Maike Buchner

Philipp Brantner

Gabriele Prinz

Meike Burger

Constance Baer

Christine Dierks

Dietmar Pfeifer

Roland Mertelsmann

John G Gribben

Hendrik Veelken

Natalie Stickel

Constance Bär

Ariane Ott

MD Katja Zirlik
email: katja.zirlik@uniklinik-freiburg.de

The microenvironment differentially impairs passive and active immunotherapy in Chronic lymphocytic leukemia -Potential therapeutic synergism of CXCR4 antagonists

Keywords: CHRONIC LYMPHOCYTIC LEUKAEMIA, STROMAL CELLS, IMMUNOTHERAPY, DRUG RESISTANCE, CHEMOKINES

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 [START_REF] Caligaris-Cappio | Role of the microenvironment in chronic lymphocytic leukaemia[END_REF][START_REF] Chiorazzi | Chronic lymphocytic leukemia[END_REF]. In contrast to the prolonged survival in vivo, CLL cells undergo rapid apoptosis in the absence of stroma in vitro. Mimicking the in vivo microenvironment by stromal cell coculture protects CLL cells from spontaneous and chemotherapy-induced apoptosis in vitro [START_REF] Burger | Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells[END_REF][START_REF] Kurtova | Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance[END_REF]. This cell contact-mediated chemoresistance is described for other malignancies as well and is termed "cell adhesion-mediated drug resistance" (CAM-DR) [START_REF] Damiano | Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines[END_REF]. Residual leukemic cells protected by stromal cells in the bone marrow or lymphatic tissue are thought to contribute to disease relapse after chemotherapy.

The homing of CLL cells towards the protective niches is mediated by the crosstalk between the chemokine CXCL12 and its receptor CXCR4. CXCR4 antagonists resensitize CLL cells to spontaneous and chemotherapy-induced apoptosis during stromal coculture in vitro [START_REF] Burger | Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells[END_REF][START_REF] Burger | Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells[END_REF]. Combination therapy with CXCR4 antagonists is therefore predicted to enhance efficacy of conventional cytoreductive treatment in CLL [START_REF] Burger | CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment[END_REF]. On the other hand, the CXCL12-CXCR4 interaction provides a physiological homing signal to the bone marrow for hematopoietic stem cells (HSCs). Thus, administration of CXCR4 antagonists mobilizes HSCs to the periphery and may render them sensitive to concomitant chemotherapy. To avoid this risk of stem cell toxicity, combination of CXCR4 antagonists with antineoplastic agents targeted to CLL cells would be conceptually advantageous.

Due to their exclusive action on cells of leukocyte lineages without decreasing HSC viability [START_REF] Lim | Effect of anti-CD52 antibody alemtuzumab on ex-vivo culture of umbilical cord blood stem cells[END_REF], passive and active immunotherapy represent promising treatment strategies in CLL [START_REF] Faderl | The evolving role of alemtuzumab in management of patients with CLL[END_REF][START_REF] Kater | Cellular immune therapy for chronic lymphocytic leukemia[END_REF][START_REF] Stanglmaier | Rituximab and alemtuzumab induce a nonclassic, caspase-independent apoptotic pathway in B-lymphoid cell lines and in chronic lymphocytic leukemia cells[END_REF]. The The graft-versus-leukemia effect observed after allogeneic hematopoietic stem cell transplantation is evidence for anti-leukemic activity of T cells against CLL (Giannopoulos andSchmitt 2006, Khouri, et al 1998). A potential CLL-associated T cell antigen is survivin, a member of the inhibitor of apoptosis (IAP) gene family [START_REF] Schmidt | Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells[END_REF]. This in vitro study was conducted to determine whether stromal contact protects CLL cells against passive and active immunotherapy. We evaluated the ex vivo cytotoxic effect of RIT and ALT and the ability of survivin-specific and allogenic CTLs generated in mixed lymphocyte reactions to lyse primary CLL cells in presence and absence of the murine stromal cell line M2-10B4 with or without the CXCR4 antagonist TN14003.

Materials and methods

Patients and healthy donors

Peripheral blood samples were obtained from 32 patients at University Medical Center Freiburg (SI Table 1) satisfying criteria for B-CLL and 10 healthy volunteers [START_REF] Cheson | National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment[END_REF].

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status, ZAP70 expression, and genomic aberrations were determined as described previously [START_REF] Hamblin | Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia[END_REF][START_REF] Osterroth | Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination[END_REF][START_REF] Pfeifer | Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays[END_REF][START_REF] Rassenti | ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia[END_REF]. 19 Patients had not been treated, 13 patients had received low-dose chlorambucil and/or fludarabine, which had been discontinued at least 6 months prior to phlebotomy.

Blood sampling had been approved by the local ethics committee and all patients gave informed consent.

Reagents and Cell lines

The highly selective tetradeca-peptide CXCR4 receptor antagonist TN14003 was a kind gift from N. Fujii, Kyoto, Japan Arabinosyl-2-fluoroadenine (F-ara-A) was purchased from Sigma-Aldrich (St. Louis, MO) and used at 20µM final concentration. ALT (Schering, Weimar, Germany) and RIT (Roche, Basel, Switzerland) were used at final concentrations of 20µg/ml and 10µg/ml, respectively.

For complement-mediated lysis, 10% fresh human serum (HS) was added.

Generation of effector CTLs

Effector CTLs generated in allogeneic mixed lymphocyte reactions (MLR) were stimulated similarly as described previously [START_REF] Kato | Gene transfer of CD40-ligand induces autologous immune recognition of chronic lymphocytic leukemia B cells[END_REF]. CLL B cells were plated into 96-well plates at 1 x 10 5 cells/well (IMDM, 10% human serum). 1 x 10 5 healthy donor CD8 + T cells/well were added and T cells were re-stimulated weekly with 2 x 10 4 CLL cells/well. For CD40 activation, CLL cells were stimulated for 48 h with 100 ng/ml sCD154 (Biosource, Camarillo, CA) before stimulation of T cells. Recombinant human IL-2 (R&D Systems, Minneapolis, MN) was added at 25 U/ml three days after each stimulation. After 1 to 4 stimulations, cytotoxicity of effector CTLs was assessed by flow cytometry or chromium ( 51 Cr) release assays. CLL cells were incubated with or without the CXCR4 blocking agent TN14003 for 30 min in a final concentration of 10 µg/ml prior to cultivation with the unlabelled stromal cell line M2-10B4 for 2 hours where indicated. Effector T cells were added to the respective culture for 4 hours. For generation of survivin-specific T cells, purified HLA-A2 + healthy CD8 + T cells were primarily stimulated with irradiated monocyte-derived, peptidepulsed dendritic cells that were pulsed prior to stimulation with the native survivin-derived peptide (Sur9: ELTLGEFLKL) for 2 hours in the presence of ß2-microglobulin (3 µg/ml, Sigma Aldrich). T cells were restimulated weekly using CD40-activated B cells pulsed with the corresponding peptide. After 3 stimulations, cytotoxicity of CTLs against primary CLL cells was assessed in chromium 51 Cr release assay. Cells were activated with IL-2 (500 U/ml) overnight and used at 30:1 (PBMC:CLL). To distinguish between PBMC and CLL cells, the latter were stained with CFSE. Cell viability was determined by propidium iodide (PI) (Sigma Aldrich) for ADCC. Otherwise, Annexin V-FITC (BD Bioscience) and PI was used. CLL cells were removed from stromal cell coculture by gentle but persistent pipetting.

F

Chromium ( 51 Cr) release assay

Chromium release assay was performed as previously described [START_REF] Zirlik | Cytotoxic T cells generated against heteroclitic peptides kill primary tumor cells independent of the binding affinity of the native tumor antigen peptide[END_REF]. In brief, survivin-specific CTLs or effector T cells from allogeneic MLR were incubated with 51 Crlabeled target cells. CTL activity was titrated at different effector-to-target ratios (30:1, 10:1, 3:1) for 4 hours and 51 Cr was measured in supernatants.

Cell polarization

For confocal analysis of cell polarization in target cells, CLL cells were stained with Mitotracker Red CM-H 2 XROS (Invitrogen, Carlsbad, CA) and treated for 30min with 10µM colchicine or control. After 3 washing steps, cells were preincubated with alemtuzumab (20µg/ml) before magnetically isolated, IL-2 activated NK cells labeled with Celltracker Green were added with a ratio of 1:1 onto adhesion slides (Marienfeld GmbH, Lauda-Königshofen, Germany), cells were fixed with 2% formaldehyde, mounted using DAPI containing mounting medium (Vector Laboratories, Burlingame, CA) and analyzed using a Leica TCS SP2 AOBS spectral confocal microscope (Leica Microsystems, Wetzlar, Germany).

Statistical analysis

Statistical analysis was performed using student`s t-test or Wilcoxon test using the Graphpad Prism Software.

Results

Stromal cells protect CLL cells from chemotherapy-induced apoptosis via CXCL12 -CXCR4 interaction, Akt activation, and upregulation of Mcl-1

To confirm the previously reported protective effect of the microenvironment on the viability of CLL cells in vitro, we assessed apoptosis of CLL cells in the presence and absence of the CXCL12-secreting stromal cell line M2-10B4 [START_REF] Burger | Small peptide inhibitors of the CXCR4 chemokine receptor (CD184) antagonize the activation, migration, and antiapoptotic responses of CXCL12 in chronic lymphocytic leukemia B cells[END_REF]. After 48 hours under standard culture conditions, only 50±7% of CLL cells remained viable. Coculture of CLL cells with stromal cells significantly enhanced viability to 81±3% (n=16, p<0.01). The CXCR4 antagonist TN14003 reversed this protective effect (Supporting Information (SI) Fig. 1A), while it did not affect CLL cell survival in the absence of stroma (shown in Fig. 1A).

Similar results were obtained for the CXCR4 antagonist AMD3100 (SI Fig 1B).

Fludarabine is the most effective single chemotherapeutic agent in CLL. In vitro, treatment of CLL cells with 20µM F-ara-A, the dephosphorylated, active fludarabine derivative, reduced absolute viability of CLL cells from 27±3% to 9±3% after 48hrs (SI Fig. 2). To compare proapoptotic F-ara-A effects under various culture conditions, we used the relative viability by defining the respective untreated control (without stroma, with stroma, with stroma and

CXCR4 antagonists increase the efficacy of CDC but not ADCC of alemtuzumab and rituximab by attenuating the protective effects of stromal cells

To investigate whether stromal cells protect CLL cells in the setting of passive Fig. 4A,C and D). Similar results for CDC were obtained when total viable cell count was determined (SI Fig. 4B). In accordance with previously published data, ALT mediated CDC in vitro more efficiently than RIT [START_REF] Voso | Rituximab reduces the number of peripheral blood B-cells in vitro mainly by effector cell-mediated mechanisms[END_REF]. Coculture with stromal cells inhibited CDC of CLL cells for both antibodies. The CXCR4 antagonist TN14003 abrogated this protective effect (Fig. 2A andB). Again, similar results were obtained when total viable cell count was determined (SI Fig. 5A andB). The viability and the capacity to secrete CXCL12 of stromal cells were not influenced by the treatment of ALT and RIT (SI Fig. 3C andD).

We next evaluated the influence of the microenvironment on ALT-and RIT-mediated ADCC of primary CLL cell using activated NK cells from healthy PBMCs as effector cells. PBMCs In contrast to CDC, we detected no increase in CLL cell viability upon coculture with stromal cells (Fig. 2C andD). This effect was independent of various effector-to-target ratios (Fig. 2C andD; E:T ratio 30:1, and SI Fig. 6A andB; E:T ratio 10:1).

Taken together, this suggests that the microenvironment exerts no protective effect against cellular effector mechanisms.

CTL-mediated killing of allogeneic CLL cells is not impaired by stromal cell contact

To examine CTL-mediated apoptosis, allogeneic CTL were incubated with target cells in the presence and absence of stromal cells and CXCR4 antagonists. To exclude differences in T cell quality as cause for variable effector function, CD8 3A). CTL-mediated apoptosis was CLL-cell specific and dose-dependent (Fig. 3B andC). At an effector-to-target ratio of 30:1, the measurable median target cell killing was 9% (range 4-36%, n=13). No differences in cytotoxicity were observed according to prognostic factors (data not shown).

Coculture of CLL cells with the stromal cell line or with the stromal cell line and TN14003 did not modulate apoptosis induced by allogeneic CTLs (Fig. 4A+B). Allogeneic CTLs were cytotoxic only for the allogeneic CLL cells against which they were originally primed, as these cells did not manifest any cytotoxicity of autologous PBMCs (Fig. 4B). However, even under conditions of prestimulation with CD40L, stromal cells did not limit CTL-induced apoptosis of CLL cells (data not shown).

To analyse possible effects of stromal contact on T cell-mediated cytotoxicity independent of the variable and rather weak allostimulatory capacity of CLL cells, we validated our results with survivin-specific, HLA-A2-restricted healthy T cells. Survivin-specific T cells effectively lysed HLA-A2 + survivin + CLL cells but not HLA-A2 -survivin -CLL cells in a dose-dependent manner (Fig. 4C). In line with a previous report [START_REF] Schmidt | Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells[END_REF], survivin was expressed in malignant B cells from a HLA-A2 positive patient with CLL as determined by quantitative real-time PCR (data not shown). Stromal cell contact did not significantly reduce antigenspecific cytotoxicity, nor did the addition of the CXCR4 antagonist increase cytotoxicity, indicating that T cell-mediated cytotoxicity is indeed independent of stromal cell contact (Fig. 4C).

Target cell polarisation is required for efficient cytotoxicity in stroma context

To functionally explain the observed differences in stroma protection capacity in terms of cellmediated or complement-mediated apoptosis, we added the toxin colchicine to the cytotoxicity assay. Colchicine destroys microtubule formation and therefore abrogates target cell polarisation and mitochondrial relocalization [START_REF] Goping | Cytotoxic T lymphocytes overcome Bcl-2 inhibition: target cells contribute to their own demise[END_REF]. Incubation with the Morphologically, colchicine treatment prevented mitochondrial accumulation towards the immunological synapse as assessed by confocal microscopy (Fig. 5B).

Discussion

Interactions between CLL cells and the microenvironment provide prolonged cell survival and various malignancies without serious side effects [START_REF] Khan | Small molecule CXCR4 chemokine receptor antagonists: developing drug candidates[END_REF][START_REF] Stone | Multiple-dose escalation study of the safety, pharmacokinetics, and biologic activity of oral AMD070, a selective CXCR4 receptor inhibitor, in human subjects[END_REF][START_REF] Voso | Rituximab reduces the number of peripheral blood B-cells in vitro mainly by effector cell-mediated mechanisms[END_REF][START_REF] Wendt | Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells[END_REF][START_REF] Yoon | CXC chemokine receptor-4 antagonist blocks both growth of primary tumor and metastasis of head and neck cancer in xenograft mouse models[END_REF].

Molecular mechanisms of the antiapoptotic function of stromal cell contact remain largely unknown. Although up-regulated in CLL, Bcl-2 does not seem to be critical for protection of CLL cells in stroma contact [START_REF] Balakrishnan | AT-101 induces apoptosis in CLL B cells and overcomes stromal cell-mediated Mcl-1 induction and drug resistance[END_REF]. We and others observed upregulation [START_REF] Awan | Mcl-1 expression predicts progression-free survival in chronic lymphocytic leukemia patients treated with pentostatin, cyclophosphamide, and rituximab[END_REF][START_REF] Johnston | Role of myeloid cell factor-1 (Mcl-1) in chronic lymphocytic leukemia[END_REF][START_REF] Pepper | Mcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers[END_REF][START_REF] Zhou | Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage[END_REF], and downregulation of Mcl-1 by siRNA enhances RIT-mediated apoptosis in vitro [START_REF] Hussain | Mcl-1 is a relevant therapeutic target in acute and chronic lymphoid malignancies: down-regulation enhances rituximab-mediated apoptosis and complement-dependent cytotoxicity[END_REF].

A strong correlation of high-affinity F C γR polymorphisms with clinical response to rituximab in follicular lymphoma (FL) [START_REF] Cartron | Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene[END_REF] With respect to active immunotherapy, B-CLL cells per se are inefficient antigen-presenting cells (APCs), largely due to an inadequate co-stimulatory capacity (Dazzi, et al 1995, Wolos and[START_REF] Wolos | Depressed stimulation in the MLR by B lymphocytes on chronic lymphocytic leukemia: failure to demonstrate a suppressor cell[END_REF] and the induction of an anergic phenotype in T cells by CLL cells. The latter phenomenon is not only induced in autologous but also in allogeneic T cells, resulting in down-modulation of genes important for proliferation and effector function [START_REF] Gorgun | Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells[END_REF]. Recently, T cells of CLL patients and also healthy T cells after direct CLL cell contact were shown to have impaired ability to form immunological synapses and subsequent polarisation events [START_REF] Ramsay | Chronic lymphocytic leukemia T cells show impaired immunological synapse formation that can be reversed with an immunomodulating drug[END_REF]. In agreement with these findings we observed efficient lysis of CLL cells in only 13 of 19 cases and overall poor cytotoxic T cell responses.

Effective cellular immune therapy, such as vaccines [START_REF] Hus | Vaccination of B-CLL patients with autologous dendritic cells can change the frequency of leukemia antigen-specific CD8+ T cells as well as CD4+CD25+FoxP3+ regulatory T cells toward an antileukemia response[END_REF] or genetically engineered T cell transfer [START_REF] Hollyman | Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy[END_REF] will have to overcome factors responsible for the disease-related immune deficiency of patients with CLL and the capacity of leukemia cells to effect cellular immune tolerance. Recent work has focused on strategies to overcome these limitations and encouraging biologic effects and clinical responses have been observed [START_REF] Biagi | Responses to human CD40 ligand/human interleukin-2 autologous cell vaccine in patients with B-cell chronic lymphocytic leukemia[END_REF][START_REF] Hus | Allogeneic dendritic cells pulsed with tumor lysates or apoptotic bodies as immunotherapy for patients with early-stage B-cell chronic lymphocytic leukemia[END_REF], Kater, et al 2004a, Kater, et al 2004b[START_REF] Mayr | Transduction of CLL cells by CD40 ligand enhances an antigen-specific immune recognition by autologous T cells[END_REF][START_REF] Zirlik | Cytotoxic T cells generated against heteroclitic peptides kill primary tumor cells independent of the binding affinity of the native tumor antigen peptide[END_REF].

Unlike in the setting of chemotherapy-and CDC-induced apoptosis, CLL-stromal cell interactions did not impair T cell-mediated cytotoxicity suggesting that the microenvironment does not limit the efficacy of active immunotherapy in B-CLL. This conclusion could be drawn from the unspecific setting in MLRs as well as from the specific [START_REF] Chu | CD40 activation does not protect chronic lymphocytic leukemia B cells from apoptosis induced by cytotoxic T lymphocytes[END_REF], Kater, et al 2004a[START_REF] Kitada | Bryostatin and CD40-ligand enhance apoptosis resistance and induce expression of cell survival genes in B-cell chronic lymphocytic leukaemia[END_REF]. In line with our findings, Mcl-1 downregulation has been shown to sensitize CLL cells against CDC but not ADCC mediated cytotoxicity of rituximab [START_REF] Hussain | Mcl-1 is a relevant therapeutic target in acute and chronic lymphoid malignancies: down-regulation enhances rituximab-mediated apoptosis and complement-dependent cytotoxicity[END_REF]. Directed exocytosis of lytic molecules such as granzymes toward the target cell is one of the major mechanisms used by CTLs an NK cells. By mitochondrial relocalization towards the site of granzyme B entry during CTL attack, activated T cells are able to overcome upregulation of Bcl-2, usually blocking Granzyme B [START_REF] Goping | Cytotoxic T lymphocytes overcome Bcl-2 inhibition: target cells contribute to their own demise[END_REF]. This suggests that cell polarization by immunological synapse formation may overcome antiapoptotic events mediated by stroma. For CLL, disruption of the microtubulus network restored to some extent the stroma protective effect, although the biological significance remains to be determined. A direct effect of microtubule disruption is unlikely as it would also affect ADCC efficacy without stroma. A potential explanation for this effect may be the missing mitochondria relocation towards the immunological synapse observed after colchicine treatment.

However, the fact that cell-mediated immunotherapy, such as active vaccinations strategies, T cell transfer or "graft vs. leukemia"-effect after allogeneic hematopoietic stem cell transplantation, is not limited by contact to stromal cells supports the application of this therapeutic strategy. Therefore, we propose this approach to be further investigated in vivo.

In conclusion, this study demonstrates that adhesion-mediated drug resistance protects CLL cells against monoclonal antibody treatment, an effect abrogated by CXCR4 antagonists.

Therefore, combined therapy with CXCR4 antagonists may represent a promising strategy to enhance the efficacy of ALT and RIT therapy in CLL. In addition, we show that CLL-stromal cell interactions do not impair T cell-mediated cytotoxicity on CLL cells.

F

Supporting information

  lymphocytic leukemia (B-CLL) represents one of the most commonly diagnosed lymphoid malignancies in Western countries, and is characterized by continuous accumulation of mature, CD5 + B cells. CLL remains incurable with cytoreductive therapy. The microenvironment in bone marrow and secondary lymphoid tissues favours survival and chemoresistance of CLL cells by upregulation of antiapoptotic molecules such as Mcl-1 and Bcl-xL

  Buchner et al. Microenvironmental impact on immunotherapy in CLL 4 monoclonal anti-CD52 and anti-CD20 antibodies (mAb) alemtuzumab (ALT, Campath-1H) and rituximab (RIT) have been approved for passive immunotherapy of relapsed or refractory CLL. Their precise mode of action is not fully understood. Monoclonal antibodies are capable to activate the classic complement pathway which results in pore formation and target cell lysis (Zipfel and Skerka 2009), known as complement dependent cytotoxicity (CDC). Furthermore, antibodies may bind to Fcγ γ γ γ receptors on various effector cells, such as natural killer (NK) cells, fixed macrophages, dendritic cells (DCs), neutrophils, and eosinophils (Nimmerjahn and Ravetch 2006). Efficient lysis of the obsoned cells by these effectors has been termed antibody-dependent cellmediated cytotoxicity (ADCC). Additional direct apoptotic effects of, e.g. RIT are under controversial discussion (Lim, et al 2010).

  (Tamamura, et al 2001). The stromal cell line M2-10B4 (ATCC; Manassas, VA), maintained in RPMI 1640 containing 10% FCS was plated on 96 well plates at 2 x 10 5 cells/well, starved overnight, and incubated with CXCR4 30min prior to coculture with CLL cells where indicated. To remove CLL cells from stromal cell coculture we performed gentle but persistent pipetting. Stromal cells were discriminated from CLL cells according to their distinct forward/sideward scatter properties in flow cytometry (Burger, et al 2005).

  indicated) as 100%[START_REF] Burger | Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells[END_REF] accounting for variabilities in spontaneous apoptosis rates in different patients' samples[START_REF] Kurtova | Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance[END_REF].Coculture with stromal cells significantly enhanced the relative viability after F-ara-A treatment from 35±9% to 59±8% after 48hrs (Fig.1A, n=8, p<0.05). The protective effect of stromal cells on chemotherapy-induced apoptosis was mainly prevented by the CXCR4 receptor antagonist TN14003 (Fig. 1A, p<0.05), in line with previous findings (Burger, et al 2005). The viability of stromal cells and their capacity to secrete CXCL12 was not influenced by 48 h F-ara-A treatment (SI Fig. 3A and B). Mechanistically, Mcl-1 protein expression was significantly increased in primary CLL cells after coculture with M2-10B4 cells, an effect abrogated by the CXCR4 antagonist TN14003 (Fig. 1B). Addition of the caspase inhibitior Z-VAD did not affect Mcl-1 expression after treatment with the CXCR4 inhibitor. Therefore, the increased apoptosis induction and emerging caspase activation does not account for the Mcl-1 downregulation. In line with previous reports, we found increased activation of Akt upon stromal cell coculture. Again, this effect could be blocked by CXCR4 inhibition (Fig. 1C). In contrast, the expression of the other proapoptotic mediators Bid and Bim was not regulated and Bad was not expressed at all (data not shown). These data suggest that stromal cell-induced apoptosis resistance is largely mediated by Mcl-1 upregulation (Balakrishnan, et al 2009).

  from healthy donors were used since NK from CLL patients are severely impaired (Gorgun, et al 2005, Katrinakis, et al 1996, Kay and Zarling 1987, Kay and Zarling 1984, Ramsay, et al 2008) and autologous NK cells from patients with CLL show inferior rituximab antibody-dependent cellular cytotoxicity than allogeneic NK cells (Weitzman, et al 2009).

  influence the survival of CLL or stromal cells in the observed time frame (Fig.5A, SI Fig.3E). NK cell activity was limited during toxin treatment (data not shown) but was maintained when the target cells were preincubated and repeatedly washed prior to addition of the activated PBMCs. In line with our previous finding (Fig.2C+D) we observed no protective effect of stroma in ADCC-mediated cytotoxicity, suggesting that effector cells may overcome stromal cell mediated protective effects. However, inhibition of microtubule formation by colchicine treatment resulted in a significant -albeit minor-protection of CLL cells only in stroma context (Fig. 5 A, p<0.01; SI Fig.7 for absolute viability).

  growth advantage and confer drug resistance to CLL cells[START_REF] Caligaris-Cappio | Role of the microenvironment in chronic lymphocytic leukaemia[END_REF][START_REF] Chiorazzi | Chronic lymphocytic leukemia[END_REF]. In vitro, the inhibition of CXCL12-CXCR4 interactions restores susceptibility of CLL cells in stromal contact to spontaneous and drug-induced apoptosis, suggesting synergistic effects of combined therapy of CXCR4 antagonists and chemotherapy in vivo.Although recent studies suggest safety and efficacy of combining cytotoxic chemotherapy with CXCR4 antagonists in acute myeloid leukemia (AML) (Uy GL 2008),this combination may provoke toxicity towards mobilized healthy hematopoietic progenitors that are normally protected in the marrow microenvironment. Therefore, we evaluated the effect of stromal coculture in the presence and absence of CXCR4 antagonists in more specific passive and active immunotherapeutic approaches targeting CLL cells but not HSCs. Indeed, stromal cell contact reduced complement-mediated cytotoxicity of alemtuzumab and rituximab. The addition of the CXCR4 antagonist TN14003 reversed the protective effect of in decreased viability of CLL cells identifying CXCR4 antagonists as potential adjuvants for selective therapeutic intervention. Promising results in preclinical tumor models indicate that CXCR4 antagonists may have anti-tumor activity in patients with

  of the anti-apoptotic Bcl-2 family member Mcl-1 upon stromal cell contact[START_REF] Balakrishnan | AT-101 induces apoptosis in CLL B cells and overcomes stromal cell-mediated Mcl-1 induction and drug resistance[END_REF]. The potential role of Mcl-1 as an oncogene in lymphoid malignancies was confirmed in transgenic mouse models in which animals with deregulated expression of Mcl-1 eventually developed widely disseminated B-cell lymphoma (Zhou, et al 1998). Interestingly, in CLL, stromal cell contact also upregulates Akt activation (Edelmann, et al 2008), an effect abrogated by CXCR4 antagonists (Fig. 1C). Since active Akt also increases Mcl-1 half-live (Maurer, et al 2006), the Akt/Mcl-1 pathway might indeed mediate antiapoptotic signals during stromal cell support. Accordingly, the inhibition of this pathway may explain the efficacy of CXCR4 antagonists in this setting. In line with our observations, Mcl-1 has been reported to mediate apoptosis resistance to fludarabine and rituximab in vivo

  setting using survivin-specific T cells. Of note, no change in stroma cell viability was observed after addition of any of the components, including activated NK and allospecific T cells (data not shown). In view of our data showing no influence on the susceptibility of B-CLL cells to ADCC and allogeneic CTL attack by stroma, it might be Buchner et al. Microenvironmental impact on immunotherapy in CLL speculated that the mode of action to induce apoptosis is critical. Allo-responsive CTLs are able to lyse resting and CD40-stimulated B-CLL cells with equal efficiency, although CD40 stimulation results in upregulation of anti-apoptotic molecules, including Mcl-1

Figure 1 :

 1 Figure 1: Stromal cells protect CLL cells from chemotherapy-induced apoptosis via CXCR4 -CXCL12 interactions and pAkt-dependent Mcl-1 upregulation. (A) Viability of CLL cells cultured with or without stromal cells and treated with 20µM F-ara-A or the combination of F-ara-A and 10µg/ml TN14003 was determined by annexin V/PI staining. Relative CLL cell viability was determined by defining the respective control as 100% (as indicated with the control bar) after 48hrs treatment. Data represent means±SEM (n=8). (B) Means (± SEM ) of mean fluorescent intensity (MFI by flow cytometry) of Mcl-1 expression in CLL cells cultured with or without stroma in the presence or absence of the CXCR4 antagonist TN14003 are displayed on the left (n=12). A representative experiment shows MFI of Mcl-1 expression in primary CLL cells cultured with or without stromal cells on the right. Below, a representative Mcl-1 immunoblot of CLL cells is shown in the presence and absence of stroma, with and without addition of TN14003 and/or the caspase inhibitor Z-VAD (20 µM). (C) Immunoblot shows pAkt and Akt of a representative CLL sample after 15min with or without coculture of stroma in the presence and absence of the CXCR4 antagonist TN14003.

Figure 2 :

 2 Figure 2: CXCR4 antagonists increase the efficacy of ALT and RIT complement induced cytotoxicity by attenuating the protective effects of stromal cells. (A+B) CLL cells (n=7) cultured with or without stromal cells in the presence or absence of the CXCR4 antagonist TN14003 treated with 20µg/ml ALT (A) or 10µg/ml RIT (B) for 12hrs. Relative viabilities were determined by staining with Annexin V and PI and presented as means±SEM.(C+D) Relative viability of CLL cells (n=12) cultured with or without stromal cells in the presence or absence of the CXCR4 antagonist TN14003 treated with 20µg/ml ALT (C) or 10µg/ml RIT (D) for 4hrs in the presence of IL-2-activated PBMC at a ratio 30:1. IgG control of the respective culture setting (w/o stroma, w/stroma, or stroma+TN14003) was defined as 100% relative viability. Using annexin V and PI staining, CLL relative cell viability was determined and presented as means±SEM.

Figure 3 :

 3 Figure 3: CTL-mediated killing of allogeneic CLL cells. (A) Killing of CLL cells of 19 CLL patients induced by effector T cells generated in allogeneic MLR with spontaneous levels of apoptosis of B-CLL cells subtracted as determined by flow cytometry (CFSE/PI staining) at an effector-to-target ratio of 30:1 (M=mutated IgV H ; UM=unmutated IgV H ). (B) Flow cytometry analysis of cytolytic activity of effector T-cells from allogeneic MLR against CLL cells by staining with CFSE/PI presented as effector-to-target ratios 3:1 and 30:1. (C) Dosedependent killing of CLL cells induced by effector T cells with spontaneous levels of apoptosis of B-CLL cells subtracted as determined by flow cytometry (CFSE/PI staining) represent means±SEM of triplicates.

Figure 4 :

 4 Figure 4: CLL -stromal cell interactions do not impair T cell mediated cytotoxicity. (A) Viability of CLL cells (n=11) in the presence or absence of stromal coculture after incubation with T cells generated in allogeneic mixed lymphocyte reaction as assessed by flow cytometry (CFSE + /PI -). Untreated control without addition of T cells of the respective culture setting was defined as 100% viability. Data represent means±SEM. (B) Cytolytic activity of effector CTLs generated in allogeneic MLR as assessed by flow cytometry against CLL cells in presence of the stromal cell line M210B4 with or without the CXCR4 inhibitor TN14003 and against autologous PMBCs at three different effector-to-target ratios. Displayed are means±SEM of PI + -values of samples from 3 representative patients. (C) Killing by allogeneic CTLs generated against a native (Sur9: ELTLGEFLKL) survivin-derived peptide of HLA-A2-positive CLL cells cultured with or without stromal cells in the presence or absence of the CXCR4 antagonist TN14003 and HLA-A2-negative CLL cells at 3 different effectortarget ratios.

Figure 5 :

 5 Figure 5: Prevention of mitochondrial relocalization in target cells restores protective stromal effect. (A) CFSE + -CLL cells were pretreated with 10µM colchicine after incubation with and without stroma before addition of ALT or IgG control and IL-2-activated PBMCs.

SIFig. 5 :

 5 Buchner et al. Microenvironmental impact on immunotherapy in CLL After 2hrs, viabililty was measured by %CFSE + /PI -and the relative viability calculated by defining the respective control culture as 100 % relative viability. Data are presented as means±SEM (n=7). (B) Confocal images of colchicine-or control-pretreated CLL cells stained for mitochondria (red) in direct contact to IL-2-activated NK cells (green) during alemtuzumab treatment. (n=7) for complement mediated cytotoxicity determined by Guava viacount. (C+D) Relative viability of CLL cells cultured with ALT (20 µg/ml) or RIT (10 µg/ml) for 4 h with IL-2 activated PBMC (n=8) for ADCC at a effector-to-target ratio of 30:1 (C) and 10:1 (D), IgG control was defined as 100% viability. Data are presented as mean ± SEM values. Viable cell count of CLL cells treated with rituximab (A) or alemtuzumab (B) in the presence of fresh human stroma, measured using the Guava viacount technology for the indicated culture conditions (n=7). Data are presented as mean ± SEM values. SI Fig. 6: (A+B) Relative viability of CLL cells (n=8) cultured with or without stromal cells in the presence or absence of the CXCR4 antagonist TN14003 treated with 20 µg/ml ALT (A) or 10 µg/ml RIT (B) for 4 hours in the presence of IL-2 activated PBMC at a ratio 10:1. IgG control of the respective culture setting (w/o stroma, w/ stroma, or stroma+TN14003) was defined as 100% relative viability. Data are presented as mean ± SEM values. SI Fig. 7: CFSE + CLL cells were pretreated with 10 µM colchicine after incubation with and without stroma before IL-2 activated PBMCs were added. After 2 hours, viabililty was measured by %CFSE + /PI - . Data are presented as mean ± SEM values.

  Figure 1 A

Z-VAD was purchased from Promega Inc and used at a final concentration of 20 µM.

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	Intracellular staining of Mcl-1
	After 48 hour culture of CLL cells with or without stromal cell contact in the presence and
	absence of CXCR4 antagonists, cells were fixed and permeabilized according to
	manufacturer's protocol (Foxp3 staining kit, eBioscience, San Diego, CA), stained with a Mcl-
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Immunoblotting Immunoblotting was performed as previously described (Buchner, et al 2009). Primary antibodies included pAkt S473 , Akt (Cell Signaling Technology), Mcl-1 (Epitomics, San Francisco, CA), or

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Sigma-Aldrich), secondary antibodies were horseradish peroxidase-conjugated from Cell Signaling Technology. Densitometric analysis was performed using ImageJ software (http://rsb.info.nih.gov/ij/). CLL cell treatment and viability assessment B-CLL cells from frozen stocks thawed prior to experiments were incubated for 48 hours with F-ara-A or with RIT and ALT for 4 h (ADCC) and 12h (CDC), respectively.

cells from a healthy donor were used as CLL-derived T-cell function is impaired (Gorgun, et al 2005,

  Overall, CLL cells had inefficient allostimulatory capacity, since we observed measurable killing of CLL cells in only 13 of 19 cases (Fig.

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	Ramsay, et al 2008).
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+ T cells from one donor were stimulated against a total of 19 different CLL samples in parallel. T

this correlation does not exist which argues against a dominant role of ADCC in CLL. Although mainly natural killer (NK) cells are established ADCC effector cells, other cells such as monocytes and

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provides evidence for an important role of ADCC in clinical lymphoma responses. In CLL, however,

macrophages may contribute to ADCC (Dalle, et al 2008, Lefebvre, et al 2006). These cell types were not analyzed in our study.

Table 1 : Clinical characteristics of CLL patients.

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British Journal of Haematology

Microenvironmental impact on immunotherapy in CLL

Acknowledgements

We thank Max Warncke, Paul Fisch and Markus Simon for helpful discussions. This work was supported by the Deutsche Krebshilfe grant 107275 (to M. Burger, H. Veelken, and K. Zirlik).

Supporting information


Paola ; Delbini

Valentina Vaja

Giovanna Graziadei

Lorena Duca

Isabella Nava

Chiara Refaldi

Maria Domenica Cappellini
email: maria.cappellini@unimi.it

Prof Domenica Maria

Cappellini

GENETIC VARIABILITY OF TMPRSS6 GENE AND ITS ASSOCIATION WITH IRON DEFICIENCY ANAEMIA

Keywords: IRON, ANAEMIA, TMPRSS6, IRIDA, haplotype TMPRSS6, anaemia, iron, IRIDA, haplotype

Transmembrane Protease, Serine 6 (TMPRSS6) has an important role in iron homeostasis and its mutations, performed in TMPRSS6-deficient mice, have been recently associated with iron-refractory iron deficiency anaemia (IRIDA). Several variants of TMPRSS6 have been already identified; however the role of polymorphisms and TMPRSS6 haplotypes, causing iron deficiency anaemia, have not yet been investigated. In this study TMPRSS6 gene was sequenced in 16 subjects with IRIDA phenotype. We identified twenty-seven DNA polymorphisms. Eight SNPs and four haplotypes were significantly associated with ironrefractory anaemia (p<0.001). Our preliminary results suggest a possible association between specific haplotypes of TMPRSS6 gene and IRIDA.

Iron deficiency anaemia is usually an acquired condition attributed to poor iron dietary intake or chronic blood loss. However, previous studies have described a familial syndrome characterized by iron deficiency anaemia not responding to oral iron and responding only partly to parenteral iron [START_REF] Hartman | Microcytic anemia with iron malabsorption: an inherited disorder of iron metabolism[END_REF][START_REF] Pearson | Ferrokinetics in the syndrome of familial hypoferremic microcytic anaemia with iron malabsorption[END_REF]. This condition, named iron-refractory iron deficiency anaemia (IRIDA) is an autosomal recessive disease characterised by congenital hypochromic, microcytic anaemia, very low mean corpuscular volume, low transferrin saturation and normal/low ferritin. IRIDA has recently been shown to be caused by mutations in the TMPRSS6 gene, which encodes a type II transmembrane serine protease, matriptase-2, that is predominantly expressed in the liver [START_REF] Finberg | Mutations in TMPRSS6 cause iron-refractory iron deficiency anaemia (IRIDA)[END_REF][START_REF] Melis | A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anaemia refractory to oral iron[END_REF]. Recent studies performed in TMPRSS6-deficient mouse model and in cell cultures have shown that matriptase-2 functions as a negative regulator of hepcidin expression [START_REF] Du | The serine protease TMPRSS6 is required to sense iron deficiency[END_REF][START_REF] Folgueras | The membrane-bound serine protease matriptase-2 (Tmprss6) is an essential regulator of iron homeostasis[END_REF]. Matriptase-2 represses hepcidin expression by cleaving membrane-bound hemojuvelin (m-HJV) [START_REF] Silvestri | The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin[END_REF], the bone morphogenetic proteins (BMPs) coreceptor, thereby reducing signalling through the SMAD proteins pathway [START_REF] Babitt | Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression[END_REF]. Cleaving m-HJV matriptase-2 inhibits hepcidin expression by reducing BMP. Although the pathogenic TMPRSS6 mutations that underlie IRIDA appear to be relatively rare, many common variants have been reported [START_REF] Benyamin | Common variants in TMPRSS6 are associated with iron status and erythrocyte volume[END_REF][START_REF] Chambers | Genome-wide association study identifies variants in TMPRSS6 a ssociated with hemoglobin levels[END_REF][START_REF] Tanaka | A genome-wide association analysis of serum iron concentrations[END_REF]. The aim of this study was to genotype for TMPRSS6 in 16 subjects with iron deficiency anaemia refractory to oral iron therapy, in order to identify possible mutations and to evaluate the role of mutations/polymorphisms and haplotype combinations of TMPRSS6 in causing IRIDA.

Materials and methods

Study subjects

Sixteen adult individuals, evocative for IRIDA, showed anaemia of varying severity (Hb=9.3±1.5 g/dL), severe microcytosis (MCV=64.2±8.8 fl), low serum iron (63.2±23.3 µg/dL), low transferrin saturation (TS=15.9±6.2%), and low serum ferritin (9.8±4.5 ng/mL). Specific tests (antigliadin antibodies, anti-transglutaminase antibodies, stool test for occult blood and endoscopy tests) excluded other causes of iron-deficiency anaemia, such as malabsorption and bleeding.

Thalassaemia, sideroblastic anaemia and the common forms of congenital dyserythropoietic anaemias were excluded. DMT1 gene mutations were excluded by direct sequencing. Oral iron treatment had no effect on reticulocytes and Hb, while intravenous iron produced a minimal response. The control population consisted of 35 non-anaemic men and 15 menstruating nonanaemic women with serum ferritin levels >20ng/ml and serum transferrin saturations >15%.

Written informed consent for genetic analysis was obtained from the 16 subjects, who were identified among subjects presenting at outpatients source of the Hereditary Anaemia Center of Fondazione IRCCS "Ca' Granda"-Ospedale Maggiore Policlinico, because of their anaemia. The study protocol was approved by the hospital ethic committee of Fondazione IRCCS "Ca' Granda"-Ospedale Maggiore Policlinico in Milan and was performed according to the tenets of the Declaration of Helsinki.

Analysis of the TMPRSS6 DNA sequence

Genomic DNA was isolated from peripheral blood lymphocytes and sequence variations in TMPRSS6 gene were evaluated by polymerase chain reaction (PCR) and direct sequencing.

Direct sequencing was performed using a fluorescence-tagged dideoxy chain terminator method in an ABI BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions.

Statistical analysis

Fisher's exact tests were used to test the allelic and genotypic associations of all the SNPs with iron deficiency anemia. Hardy-Weinberg equilibrium (HWE) of the genotypic frequencies among cases and controls was examined. To determine statistically significant differences in genotype and allele frequencies between the two groups, the χ2 test or Fisher's exact test for the 2×2 contingency table file was used. The strength of the association was estimated by odds ratio of risk (OR) and 95% confidence intervals (CI). Linkage disequilibrium (LD) calculations were done using the Haploview package. Haplotype frequency estimation and association analysis using the permutation test were performed with Haploview version 4.0 software (Broad Institute of MIT and Harvard, Cambridge, MA, USA). Haplotype distributions were evaluated by the permutation test on the basis of 10,000 replications to obtain the empirical significance [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF]. A cut off criterion of p<0.01 for a significance test on the contingency table of all possible simulated haplotypes within possible SNP combination in cases and controls was used.

Results and Discussion

Complete sequencing of TMPRSS6 gene (Reference Sequence NM_153609) from 16 subjects with iron deficiency anaemia was performed. Two novel single nucleotide substitutions were found in two unrelated IRIDA subjects: nt.35801185A>G which predicts the amino acid substitution H448R and nt.35829153C>T which predicts a synonymous change F5F. Coding coordinates for both nucleotides and amino acids are numbered from the start of translation. Two rare known nonsynonymous polymorphisms, nt.35815670A>G (rs2235324) and nt.35793530G>A (rs2235979) were found in different subjects. Twenty-seven different DNA polymorphisms, eight of which are novel, were identified. Sixteen different DNA sequence variations were identified in controls. None of these sequence variations was novel. The distribution of the allele frequencies for SNPs are shown in Fig. 1. The overall genotype call rate was 95% (range, 92-96%), and the accuracy was 100% according to duplicate genotyping of >5% of samples. Significant deviation from HWE was observed in controls for three SNPs, rs5756515, rs5756516 and rs4824454. The use of high stringency cutoffs resulted in the loss of some genotyping data and reduction in the number of patients studied from the original sample set. Other SNPs evaluated in the final analysis were in HWE in controls, they had high call rates and concordant duplicates by genotyping, suggesting that this study was not likely to be influenced by genotyping errors. In the screen of twenty-seven SNPs of TMPRSS6, allele frequencies of rs855791C and rs2235321T, already reported in LD [START_REF] Benyamin | Common variants in TMPRSS6 are associated with iron status and erythrocyte volume[END_REF][START_REF] Tanaka | A genome-wide association analysis of serum iron concentrations[END_REF], were significantly increased in subjects with iron deficiency anaemia compared with healthy controls (p<0.0001, OR=5.18, 95%CI 2.21<12.11 and p=0.011, OR=3.33, 95%CI 1.27<8.71, respectively). These TMPRSS6 SNPs are missense (rs855791) and synonymous (rs2235321) SNPs within the trypsin-like serine protease domain. Comparison with other serine proteases showed that the amino acid altered by rs855791 is located close to both the catalytic and the specificity site of the serine protease, suggesting that rs855791 may be a causal variant, acting through altered protease activity or substrate binding. The rs2235321, on exon 17, was in LD with rs855791 (D'=1.0, r 2 =0.444, CI=0.8-1.0), representing probably the same signal. Among the common non-synonymous polymorphisms, rs11704654, rs4820268 and rs2543519 were most significantly associated with iron-refractory anaemia (p<0.0001). However rs2543607 and rs2543633 were also associated with iron-refractory anaemia (p<0.001, OR=6.90, 95%CI 2.49<19.07) and in LD with each other (D'=1.0, r 2 =0.211, CI=0.73-1.0). The polymorphisms rs732733, rs732734 and rs2238761 were not significantly different between the groups (p = 0.076), although a different trend was observed between anaemic and control subjects. A larger sample size is probably required considering our strict cut-off for significance.

To determine whether specific TMPRSS6 alleles might be associated with iron-refractory anaemia, and therefore overrepresented in the anaemic group, haplotypes and haplotype blocks within TMPRSS6 were derived using HaploView 4.0 software (http://www.broad.mit.edu/mpg /haploview) [START_REF] Barrett | Haploview: analysis and visualization of LD and haplotype maps[END_REF]. Based on the haplotype reconstruction results calculated by PHASE2.0, Haploview used the confidence interval method to perform LD assessment and defining of haplotype blocks. The r 2 threshold for linkage disequilibrium was set at 0.8 and the multiple loci LOD threshold as 3.0. Only common haplotypes (frequency >5%) were retained in this study. In the haplotype analysis, we identified three haplotype block sets, containing 6, 9 and 2 SNPs respectively (Tab.1). Strong LD was observed within each block, meaning that each block is a smallest evolutionary unit. The CCCTTG, TCCTTG and CCCTCG haplotypes in Block 1 seemed to be risk haplotypes (p<0.0001, p<0.001 and p=0.0020, respectively), while the CTCCCG haplotype was observed to be protective (p=0.0052). In Block 2, the GGGGGCTTA haplotype was observed only in anaemic subjects (0.085, p=0.0033), while the AAAGATCTG haplotype was significantly less common in anaemic subjects (0.094) than in control group (0.385, p<0.0020).

Since the GGGGGCTTA haplotype was rare in all anaemic patients and absent in the controls, we think that the significant association between this haplotype and iron deficiency anaemia is not reliable. In Block 3 the predominant TMPRSS6 haplotype observed in subjects with iron deficiency anaemia was also the predominant haplotype observed among controls. The haematological and iron parameters of patients haplotype bloks, that show phenotype evocative for IRIDA, are reported in Table 2. Although we noted a significant association of these haplotypes with disease, none significant haematological differences among blocks were found. Our strict cut-off for significance indicates that it may require a much larger sample size to be confident of such an association. The low number of patients studied is one of the limitations of our study. Studies analyzing additional iron deficiency anaemia phenotypes in larger samples would enable us to confirm possible associations between the genetic TMPRSS6 polymorphisms and the iron deficiency anaemia phenotype.

In conclusion, our preliminary results suggest a possible association of genetic variants and specific haplotypes of TMPRSS6 gene with IRIDA.

Fig 1 .

 1 Fig 1. Linkage disequilibrium plot of the TMPRSS6 region and first screening of 24 tag single nucleotide polymorphisms (SNPs). In the upper panel, P values for differences in allele frequencies were calculated by chi-square test using two-by-two contingency tables. The -log P value for each SNP is shown. In the lower panel, r 2 values calculated using Haploview version 4.0 software based on data from anaemic subjectscontrols cohort are shown. The location and direction of transcription of TMPRSS6 are indicated by arrows.

  Association analysis results based on haplotype blocks Tab 2. Haematological and Iron Parameters in patients haplotype blocks

	Tab 1. Block Block	rs855791 rs4820268 Haplotype rs2543519 Frequency Haemoglobin Mean Corpuscolar (g/dL) Volume (fL)	rs11704654 Case, control ratios Serum Ferritin Serum Iron χ χ χ χ2 (ng/mL) (µg/dL)	p value Transferrin Saturation %
	Block 1 Block 2 Block 3		CTCCCG 9.5+1.5 CTCTTG 10.1+1.2 CCCCTG 9.5+1.7	0.541 58.9+6.3 0.092 61.0+8.1 rs2543633 0.061 59.5+5.9	10.5:21.5, 61.0:39.0 9+5 rs2543607 2.1:29.9, 10.0:90.0 10+4 0.0:32.0, 8.0:92.0 9+4	p=0.001 p=0.0001 7.821 49+20 0.357 50+21 2.725 49+20	<0.001 14+11 n.s. 15+10 n.s. 14+11
	TCCTTG rs2235321 CTTCCG CCCTTG (values expressed as mean + standard deviation) Block 1	0.033 0.056 0.045		4.4:27.6, 0.0:100.0 0.5:31.5, 7.0:93.0 6.0:26.0, 0.0:100.0	p=0.01 14.146 1.413 19.643	<0.001 n.s. <0.0001
			CCCTCG		0.023		3.0:29.0, 0.0:100.0	9.593	0.0020
			CCTCTG		0.015		0.0:32.0, 2.0:98.0	p=0.05 0.65	n.s.
			AAAGATCTG		0.314		3.0:29.0, 38.5:61.4	9.563	0.0020
	Block 2		357.92 o F r GAAGATCTG GAGGATCTG GAAAATCTG GGGGGCTTA F o r F o r F o r	0.252 0.064 0.030 0.021		358.21 10.7:21.3, 22.6:77.3 4.0:28.0, 4.5:95.4 0.0:32.0, 4.0:95.9 2.7:29.3, 0.0:99.9	358.41 1.516 2.581 1.319 8.658	(Mb) n.s. n.s. n.s. 0.0033
	Block 3 n.s., not significant	CT	TMPRSS6 P 0.992 P P P	31.0:1.0, 100.0:0.0	3.149	n.s.
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