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Mesenchymal stromal cells (MSCs) support hematopoiesis and exert immunoregulatory activities. Here, we analyzed the functional outcome of the interactions between MSCs and monocytes/macrophages. We showed that MSCs supported the survival of monocytes that underwent differentiation into macrophages, in the presence of macrophage colony-stimulating factor. However, MSCs skewed their polarization toward a peculiar M2-like functional phenotype (MMSC), through a prostaglandin E2-dependent mechanism. MMSC were characterized by high expression of scavenger receptors, increased phagocytic capacity, and high production of interleukin (IL)-10 and transforming growth factor-β. These cytokines contributed to the immunoregulatory properties of MMSC, which differed from those of typical IL-4-induced macrophages (M2). In particular, interacting with activated natural killer (NK) cells, MMSC inhibited both the expression of activating molecules such as NKp44, CD69, and CD25 and the production of IFNγ, while M2 affected only IFNγ production. Moreover, MMSC inhibited the proliferation of CD8+ T cells in response to allogeneic stimuli and induced the expansion of regulatory T cells (Tregs). Toll-like receptor engagement reverted the phenotypic and functional features of MMSC to those of M1 immunostimulatory/proinflammatory macrophages. Overall our data show that MSCs induce the generation of a novel type of alternatively activated macrophages capable of suppressing both innate and adaptive immune responses. These findings may help to better understand the role of MSCs in healthy tissues and inflammatory diseases including cancer, and provide clues for novel therapeutic approaches.

NK cells are innate lymphocytes characterized by the expression of nuclear factor interleukin 3 regulated (NFIL3 or E4BP4), eomesodermin (EOMES) transcription factors (TFs), and by the ability to exert cytolytic activity and release IFN-γ. In the haploidentical-hematopoietic stem cell transplantation (haplo-HSCT) setting, CD34(+) donor derived NK cells play a major role in the control of leukemic relapses. Therefore, it is important to better define cytokines that influence NK-cell differentiation from CD34(+) precursors. We analyzed the effects of IL-1β on NK-cell differentiation from umbilical cord blood (UCB) CD34(+) cells. While IL-1β inhibited CD161(+) CD56(+) cell proliferation, an increased expression of LFA-1, CD94/NKG2A, KIRs, and perforin on CD56(+) cells was detected. In addition, within the CD161(+) CD56(+) IL-1RI(+) LFA-1(-) cell fraction (representing group 3 innate lymphoid cells, ILC3-like cells), a significant increase of EOMES, NKp46, and CD94/NKG2A receptors, cytolytic granules, and IFN-γ was detected. This increase was paralleled by a decrease of related orphan receptors (RORγt) TF, NKp44 expression, and IL-22 production. These data suggest that IL-1β inhibits ILC3- while favoring NK-cell maturation. Since in haplo-HSCT conditioning regimen, infections or residual leukemia cells may induce IL-1β production, this may influence the NK/ILC3 development from donor-derived CD34(+) precursors.

Human natural killer (NK) cell development is a step-by-step process characterized by phenotypically identified stages. CD161 is a marker informative of the NK cell lineage commitment, whereas CD56, CD117, and CD94/NKG2A contribute to define discrete differentiation stages. In cells undergoing in vitro differentiation from CD34+ umbilical cord blood (UCB) progenitors, LFA-1 expression allowed to discriminate between immature noncytolytic CD161+CD56+LFA-1− and more differentiated cytolytic CD161+CD56+LFA-1+ NK cells. CD161+CD56+LFA-1− NK cells produce large amounts of CXCL8 after phorbol myristate acetate (PMA) or cytokine treatment. Remarkably, CXCL8 mRNA expression was also detected in fresh stage III immature NK cells isolated from tonsils and these cells expressed CXCL8 protein on PMA stimulation. Within in vitro UCB-derived CD161+CD56+LFA-1− NK cells, CXCL8 release was also induced on antibody-mediated cross-linking of NKp44 and CD161. Such unexpected activating function of CD161 was confined to the CD161+CD56+LFA-1− subset, because it did not induce cytokine release or CD107a expression in CD161+CD56+LFA-1+ cells or in mature peripheral blood NK cells. Anti-CXCL8 neutralizing antibody induced a partial inhibition of NK cell differentiation, which suggests a regulatory role of CXCL8 during early NK cell differentiation. Altogether, these data provide novel information that may offer clues to optimize NK cell maturation in hematopoietic stem cell transplantation.

Tertiary lymphoid structures (TLSs) are a common finding in non-small cell lung cancer (NSCLC) and are predictors of favourable clinical outcome. Here we show that NCR(+) innate lymphoid cell (ILC)-3 are present in the lymphoid infiltrate of human NSCLC and are mainly localized at the edge of tumour-associated TLSs. This intra-tumoral lymphocyte subset is endowed with lymphoid tissue-inducing properties and, on activation, produces IL-22, TNF-α, IL-8 and IL-2, and activates endothelial cells. Tumour NCR(+)ILC3 may interact with both lung tumour cells and tumour-associated fibroblasts, resulting in the release of cytokines primarily on engagement of the NKp44-activating receptor. In patients, NCR(+)ILC3 are present in significantly higher amounts in stage I/II NSCLC than in more advanced tumour stages and their presence correlate with the density of intratumoral TLSs. Our results indicate that NCR(+)ILC3 accumulate in human NSCLC tissue and might contribute to the formation of protective tumour-associated TLSs.

An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.

Natural killer (NK) cell recognition and function in humans is regulated by multiple cell surface receptors. The "on" signal leading to NK cell triggering is primarily mediated by natural cytotoxicity receptors (NCR). Analysis of NK cells in primate animal models is of particular relevance because NK cells may play an essential role in host defenses against infections. We analyzed Macaca fascicularis PBMC and in vitro-derived NK cell populations and clones by cytofluorometry, using a wide panel of mAb, and by cytolytic activity assays. In addition, RT-PCR strategy and transient transfections were used to isolate M. fascicularis NCR. NCR-specific mAb reactivity (anti-NKp46 and anti-NKp30) was present on M. fascicularis PBMC and on NK cell cultures. Macaque NCR were functional in both redirected killing and in mAb-mediated masking assays. Cloning of macNKp46 and macNKp30 NCR homologous genes showed a high sequence similarity (86 % and 88 %, respectively) with their human counterparts. Attempts at identifying NKp44 surface reactivity and at cloning the macaque homologue were unsuccessful. NKp46 and NKp30 NCRs, but not NKp44, are highly conserved in M. fascicularis NK cells. This suggests the possibility of a staged appearance of the NCR during phylogenesis and provides a useful tool for the study of natural immunity correlates of protection in primate SIV/SHIV infection models.

The degree of matching of HLA genes between the selected donor and recipient is an important aspect of the selection of unrelated donors for allogeneic bone marrow transplantation (UBMT). The most sensitive methods currently used are serological typing of HLA class I genes, mixed lymphocyte culture (MLC), IEF and molecular genotyping of HLA class II genes by direct sequencing of PCR products. Serological typing of class I antigenes (A, B and C) fails to detect minor differences demonstrated by direct sequencing of DNA polymorphic regions. Molecular genotyping of HLA class I genes by DNA analysis is costly and work-intensive. To improve compatibility between donor and recipient, we have set up a new rapid and non-radioisotopic application of the 'fingerprinting PCR' technique for the analysis of the polymorphic second exon of the HLA class I A, B and C genes. This technique is based on the formation of specific patterns (PCR fingerprints) of homoduplexes and heteroduplexes between heterologous amplified DNA sequences. After an electrophoretic run on non-denaturing polyacrylamide gel, different HLA class I types give allele-specific banding patterns. HLA class I matching is performed, after the gel has been soaked in ethidium bromide or silver-stained, by visual comparison of patients' fingerprints with those of donors. Identity can be confirmed by mixing donor and recipient DNAs in an amplification cross-match. To assess the technique, 10 normal samples, 22 related allogeneic bone marrow transplanted pairs and 10 unrelated HLA-A and HLA-B serologically matched patient-donor pairs were analysed for HLA class I polymorphic regions. In all the related pairs and in 1/10 unrelated pairs, matched donor-recipient patterns were identified. This new application of PCR fingerprinting may confirm the HLA class I serological selection of unrelated marrow donors.

Natural killer cells express clonally distributed receptors specific for major histocompatibility complex class I molecules. The human leukocyte antigen (HLA)-C-specific receptors have been molecularly identified and cloned. They exist not only as inhibitory (p58) but also as activatory (p50) receptors. Here we show that p50 and p58 are highly homologous in their extracellular regions formed by two Ig-like domains. In contrast, major differences exist in their transmembrane and cytoplasmic portions. Whereas p 58 displays a 76-84-amino acid cytoplasmic tail containing an unusual antigen receptor activation motif, p50 is characterized by a shorter 39-amino acid tail. In addition, whereas p58 has a nonpolar transmembrane portion, p50 contains the charged amino acid Lys. These data strongly suggest that receptors with identical HLA-C allele specificity can mediate functions of opposite sign owing to their different transmembrane/cytoplasmic portions.

Dendritic cells (DCs) migrate from peripheral tissues to secondary lymphoid organs (SLOs) through the afferent lymph. Owing to limitations in investigating human lymph, DCs flowing in afferent lymph have not been properly characterized in humans until now. In this study, DCs present in seroma, an accrual of human afferent lymph occurring after lymph node surgical dissection, were isolated and analyzed in detail. Two main DC subsets were identified in seroma that corresponded to the migratory DC subsets present in lymph nodes, that is, CD14+ and CD1a+. The latter also included CD1abright Langerhans cells. The two DC subsets appeared to share the same monocytic precursor and to be developmentally related; both of them spontaneously released high levels of TGF-β and displayed similar T cell–activating and –polarizing properties. In contrast, they differed in the expression of surface molecules, including TLRs; in their phagocytic activity; and in the expression of proteins involved in Ag processing and presentation. It is worth noting that although both subsets were detected in seroma in the postsurgical inflammatory phase, only CD1a+ DCs migrated via afferent lymph under steady-state conditions. In conclusion, the high numbers of DCs contained in seroma fluids allowed a proper characterization of human DCs migrating via afferent lymph, revealing a continuous stream of DCs from peripheral regions toward SLOs under normal conditions. Moreover, we showed that, in inflammatory conditions, distinct subsets of DCs can migrate to SLOs via afferent lymph.

Natural killer (NK) cells have been shown to express a clonally distributed ability to recognize HLA class I alleles. The previously defined NK clones belonging to "group 1" recognize HLA-C*0401 (Cw4) and other HLA-C alleles sharing Asn at position 77 and Lys at position 80. Conversely, the "group 2" NK clones recognize HLA-Cw*0302 (Cw3) and other HLA-C alleles characterized by Ser at position 77 and Asn at position 80. We assessed directly the involvement of these two residues in the capacity of NK cell clones to discriminate between the two groups of HLA-C alleles. To this end, Cw3 and Cw4 alleles were subjected to site-directed mutagenesis. Substitution of the amino acids typical of the Cw3 allele (Ser-77 and Asn-80) with those present in Cw4 (Asn-77 and Lys-80) resulted in a Cw3 mutant that was no longer recognized by group 2 NK cell clones, but that was recognized by group 1 clones. Analysis of Cw3 or Cw4 molecules containing single amino acid substitutions indicates roles for Lys-80 in recognition mediated by group 1 clones and for Ser-77 in recognition mediated by group 2 clones. These results demonstrate that NK-mediated specific recognition of HLA-C allotypes is affected by single natural amino acid substitutions at positions 77 and 80 of the heavy chain.

Natural killer (NK) cells are the main lymphoid population in the maternal decidua during the first trimester of pregnancy. Decidual NK (dNK) cells display a unique functional profile and play a key role in promoting tissue remodeling, neoangiogenesis, and immune modulation. However, little information exists on their origin and development. Here we discovered CD34 + hematopoietic precursors in human decidua (dCD34 + ). We show that dCD34 + cells differ from cord blood- or peripheral blood-derived CD34 + precursors. The expression of IL-15/IL-2 receptor common β-chain (CD122), IL-7 receptor α-chain (CD127), and mRNA for E4BP4 and ID2 transcription factors suggested that dCD34 + cells are committed to the NK cell lineage. Moreover, they could undergo in vitro differentiation into functional (i.e., IL-8– and IL-22–producing) CD56 bright CD16 − KIR +/− NK cells in the presence of growth factors or even upon coculture with decidual stromal cells. Their NK cell commitment was further supported by the failure to undergo myeloid differentiation in the presence of GM-CSF. Our findings strongly suggest that decidual NK cells may directly derive from CD34 + cell precursors present in the decidua upon specific cellular interactions with components of the decidual microenvironment.

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells endowed with the capacity of producing large amounts of IFNα. Here we show that the Leukocyte-Associated Ig-like Receptor-1 (LAIR-1) is abundantly expressed on pDCs (the highest expression among all leukocytes) and its cross-linking inhibits IFNα production in response to Toll-like receptor ligands. Remarkably, LAIR-1 expression in pDCs is down-regulated in the presence of interleukin (IL)-3, thus indicating coordinated functions with NKp44, another pDC inhibitory receptor, which is conversely induced by IL-3. Nevertheless, the expression of NKp44 in pDCs isolated from secondary lymphoid organs, which is thought to be influenced by IL-3, is not coupled to a decreased expression of LAIR-1. Interestingly, pDCs isolated from peripheral blood of systemic lupus erithematosus (SLE) patients express lower levels of LAIR-1 while displaying slight but consistent expression of NKp44, usually undetectable on pDCs derived from healthy donors. Using sera derived from SLE patients, we show that LAIR-1 and NKp44 display synergistic inhibitory effects on IFNα production by interleukin IL-3 cultured pDCs stimulated with DNA immunocomplexes. In conclusion, our results indicate that the inhibitory function of LAIR-1 may play a relevant role in the mechanisms controlling IFNα production by pDCs both in normal and pathological innate immune responses.

In this study, we have evaluated 14 large granular lymphocyte (LGL) expansions, 11 of which were CD8+. Analysis of the membrane expression of the alpha and beta chains of the CD8 antigen, using specific monoclonal antibodies (MoAbs), has shown that LGL expansions with the CD3+, CD4+, CD8+, CD57+ T-cell receptor (TcR) alpha beta phenotype bear the CD8 alpha/alpha isoform, while the CD3+, CD4-, CD8+, CD57+ TcR alpha beta samples were positive for both the CD8 alpha and CD8 beta chains. These data were confirmed also by messenger RNA analysis. One additional case, with a peculiar phenotype (CD3-, CD2-, CD4-, CD8+, CD57-) and a germline configuration of the TcR beta and gamma chain genes, expressed only the CD8 alpha chain. After additional phenotypic analysis with a wider panel of MoAbs, it was found that the beta chain of the interleukin-2 receptor was constitutively expressed on the majority of the samples tested, and that most of the monoclonal samples coexpressed CD45RA/R0 antigens. Using MoAbs directed against the variable regions of the TcR beta chain, we could show a preferential V beta region restriction in the CD8+ monoclonal cases. This more extensive characterization of CD8+ LGL expansions has further documented the marked heterogeneity within this rare condition and allowed a better phenotypic dissection between the monoclonal and polyclonal cases.

Human NK cells can be divided into CD56dimCD16+ killer Ig-like receptors (KIR)+/− and CD56brightCD16− KIR− subsets that have been characterized extensively regarding their different functions, phenotype, and tissue localization. Nonetheless, the developmental relationship between these two NK cell subsets remains controversial. We report that, upon cytokine activation, peripheral blood (PB)-CD56bright NK cells mainly gain the signature of CD56dim NK cells. Remarkably, KIR can be induced not only on CD56bright, but also on CD56dim KIR− NK cells, and their expression correlates with lower proliferative response. In addition, we demonstrate for the first time that PB-CD56dim display shorter telomeres than PB- and lymph node (LN)-derived CD56bright NK cells. Along this line, although human NK cells collected from nonreactive LN display almost no KIR and CD16 expression, NK cells derived from highly reactive LN, efferent lymph, and PB express significant amounts of KIR and CD16, implying that CD56bright NK cells could acquire these molecules in the LN during inflammation and then circulate through the efferent lymph into PB as KIR+CD16+ NK cells. Altogether, our results suggest that CD56brightCD16− KIR− and CD56dimCD16+KIR+/− NK cells correspond to sequential steps of differentiation and support the hypothesis that secondary lymphoid organs can be sites of NK cell final maturation and self-tolerance acquisition during immune reaction.

Tryptophan (Trp) catabolism mediated by indoleamine 2,3-dioxygenase (IDO) plays a central role in the regulation of T-cell–mediated immune responses. In this study, we also demonstrate that natural killer (NK)–cell function can be influenced by IDO. Indeed, l-kynurenine, a Trp-derived catabolite resulting from IDO activity, was found to prevent the cytokine-mediated up-regulation of the expression and function of specific triggering receptors responsible for the induction of NK-cell–mediated killing. The effect of l-kynurenine appears to be restricted to NKp46 and NKG2D, while it does not affect other surface receptors such as NKp30 or CD16. As a consequence, l-kynurenine–treated NK cells display impaired ability to kill target cells recognized via NKp46 and NKG2D. Instead, they maintain the ability to kill targets, such as dendritic cells (DCs), that are mainly recognized via the NKp30 receptor. The effect of l-kynurenine, which is effective at both the transcriptional and the protein level, can be reverted, since NK cells were found to recover their functional competence after washing.

Treatment of melanoma cell lines with IFN-gamma induces the switch from proteasome (PS) to immunoproteasome (iPS). This finding has profound implications for the immunobiology of melanoma cells since certain peptides (such as Melan-A(mart1)(27-35)) are cleaved differently by iPS, thus implying a different ability to be presented by HLA class I molecules. IFN-alpha is a cytokine not only produced during infectious diseases, but also used in the treatment of certain cancers. Nevertheless, the effects of IFN-alpha on the switch of PS to iPS are largely unknown. A comparison of the effect of both IFN-alpha and IFN-gamma was thus carried out on melanoma cell lines. RT-PCR showed that mRNA for iPS subunits (i.e. LMP-2, LMP-7 and MECL-1) was detectable both in untreated and IFN-treated melanoma cells. Immunoblotting analysis revealed that while IFN-gamma was able to consistently induce the switch from PS to iPS, IFN-alpha treatment did not, possibly due to post-transcriptional event(s) blocking the expression of iPS-specific subunits. Finally, Melan-A(mart1)(27-35) peptide was found only in the HPLC-MS spectra from both untreated and IFN-alpha-treated cells, but not upon IFN-gamma treatment. Altogether, these data demonstrate that IFN-alpha does not induce the switch from PS to iPS.

The NK cell maturation from CD34(+) Lin(-) hematopoietic cell precursors is a complex process that requires the direct contact with stromal cells and/or the synergistic effect of different cytokines. In this study we show that IL-21 is capable of inducing an accelerated NK cell maturation when added to cultures of CD34(+) Lin(-) cells isolated from human cord blood supplemented with IL-15, Flt3-L and SCF. After 25 days of culture, 50% of CD56(+) cells expressed various NK cell markers including the NKp46 and NKp30 triggering receptors, the CD94/NKG2A inhibitory receptor and CD16. At day 35, substantial fractions of NK cells expressed KIR, CD8 and CD2, i.e. surface markers expressed by mature NK cells, that are virtually undetectable in developing NK cells cultured in the absence of IL-21. Remarkably, similar to mature NK cells all these markers were included in the CD56(dim) cell fraction, while the CD56(bright) population was only composed of CD94/NKG2A(-) and CD94/NKG2A(+) cells. Thus, IL-21 allows the induction of a full NK cell maturation in vitro and offers an important tool for dissecting the molecular mechanisms involved in different steps of NK cell maturation and in the acquisition of a mature KIR repertoire.

NK cells display several killer inhibitory receptors (KIR) specific for different alleles of MHC class I molecules. A family of KIR are represented by type I transmembrane proteins belonging to the immunoglobulin superfamily (Ig-SF). Besides cDNA encoding for these KIR, additional cDNA have been identified which encode for Ig-SF receptors with still undefined specificity. Here we analyze one of these cDNA, termed cl.15.212, which encodes a type I transmembrane protein characterized by two extracellular Ig-like domains and a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) which is typical of KIR. cl.15.212 cDNA displays approximately 50 % sequence homology with other Ig-SF members. Different from the other KIR, cl.15.212 mRNA is expressed by all NK cells and by a fraction of KIR+ T cell clones. cl.15.212 cDNA codes for a membrane-bound receptor displaying an apparent molecular mass of 49 kDa, thus termed p49. To determine the specificity of the cl.15.212-encoded receptor, we generated soluble fusion proteins consisting of the ectodomain of p49 and the Fc portion of human IgG1. Soluble molecules bound efficiently to 221 cells transfected with HLA-G1, -A3, -B46 alleles and weakly to -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3 or -Cw4. The binding specificity of soluble p49-Fc was confirmed by competition experiments using an anti-HLA class I-specific monoclonal antibody. Finally, different cDNA encoding for molecules homologous to cl.15.212 cDNA have been isolated, two of which lack the sequence encoding the transmembrane portion, thus suggesting they may encode soluble molecules.

Stromal cells (SC) are an important component of decidual tissues where they are in strict proximity with both NK and CD14(+) myelomonocytic cells that play a role in the maintenance of pregnancy. In this study we analyzed whether decidual SC (DSC) could exert a regulatory role on NK and CD14(+) cells that migrate from peripheral blood (PB) to decidua during pregnancy. We show that DSCs inhibit the IL15-mediated up-regulation of major activating NK receptors in PB-derived NK cells. In addition, the IL15-induced NK cell proliferation, cytolytic activity and IFN-γ production were severely impaired. DSCs sharply inhibited dendritic cells differentiation and their ability to induce allogeneic T cell proliferation. Indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) mediated the inhibitory effect of DSCs. Our results strongly suggest an important role of DSCs in preventing potentially dangerous immune response, thus contributing to maintenance of pregnancy.

Page 1980 Due to a printing error the affiliation given for Michele Cilli was wrongly indicated; the correct affiliation is: Istituto Scientifico Tumori and Centro Biotecnologie Avanzate, Genova, Italy

It is well known that patients subjected to ABMT present a profound and long lasting immunological impairment. Although the pattern of the phenotypic expression and the function of T cells, as well as the immunoglobulin levels and the capacity to produce specific humoral responce after ABMT have been extensively studied (1), few informations have been produced on the B-cell lineage. In this study we analized the pattern of B-cell reconstitution in the bone marrow (BM) and peripheral blood (PB) in the firtst 3 months after ABMT.

Allogeneic haematopoietic stem cell transplants might induce immunological alterations leading to autoimmune-like syndromes. In particular neutrophil-associated antigens could represent the target for autoantibodies against neutrophils in patients receiving an allogeneic peripheral stem cell or bone marrow transplantation, giving rise to granulocytopenia. With this aim we studied prospectively 43 allotransplanted patients for the presence of antibodies reacting with neutrophils (ARN), looking for a correlation with a post-engraftment neutropenia. Our data showed that the direct test for ARN was positive in 30 patients. Interestingly, 7/7 patients who received a T-cell-depleted marrow transplant developed ARN. Antibodies with a specific neutrophil-antigen reactivity were detected in 4 patients, 1 with an anti-CD16/FcbetaRIIIb receptor reactivity and 3 with anti-NA 1 reacting patterns, respectively. From a clinical point of view, it was not possible to demonstrate a close and significant relationship between neutropenia and ARN, although patients showing ARN had slightly lower absolute levels of peripheral neutrophils until 6 months after BMT. In conclusion, ARN may be detected in the majority of patients following allogeneic stem cell transplantation; in addition, since ex vivo or in vivo T-cell-depletion leads to a higher percentage of patients positive for ARN, it could be hypothesized that "autoimmune-like" disorders in transplanted patients might be related to a T-cell derangement due to different numbers and subsets of T lymphocytes.