engraftment. The mechanism by which this occurs remains unknown and recipient cells may be killed, out-competed for homeostatic niches or simply diluted out by expanding donor T cells. The skin, a target organ of GVHD, normally contains T cells which enter from the blood in the steady state. Studies in mice have highlighted the gate-keeping function of inflammation in allowing trafficking of host-reactive donor T cells into tissues during conversion from mixed to full donor chimerism in blood. This implies that the equilibration of donor engraftment in the blood and tissue may occur more rapidly in patients at risk for GVHD. To test this hypothesis, we set out to define the relationship between skin and blood donor T cell engraftment in patients with and without GVHD. Methods: We studied a group of 51 patients receiving fludarabine melphalan (FM) conditioning with alemtuzumab 30mg for matched related donors and 60mg for matched unrelated donors. Skin biopsies were obtained at 28 and 100 days post transplant, dermal T cells isolated by migration and chimerism assessed in sex-mismatched transplants by combined immunofluorescence/in situ hybidization for XY chromosomes. Peripheral blood myeloid (CD15+) and T cell (CD3+) chimerism was determined by short tandem repeat amplification at monthly intervals after transplantation. All patients gave consent for clinical follow up and post transplant blood and skin sampling for research purposes, according to protocols approved by the local research ethics committee of Northumberland and North Tyneside. Results: All patients achieved >95% myeloid engraftment by day 100. Median (range) T cell engraftment was variable and significantly higher after MUD transplants: 70% (9-99%) than MRD transplants: 21% (5-85%; Mann Witney p Conclusion: This analysis supports the hypothesis that the equilibration of blood and tissue donor T cells is influenced by GVHD and may offer a means to predict patients at risk of GVHD after withdrawal of immunosuppression or donor lymphocyte infusion. Disclosures: No relevant conflicts of interest to declare.