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In paroxysmal nocturnal hemoglobinuria (PNH), hematopoietic cells lacking glycosylphosphatidylinositol (GPI)-linked proteins on their surface (GPI(neg)) exist alongside normal (GPI+) cells. Analysis of natural killer (NK) cell subsets in 47 PNH patients revealed that the ratio of CD56(bright):CD56(dim) NK cells differed in the GPI+ and GPI(neg) populations, with GPI(neg)CD56(bright) NK cells significantly more abundant in peripheral blood than their normal GPI+ counterparts. Indeed, GPI+CD56(bright) NK cells were not detected in the peripheral blood of some patients, suggesting their trafficking to a niche unavailable to the GPI(neg)CD56(bright) NK cell population. Defective cellular trafficking in this disease was supported by findings showing differential chemokine receptor expression between GPI+ and GPI(neg) NK cells and impaired stromal cell-derived factor 1 (SDF-1)-induced chemotaxis of GPI(neg) NK cells. Our results indicate a role for GPI-linked proteins in NK cell subset homeostasis and suggest that differential chemokine responses might contribute to the balance of GPI+ and GPI(neg) populations in this disease.

Activation of granzyme B, a key cytolytic effector molecule of natural killer (NK) cells, requires removal of an N-terminal pro-domain. In mice, cathepsin C is required for granzyme processing and normal NK cell cytolytic function, whereas in patients with Papillon-Lefèvre syndrome (PLS), loss-of-function mutations in cathepsin C do not affect lymphokine activated killer (LAK) cell function. Here we demonstrate that resting PLS NK cells do have a cytolytic defect and fail to induce the caspase cascade in target cells. NK cells from these patients contain inactive granzyme B, indicating that cathepsin C is required for granzyme B activation in unstimulated human NK cells. However, in vitro activation of PLS NK cells with interleukin-2 restores cytolytic function and granzyme B activity by a cathepsin C-independent mechanism. This is the first documented example of a human mutation affecting granzyme B activity and highlights the importance of cathepsin C in human NK cell function.

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in ApcMin/+ mice, which carry a heterozygous germ line mutation at codon 850 of Apc, there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition, ApcMin/+ mice show parallel depletion of splenic natural killer (NK) cells, immature B cells, and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients, we have shown that the capacity of transplanted ApcMin/+ bone marrow cells for T- and B-cell development appears normal. In contrast, although the ApcMin/+ bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow, ApcMin/+ animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of ApcMin/+ mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. (Blood. 2004;103:1050-1058)

Introduction Immune dysfunction in multiple myeloma (MM) is now a well accepted, if not yet completely understood, phenomenon contributing to both an increased incidence of infection and suboptimal responses to immunotherapies. The recently described Th17 subset of T helper cells is expanded in a range of cancers, and may contribute either to the evolution or control of tumours, but its role in myeloma pathogenesis remains unclear. We therefore investigated the presence, phenotype and function of this novel immune subset in a cohort of MM patients and healthy donors. Methods Peripheral blood (PB) samples were taken from healthy donors (HC, n=27) and MM patients (n=31); mononuclear cells (PBMC) were isolated by density centrifugation. Bone marrow aspirate (BM) samples were taken from MM patients (n=7) and HC (n=3); mononuclear cells were isolated by red cell lysis (BioLegend). For phenotyping, cells were treated with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A and assessed by flow cytometry. Where stated, cells were stimulated with candidal wall mannoprotein MP65 (Peptivator, Miltenyi Biotec). For coculture experiments, CD4+T cells were isolated from PBMC using magnetic bead separation (MACS, Miltenyi Biotec) and cultured with human myeloma cell lines (HMCLs: U266B, JIM3, KMS11) with or without human bone marrow stromal cell lines (BMSC: HS-5, HS-27). Statistical analysis was performed using GraphPad Prism v6. Results As noted in previous studies, patients with myeloma have a marked CD4+ T-cell lymphopenia. Within the CD4+ population, MM patients also had a reduced frequency of IL-17+ cells (Th17) compared to HC: 0.41% (95% CI 0.27-0.56) vs. 0.81% (95% CI 0.47-1.15), p=0.0241 (student’s t-test). Using the mean fluorescence intensity for IL-17 as a surrogate indicator of cytokine production, IL-17 production in Th17 from MM patients was significantly reduced compared to HC (p=0.0195). Advancing disease stage correlated significantly with a reduction in PB Th17 frequency (p PB Th17 cells from myeloma patients showed a marked reduction in expression of CD161 compared with HC: 25.5% vs. 54.2%; this was again a stage-related phenomena (p=0.0019, one-way ANOVA). CCR6 expression on Th17s was also reduced in MM compared to controls in a stage-related manner (p=0.0036). Furthermore, PB Th17 cells in MM had impaired capacity to produce IL-17 in response to stimulation with a fungal antigen: 7% responded cf. 21% in HC (p=NS). To determine what, if any, effect MM tumour cells exerted on Th17 homeostasis, we employed a coculture model. CD4+ T cells from HC were cultured with mitomycin C-treated HMCLs for 7 days. Presence of HMCLs resulted in an expanded Th17 population compared to control cultures of CD4+ alone: 2.4% (95% CI: 0.13-1.30) vs..0.7% (95% CI: -0.20-5.07), p=0.09. The expansion of Th17 cells was augmented when BMSC were included (p=0.0476). We observed higher CD161 expression on coculture-derived Th17s when stromal cells were present. Conclusions Multiple abnormalities are apparent in Th17 immunity in MM: their frequency is decreased in PB and increased in BM, in a stage-related manner suggestive of either a homing defect or a tumour microenvironmental expansion. Our in vitro co-culture data suggest tumour-derived Th17 expansion may be responsible. The Th17 phenotype in MM is aberrant, with the relative absence of key lineage markers and attenuated functional responses to fungal antigens. Since IL-17 has been reported to be a growth factor for MM cells, these abnormalities may contribute both to the propagation of the malignant clone and the impaired anti-infective immunity seen in this disease. Disclosures No relevant conflicts of interest to declare.

The use of GSCF-mobilised Peripheral Blood Stem Cells (PBSC) for unrelated donor (UD) transplantation has increased dramatically since 2000. The association of PBSC with more rapid engraftment and with an increase in chronic Graft versus Host Disease (GvHD), compared to bone marrow (BM) has been reported in a number of studies. More recently the use of PBSC has been associated with an increase in transplant related mortality (TRM) and decrease in survival (OS) in T-cell replete transplants. We sought to analyse the impact of PBSC compared to BM in a cohort of UD transplant recipients, where T-cell depleting agents (in-vivo campath in >90%) were included in the transplant conditioning. The study included 145 patients transplanted between January 2000 and March 2006: CML- 35 in 1CP; acute leukaemia (AML in 61, ALL in 49)-110 in CR1 or 2. All had myeloablative conditioning regimens and received grafts with 9–10/10 matched HLA alleles. 86 patients received BM and 59 PBSC. There were no associations between the stem cell source and any transplant variable (including disease and stage). There was a trend to an increased use of PBSC in patients with a single antigen mismatch (p=0.052). All evaluable patients achieved neutrophil engraftment, with a significantly faster time to engraft in recipients of PBSC compared to BM (16 vs 20 days; p=0.0003). The incidence of acute GvHD was 46% (grade I in 50%, II in 41%, III in 8%, IV in 2%). This was significantly higher in recipients of PBSC (60%) compared to BM (36%; p=0.006), however there was no increase in either II/IV (p=0.69) or III/IV (p=0.18) disease in PBSC recipients. In univariate analysis, the presence of a single HLA mismatch (p=0.026) was the only other variable to be associated with an increase in acute GvHD. In a logistic regression model including both these variables, the use of PBSC remained significantly associated with an increase in aGvHD (OR=2.3; 95% CI 1.1,4.7;p=0.020). The TRM was 14%, 27% and 39% at 100 days, 1 and 5 years respectively. At none of these time points was the stem cell source associated with a significant difference in TRM. The 5-year incidence of chronic GvHD was 58% (BM 55%, PBSC 60%; NS), extensive disease in one third, and of relapse was 61% (BM 60%, PBSC 62%; NS). The 5-years OS was 41% with a median follow-up of 3.4 years (0.5–7.1). This was 44% using PBSC and 40% using BM (NS). In conclusion, although we observed an increase in acute GVHD with PBSC this was only of grade 1 disease. We found no association between the use of PBSC and an increased risk of chronic GVHD or of a worse transplant outcome, when compared to BM, in recipients of T-cell depleted myeloablative transplants for leukaemia.