Immune-regulatory properties carried by human amnion epithelial cells: Focus on the role of HLA-G and adenosinergic ectoenzymes.

Human amnion epithelial cells (hAEC) can be efficiently isolated from full-term amnion membrane and have been gaining recognition as advanced medical products. Such cells originate directly from the embryo during the early phase of development and exert a crucial function in the establishment of a tolerogenic environment, to avoid maternal immune rejection. Amnion cell immuno-modulation may be exploited, but additional efforts are required to establish the mechanisms underlying such capacity. The way to fully clarify such an issue is so far long. Here we overview current knowledge on the effects on innate or adaptive immune cells offered by intact hAEC or secreted mediators, pinpointing the mechanisms to date elucidated by our group and others. We move from the description of hAEC general features to molecular intermediaries generating effects directly or indirectly on immune cells. We focus on the role of non-canonical HLA class I molecules, with emphasis on HLA-G, but expand such analysis on adenosinergic mediators, cytokines, and hAEC-derived microvesicles. Finally, we report the ongoing clinical trials exploiting hAEC multipotency and immune modulation.

Zoledronic acid boosts γδ T-cell activity in children receiving αß+ T and CD19+ cell-depleted grafts from an HLA-haplo-identical donor.

We demonstrated that γδ T cells of patients given HLA-haploidentical HSCT after removal of αß+ T cells and CD19+ B cells are endowed with the capacity of killing leukemia cells after ex vivo treatment with zoledronic acid (ZOL). Thus, we tested the hypothesis that infusion of ZOL in patients receiving this type of graft may enhance γδ T-cell cytotoxic activity against leukemia cells. ZOL was infused every 28 d in 43 patients; most were treated at least twice. γδ T cells before and after ZOL treatments were studied in 33 of these 43 patients, till at least 7 mo after HSCT by high-resolution mass spectrometry, flow-cytometry, and degranulation assay. An induction of Vδ2-cell differentiation, paralleled by increased cytotoxicity of both Vδ1 and Vδ2 cells against primary leukemia blasts was associated with ZOL treatment. Cytotoxic activity was further increased in Vδ2 cells, but not in Vδ1 lymphocytes in those patients given more than one treatment. Proteomic analysis of γδ T cells purified from patients showed upregulation of proteins involved in activation processes and immune response, paralleled by downregulation of proteins involved in proliferation. Moreover, a proteomic signature was identified for each ZOL treatment. Patients given three or more ZOL infusions had a better probability of survival in comparison to those given one or two treatments (86% vs. 54%, respectively, p = 0.008). Our data indicate that ZOL infusion in pediatric recipients of αß T- and B-cell-depleted HLA-haploidentical HSCT promotes γδ T-cell differentiation and cytotoxicity and may influence the outcome of patients.

Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells.

BACKGROUND: Follicular center lymphoma displays widespread lymph node involvement at diagnosis. The chemoattractants that control the locomotion of follicular center lymphoma B cells have not been established. Stromal cell-derived factor-1 (SDF-1) is a CXC-class chemokine that enhances the migration of normal human B cells and is expressed in peripheral lymphoid tissues. Here we have investigated 1) whether SDF-1 stimulates the in vitro locomotion of follicular center lymphoma B cells and of their presumed normal counterparts (i. e., germinal center B cells) and 2) whether the same cells express SDF-1 transcripts. METHODS: B cells were purified by immunomagnetic bead manipulation. Messenger RNA was detected by reverse transcription-polymerase chain reaction. Migration was assessed by the filter and collagen invasion assays. All P values were two sided. RESULTS: Follicular center lymphoma B lymphocytes showed a statistically significant migratory response to 300 ng/mL SDF-1, both in the filter and in the collagen assays (P =.002 for each). Such response was mediated by the SDF-1 receptor, CXCR4. CD40 monoclonal antibody (MAb) and tonsillar germinal center B cells treated with CD40 MAb and recombinant interleukin 4, but not freshly isolated, migrated statistically significantly faster in the presence than in the absence of SDF-1 (P =.002 in both filter and collagen assays). Freshly isolated follicular center lymphoma and germinal center B cells expressed SDF-1 transcripts. CONCLUSIONS: This study shows that SDF-1 substantially enhances the migration of follicular center lymphoma B cells but not the migration of freshly purified germinal center B cells. This difference may be related to the extended survival of follicular center lymphoma versus germinal center B cells. SDF-1 produced in follicular center lymphoma lymph nodes may play a role in the local dissemination of tumor cells.

Cytokine gene expression in neoplastic B cells from human mantle cell, follicular, and marginal zone lymphomas and in their postulated normal counterparts.

Cytokines may promote tumor growth by paracrine and/or autocrine pathways. Little information is available because malignant cells differ from their normal counterparts for the cytokine repertoire they express. Here we have investigated by reverse transcription-PCR the expression of 22 cytokine genes in neoplastic B lymphocytes from six patients with mantle cell lymphoma, 10 with follicular lymphoma, and 5 with marginal zone lymphoma and in their normal counterparts, i.e., naive, germinal center, and memory B cells, purified from tonsils. The overall profiles of cytokine gene expression in neoplastic B cells and in the corresponding normal B-cell subsets were similar, but some "holes" in the repertoire of malignant versus normal B lymphocytes were detected. Different "hole" combinations were identified consistently in mantle cell lymphoma, follicular lymphoma, and marginal zone lymphoma, thus representing molecular fingerprints of each individual lymphoma entity.

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

Hypoxia-inducible factor (HIF)-1α suppression in myeloma cells blocks tumoral growth in vivo inhibiting angiogenesis and bone destruction.

Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1α inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1α inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1α. The effect of HIF-1α inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1α inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.

Complementary IL-23 and IL-27 anti-tumor activities cause strong inhibition of human follicular and diffuse large B-cell lymphoma growth in vivo.

Interleukin (IL)-23 and IL-27 are pro-inflammatory cytokines that share functional and structural similarities and may exert anti-tumor activities against solid and hematological malignancies. Here, we asked whether IL-23 and IL-27, alone or in combination, may act directly against human follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cells. In this study, we demonstrated for the first time that human primary FL and DLBCL cells expressed complete and functional IL-23 and IL-27 receptors (R) and that IL-23 and IL-27 exerted anti-tumor activities in vitro and in vivo through different and complementary mechanisms. In vivo studies using severe combined immunodeficiency /non-obese diabetic mice-injected subcutaneously with human SU-DHL-4 cell line revealed that IL-23 inhibited directly tumor-cell proliferation, whereas IL-27 impaired the angiogenic program of lymphoma cells resulting in strong reduction of cell growth. In addition, combined treatment of IL-23 and IL-27 amplified the anti-tumor effects in vivo as compared with administration of each cytokine alone. These anti-tumor mechanisms were confirmed by in vitro experiments performed with primary lymphoma cells and cell lines. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of the IL-23 and IL-27 in lymphoma patients.

Absence of IL-12Rß2 in CD33(+)CD38(+) pediatric acute myeloid leukemia cells favours progression in NOD/SCID/IL2RγC-deficient mice.

Childhood acute myeloid leukemia (AML) is a hematological malignancy in which tumor burden is continuously replenished by leukemic-initiating cells (ICs), which proliferate slowly and are refractory to chemotherapeutic agents. We investigated whether interleukin (IL)-12, an immuno-modulatory cytokine with anti-tumor activity, may target AML blasts (CD45(+)CD33(+)) and populations known to contain leukemia ICs (that is, CD34(+)CD38(-), CD33(+)CD38(+) and CD44(+)CD38(-) cells). We demonstrate for the first time that: i) AML blasts and their CD34(+)CD38(-), CD33(+)CD38(+), CD44(+)CD38(-) subsets express the heterodimeric IL-12 receptor (IL-12R), ii) AML cells injected subcutaneously into NOD/SCID/Il2rg(-/-) (NSG) mice developed a localized tumor mass containing leukemic ICs and blasts that were virtually eliminated by IL-12 treatment, iii) AML cells injected intravenously into NSG mice engrafted within the first month in the spleen, but not in bone marrow or peripheral blood. At this time, IL-12 dramatically dampened AML CD45(+)CD33(+), CD34(+)CD38(-), CD33(+)CD38(+) and CD44(+)CD38(-) populations, only sparing residual CD33(+)CD38(+) cells that did not express IL-12Rß2. From 30 to 60 days after the initial inoculum, these IL-12-unresponsive cells expanded and metastasized in both control and IL-12-treated NSG mice. Our data indicate that the absence of IL-12Rß2 in pediatric AML cells favours leukemia progression in NOD/SCID/IL2Rγc-deficient mice.

Cytokines as anti-angiogenic agents in haematological malignancies.

The role of angiogenesis in haematological malignancies has been recently recognized. In these tumors, angiogenesis has been investigated predominantly in the bone marrow (BM) compartment where it appears to be regulated by multiple interactions between malignant cells and different cell populations present in the tumor microenvironment. Thus, angiogenesis represents a therapeutic target that opens new perspectives for the treatment of haematological malignancies. Cytokines are small proteins that mediate intercellular communications, thus regulating important cellular functions, such as immune responses and angiogenesis. Some cytokines show anti-angiogenic properties through different mechanisms; these cytokines can interfere directly with biological functions of endothelial cells and/or target tumor cells inhibiting their capability to stimulate formation of new microvessels that are essential for tumor growth and dissemination. In this review we will summarize the current knowledge about the role of cytokines as anti-angiogenic agents in cancer, focusing our attention on the anti-angiogenic activity of IL-12 family members in haematological malignancies.

Interleukin-27 inhibits pediatric B-acute lymphoblastic leukemia cell spreading in a preclinical model.

B-acute lymphoblastic leukemia (B-ALL) represents the most common pediatric hematological tumor that derives from the aberrant proliferation of early B lymphocytes in the bone marrow. Although most of the B-ALL children take advantage from current therapeutic protocols, some patients relapse and need alternative therapies. With this background, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine with antitumor properties, may function as an antitumor agent against pediatric B-ALL cells. Here we show for the first time that pediatric B-ALL cells functional IL-27R and that IL-27 dampens directly tumor growth in vivo and in vitro through mechanisms elucidated in this study. The novelty of these results deals with the first demonstration that (1) B-ALL cells from pediatric patients injected intravenously (i.v.) into NOD/SCID/Il2rg(-/-) (NSG) mice gave rise to leukemic spreading that was severely hampered by IL-27; (2) IL-27-treated mice, compared with controls, showed significant reduction of putative B-ALL-initiating cells and blasts in the peripheral blood (PB), bone marrow (BM) and spleen; and that (3) IL-27 reduced in vitro B-ALL cell proliferation and angiogenesis, induced apoptosis and downregulated miR-155. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of IL-27 in childhood B-ALL patients.

HOXB7 expression by myeloma cells regulates their pro-angiogenic properties in multiple myeloma patients.

The deregulation of the homeobox genes as homeoboxB (HOXB)-7 has been previously associated to tumor progression and angiogenesis; here we investigated the potential role of HOXB7 in the pro-angiogenic properties of multiple myeloma (MM) cells. We found that HOXB7 was expressed in 10 out of 22 MM patients analyzed at the diagnosis related to high bone marrow angiogenesis and overexpressed in about 40% of myeloma cell lines compared with normal plasma cells. Enforced HOXB7 expression in MM cells by a lentiviral vector significantly modified their transcriptional and angiogenic profile, checked by combined microarray and angiogenesis PCR analyses, upregulating VEGFA, FGF2, MMP2, WNT5a and PDGFA and downregulating thrombospoindin-2. The pro- and anti-angiogenic HOXB7-related gene signature was also validated in a large independent dataset of MM patients. Accordingly, MM-induced vessel formation was significantly increased by HOXB7 overexpression both in vitro angiogenic and chorioallantoic membrane assays, as well as the HOXB7 silencing by small interfering RNA inhibited the production of angiogenic factors, and the pro-angiogenic properties of MM cells. Finally, in SCID-NOD mice we confirmed that HOXB7 overexpression by MM cells stimulated tumor growth, increased MM-associated angiogenesis and the expression of pro-angiogenic genes by microarray analysis supporting the critical role of HOXB7 in the angiogenic switch in MM.

Expression and function of IL-12 and IL-18 receptors on human tonsillar B cells.

IL-12 activates murine and human B cells, but little information is available as to the expression and function of IL-12R on human B lymphocytes. Here we show that the latter cells, freshly isolated from human tonsils, expressed the transcripts of both beta1 and beta2 chains of IL-12R and that beta2 chain mRNA was selectively increased (4- to 5-fold) by incubation with Staphylococcus aureus Cowan I bacteria or IL-12. B cell stimulation with IL-12 induced de novo expression of the transcripts of the two chains of IL-18R, i.e., IL-1 receptor-related protein and accessory protein-like. Functional studies showed that both IL-12 and IL-18 signaled to B cells through the NF-kappaB pathway. In the case of IL-12, no involvement of STAT transcription factors, and in particular of STAT-4, was detected. c-rel and p50 were identified as the members of NF-kappaB family involved in IL-12-mediated signal transduction to B cells. IL-12 and IL-18 synergized in the induction of IFN-gamma production by tonsillar B cells, but not in the stimulation of B cell differentiation, although either cytokine promoted IgM secretion in culture supernatants. Finally, naive but not germinal center or memory, tonsillar B cells were identified as the exclusive IL-12 targets in terms of induction of NF-kappaB activation and of IFN-gamma production.

The homeotic gene products in the control of cell differentiation and proliferation.

Embryo development is controlled by a successive series of genes that provide each cell in the growing embryo with precise position information. Knowledge of these genes is known most completely from Drosophila, where a simple initial pattern generated by maternal effect genes is gradually segmented by the sequential transient expression of three successive series of genes: the gap genes, the pair-rule genes, and the segment polarity genes. Superimposing their action on the preexisting segments, these homeotic genes cause unique and often very distinctive patterns of differentiation for different segments. In humans, about 40 homeotic genes have been identified and are grouped into four clusters on chromosomes 2, 7, 12, and 17, whose organization reflects their spatial and temporal expression. Several homeotic genes have been found expressed not only in embryonic tissues, but also in adult tissues, most notably in the hematopoietic lineage, and also in tumors, especially leukemia, teratocarcinoma, and neuroblastoma, wherein their expression pattern is modified in a complex manner by retinoic acid. The target genes of the transcription factors encoded by the homeotic genes are largely unknown, but recent reports indicate that they may regulate the expression of adhesion molecules on the membrane and the production of components of the extracellular matrix. Abnormal expression of these genes can therefore affect not only cell proliferation, but also the spread of cells to aberrant locations.

Transcriptional repression by the human homeobox protein EVX1 in transfected mammalian cells.

The human homeobox protein EVX1 (EVX1) is thought to play an important role during embryogenesis. In this study, the effect of EVX1 on gene transcription has been investigated in transfected mammalian cells. EVX1 expression represses transcription of a reporter gene directed by either cell-specific or viral promoter/enhancer sequences in a variety of mammalian cell lines and in a concentration-dependent manner. Transcriptional repression is independent of the presence of DNA-binding sites for EVX1 in all the promoters we tested. Furthermore, repression by EVX1 is evident also using a TATA-less minimal promoter in the reporter construct. A carboxyl-terminal proline/alanine-rich region of EVX1 seems to be responsible for the transcriptional repression activity, as suggested by transfection of EVX1 mutants. We speculate that the repressor function of EVX1 contributes to its proposed role in embryogenesis.

Expression of HOXC4 homeoprotein in the nucleus of activated human lymphocytes.

We have analyzed the expression of homeoproteins of the HOX family in resting and activated lymphoid cells and in neoplastic lymphoid cell lines by the use of monoclonal antibodies (MoAbs) already shown to react with the homeoproteins HOXA10, HOXC6, and HOXD4, respectively. Anti-HOXA10 and C6 MoAbs DIDi not show any reactivity with the lymphoid cells tested, whereas anti-HOXD4 MoAb stained few resting peripheral blood lymphocytes (PBLs) and most phytohemagglutinin (PHA)-stimulated PBLs as early as 6 hours after stimulation. The pattern of staining of PHA-activated PBLs is reminiscent of the stages of nucleolar fragmentation in different phases of the cell cycle. The MoAb reacted also with activated or Epstein-Barr virus-transformed B cells, with clonal or polyclonal T and natural killer (NK) cells, with leukemic T-cell lines, and with a Burkitt's lymphoma cell line. RNAse protection experiments, per formed with probes specific for HOXD4 or for the highly homologous HOXA4, HOXB4, and HOXC4, belonging to the same paralogy group, indicated that only HOXC4 mRNA is present in resting or activated PBLs. Northern blot analysis on polyA+ RNA from activated PBLs or Raji cells showed the presence of two different HOXC4 transcripts of 2.8 and 1.9 kb. Gel retardation and Southwestern blot assays showed the presence of a 32-kD homeoprotein with DNA-binding properties typical of a HOX4 homeoprotein in nucleolar extracts of PHA-activated, but not of resting, lymphocytes. Taken together, these data indicate that the HOXC4 homeoprotein is expressed in activated and/or proliferating lymphocytes of the T-, B-, or NK-cell lineage, whereas it is weakly expressed in a minority of resting cells. The early expression and the nucleolar localization suggest an involvement of HOXC4 in the regulation of genes controlling lymphocyte activation and/or proliferation.

Expression of costimulatory molecules in human neuroblastoma. Evidence that CD40+ neuroblastoma cells undergo apoptosis following interaction with CD40L.

Tumour cells display low to absent expression of costimulatory molecules. Here, we have investigated the expression of costimulatory molecules (CD40, CD80, CD86, PD-1L, B7H2, OX40L and 4-1BBL) in human neuroblastoma (NB) cells, since virtually no information is available on this issue. Both established NB cell lines and primary tumours were tested by RT-PCR and flow cytometry. Neuroblastoma cell lines expressed the transcripts of all costimulatory molecule genes, but not the corresponding proteins. Culture of NB cell lines with human recombinant (r)IFN-gamma induced surface expression of CD40 in half of them. Primary NB cells showed CD40, CD80, CD86, OX40L, 4-1BBL, but not PD-1L and B7H2, mRNA expression. Surface CD40 was consistently detected on primary NB cells by flow cytometry. Interferon-gamma gene-transfected NB cells expressed constitutively surface CD40 and were induced into apoptosis by incubation with rCD40L through a caspase-8-dependent mechanism. CD40 may represent a novel therapeutic target in NB.

Ultrastructural and functional studies of the interaction between IL-12 and IL-2 for the generation of lymphokine-activated killer cells.

IL-12 promotes generation of LAK activity in short-term-cultured NK cells, but information on the structure and function of IL-12-induced LAK cells is not yet available. The latter issues have been here investigated with emphasis on interactions between IL-12 and IL-2. Peripheral blood mononuclear cells (MNC) exposed to IL-12 for 5-7 days displayed a decrease in the amount and density of the matrix of large granular lymphocyte (LGL)-associated granules. In cells cultured with IL-12 and IL-2 for 5-7 days, empty vacuoles were predominant and the electron-dense matrix was scanty. In MNC incubated with IL-2 for 5-7 days, most granules were loaded with electron-dense matrix. IL-12 and IL-2 displayed an additive effect on LAK cell cytotoxicity until approximately 48 h in culture which was followed by a sharp decline. Immunocytochemical and biochemical studies demonstrated that MNC cultured for 5-7 days with IL-12 and IL-2 displayed downregulated perforin expression and upregulated granzyme B expression. Fas ligand expression was virtually undetectable in MNC cultured for 5-7 days with or without cytokines. It appears that perforin downregulation plays a major role in the reduced cytotoxicity of MNC cultured with IL-12 and IL-2 for 5-7 days.

Low-dose interferon-gamma-producing human neuroblastoma cells show reduced proliferation and delayed tumorigenicity.

Interferon-gamma (IFN-gamma) directs T helper-1 cell differentiation and mediates antitumour effects in preclinical models. However, high-dose IFN-gamma is toxic in vivo, and IFN-gamma-transfected neuroblastoma (NB) cells secreting high amounts of the cytokine may be lost due to cell apoptosis or differentiation. Two human NB cell lines (ACN and SK-N-BE2(c)) differing as to genetic and phenotypic features were transfected with the human IFN-gamma gene and selected on the grounds of the low concentrations of IFN-gamma produced. In both IFN-gamma-transfected cell lines, autocrine and paracrine activation of IFN-gamma-mediated pathways occurred, leading to markedly reduced proliferation rate, to increased expression of surface HLA and CD40 molecules and of functional TNF binding sites. ACN/IFN-gamma cells showed a significantly delayed tumorigenicity in nude mice as compared to parental cells. ACN/IFN-gamma tumours were smaller, with extensive necrotic area as a result of a damaged and defective microvascular network. In addition, a significant reduction in the proliferation index was observed. This is the first demonstration that IFN-gamma inhibits in vivo proliferation of NB cell by acting on the tumour cell itself. This effect adds to the immunoregulatory and antiangiogenic activities operated by IFN-gamma in syngeneic tumour-bearing hosts.

Differential DNA binding properties of three human homeodomain proteins.

The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes.