A Platelet's Guide to Synovitis.

BACKGROUND: Platelets have the ability to influence the immune system and the inflammatory process and may be strongly involved in the whole pathogenic process of chronic inflammatory joint diseases, such as rheumatoid arthritis. They may play a significant role even before the clinical onset of the disease, contributing to the loss of tolerance of the immune system and the induction of autoimmunity. Subsequently, they can interact with the most important cellular players involved in autoimmunity and inflammation, namely innate immunity cells and T cells and eventually contribute to the building of inflammation in the synovium, thus inducing the activation, migration, and proliferation of fibroblasts that eventually lead to joint damage. Due to their peculiar features, studying the behavior of platelets is a challenging task; however, platelets may prove to be valuable therapeutic targets in the future.

Angiogenic T cells in primary Sjögren's syndrome: a double-edged sword?

OBJECTIVES: The mechanisms underlying increased cardiovascular risk in primary Sjögren's syndrome (pSS) remain unclear. Since the recently discovered angiogenic T cells (Tang) may participate in endothelial repair by cooperating with endothelial progenitor cells (EPC), we aimed to quantify and characterise Tang in the peripheral blood and minor salivary glands (MSG) of pSS patients. METHODS: Tang (CD3+CD31+CXCR4+) and EPC (CD34+CD133+VEGFR-2+) were quantified by flow cytometry in peripheral blood samples from 36 pSS patients and 20 healthy donors. Tang subsets were assessed on the basis of CD4, CD8 and CD28 expression. Labial MSG sections from 10 pSS patients and 12 non-pSS sicca syndrome controls were subjected to immunofluorescence staining to investigate the presence of Tang and the expression of the CXCR4-ligand stromal cell-derived factor-1 (SDF-1)/CXCL12. RESULTS: Circulating Tang cells were expanded and directly correlated to EPC in pSS. Both Tang and EPC directly correlated with disease activity as calculated with the EULAR Sjögren's syndrome disease activity index (ESSDAI). In pSS, the majority of Tang cells were CD4-CD8- double negative (DN) and lacked CD28 revealing a senescent phenotype. A subset of CD4+, CD8+ and DN Tang cells produced interleukin-17. Immunohistology revealed the exclusive presence of periductal and perivascular infiltrating Tang cells along with increased SDF-1/CXCL12 expression in pSS MSG compared to non-pSS sicca syndrome controls. CONCLUSIONS: In pSS, Tang cells are expanded in peripheral blood and infiltrate MSG. Tang may be novel actors in pSS-related endothelial dysfunction and glandular neo-angiogenesis and inflammation.

Effects of anti-NKG2A antibody administration on leukemia and normal hematopoietic cells.

Natural killer cells are key cells of the innate immune system. Natural killer cell receptor repertoires are diversified by a stochastic expression of killer-cell-immunoglobulin-like receptors and lectin-like receptors such as NKG2 receptors. All individuals harbor a subset of natural killer cells expressing NKG2A, the inhibitory checkpoint receptor for HLA-E. Most neoplastic and normal hematopoietic cells express HLA-E, the inhibitory ligand of NKG2A. A novel anti-human NKG2A antibody induced tumor cell death, suggesting that the antibody could be useful in the treatment of cancers expressing HLA-E. We found that immunodeficient mice, co-infused with human primary leukemia or Epstein-Barr virus cell lines and NKG2A(+) natural killer cells, pre-treated with anti-human NKG2A, were rescued from disease progression. Human NKG2A(+) natural killer cells reconstituted in immunodeficient mice after transplantation of human CD34(+) cells. These natural killer cells are able to kill engrafted human primary leukemia or Epstein-Barr virus cell lines by lysis after intraperitoneal administration of anti-human NKG2A. Thus, this anti-NKG2A may exploit the anti-leukemic action of the wave of NKG2A(+) natural killer cells recovering after hematopoietic stem cell transplants or adoptive therapy with natural killer cell infusions from matched or mismatched family donors after chemotherapy for acute leukemia, without the need to search for a natural killer cell alloreactive donor.

Haploidentical hematopoietic transplantation from KIR ligand-mismatched donors with activating KIRs reduces nonrelapse mortality.

Because activating killer cell immunoglobulinlike receptors (KIRs) are heterogeneously expressed in the population, we investigated the role of donor activating KIRs in haploidentical hematopoietic transplants for acute leukemia. Transplants were grouped according to presence vs absence of KIR-ligand mismatches in the graft-vs-host direction (ie, of donor-vs-recipient natural killer [NK]-cell alloreactivity). In the absence of donor-vs-recipient NK-cell alloreactivity, donor activating KIRs had no effects on outcomes. In the 69 transplant pairs with donor-vs-recipient NK-cell alloreactivity, transplantation from donors with KIR2DS1 and/or KIR3DS1 was associated with reduced risk of nonrelapse mortality, largely infection related (KIR2DS1 present vs absent: hazard ratio [HR], 0.25; P = .01; KIR3DS1 present vs absent: HR, 0.18; P = .006), and better event-free survival (KIR2DS1 present vs absent: HR, 0.31; P = .011; KIR3DS1 present vs absent: HR, 0.30; P = .008). Transplantation from donors with KIR2DS1 and/or KIR3DS1 was also associated with a 50% reduction in infection rate (P = .003). In vitro analyses showed that KIR2DS1 binding to its HLA-C2 ligand upregulated inflammatory cytokine production by alloreactive NK cells in response to infectious challenges. Because ∼40% of donors able to exert donor-vs-recipient NK-cell alloreactivity carry KIR2DS1 and/or KIR3DS1, searching for them may become a feasible, additional criterion in donor selection.

Differences in Aspergillus-specific immune recovery between T-cell-replete and T-cell-depleted hematopoietic transplants.

BACKGROUND: After hematopoietic stem cell transplantation, invasive aspergillosis remains one of the most lethal infections. Susceptibility may be due to prophylaxis and treatment of graft-vs.-host disease in T-cell-replete transplants, and delayed immune rebuilding due to T-cell depletion in haploidentical transplantation. METHODS: We monitored CD4(+) T-cell recovery and anti-Aspergillus immune competence in pediatric recipients of T-cell-replete matched transplants and of prevalently adult recipients of T-cell-depleted matched or haploidentical transplants for hematological malignancies. RESULTS: Although CD4(+) T-cell counts were higher in T-cell-replete transplant recipients at all post-transplant time points, Aspergillus-specific T cells were first detected 15-18 months after T-cell-replete matched, 7-9 months after T-cell-depleted matched, and 9-12 months after haploidentical transplantation, respectively. Incidence of invasive aspergillosis was 22% with 10% mortality after T-cell-replete transplants, 0% after T-cell-depleted matched, and 7% with 4% mortality after haploidentical transplants. CONCLUSIONS: Although T-cell counts were significantly higher after T-cell-replete transplants, post-transplant immune suppression/GvHD appeared to impair their function. Specific Aspergillus immune competence recovered faster after T-cell-depleted transplants, whether matched or haploidentical. T-cell-replete transplants were associated with a higher incidence of invasive aspergillosis and Aspergillus-related deaths. These results showed that T-cell depletion without post-transplant immunosuppression is associated to a faster immune recovery than T-cell-replete transplantation.

Thymosin alpha1 to harness immunity to pathogens after haploidentical hematopoietic transplantation.

We designed a phase I/II clinical study to determine safety and efficacy of thymosin alpha1 (Talpha1) administration in recipients of one HLA haplotype (haploidentical) stem cell transplants for hematologic malignancies. Talpha1 administration did not cause acute or chronic graft versus host disease and was associated with significant improvement in polymorphonuclear (phagocytosis) and dendritic cell (phagocytosis, expression of costimulatory molecules, and cytokine production) functions. It was also associated with increased T-cell counts and earlier appearance of functional pathogen-specific T cell responses (by a sensitive limiting dilution assay that detects frequency of T cells specific for Aspergillus, Candida, CMV, ADV, VZV, HSV, Toxoplasma). Five of six haploidentical transplant recipients who received Talpha1 are alive and disease free at a median follow-up of 10 months after transplantation (range: 5-20). They experienced only a single nonlethal infectious episode and one patient developed fatal immune hemolytic anemia. At this very early stage of the clinical trial, we conclude Talpha1 administration is safe and may impact favorably on immune function. Larger numbers of patients and longer follow-up are, of course, needed to assess its impact on survival.

Photodynamic purging of alloreactive T cells for adoptive immunotherapy after haploidentical stem cell transplantation.

After haploidentical stem cell transplantation immune recovery is inevitably slow and infectious related mortality is about 30-40%. Immune reconstitution could be improved by infusing donor T cells, but the obstacle is graft-versus-host disease. In a mixed lymphocyte reaction, alloantigen-stimulated T cells uptake 4,5-dibromorhodamine methyl ester (TH9402), a compound that is structurally similar to rhodamine. TH9402 preferentially localizes in mitochondria and when exposed to 500- to 600-nm wavelength visible light delivered through the Theralux device (Kiadis Pharma, Amsterdam, The Netherlands), it becomes highly cytotoxic through oxidative damage. This study investigated a range of parameters, and combinations thereof, with the aim of achieving optimal T cell allodepletion and preservation of pathogen-specific responses. We report on 11 clinical scale dry runs which reproducibly yielded the following results. Blood mononuclear cells were stimulated with haploidentical irradiated (20 Gy). Blood mononuclear cells in a mixed lymphocyte reaction. Cells were then incubated with TH9402 and exposed to light delivered through the Theralux device. Optimal conditions for T cell allodepletion emerged as (1) duration of mixed lymphocyte reaction: 24 h; (2) responder cell concentration: 3-5x10(6)/ml; (3) TH9402 concentration: 5 microM; (4) quantity of internalized TH9402, as measured by mean fluorescence intensity (MFI): 20,000-25,000 MFI; (5) energy delivered: 0.1 J/cm(2). Only under these conditions were the frequencies (by limiting dilution analyses) of alloantigen-specific T cells maximally reduced, i.e., 2467+/-639 (mean+/-SD) times, when compared with non-TH9402-treated cells. Pathogen-specific responses to pathogen antigens such as Cytomegalovirus, Adenovirus, Varicella Zoster Virus, Herpes Simplex Virus, Aspergillus fumigatus, Candida albicans, Toxoplasma gondii were retained, although with a 19+/-9.7 times reduction in frequency. This remarkable drop in frequency of alloreactive T cells is expected to allow safe infusion of relatively large numbers of T cells across histocompatibility barriers for adoptive transfer of donor immunity. Consequently, a clinical trial is planned to incorporate infusion of photo-allodepleted donor T cells after haploidentical stem cell transplantation with the aim of decreasing infection-related mortality.

Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value.

We analyzed 112 patients with high-risk acute myeloid leukemia (61 in complete remission [CR]; 51 in relapse), who received human leukocyte-antigen (HLA)-haploidentical transplants from natural killer (NK) alloreactive (n = 51) or non-NK alloreactive donors (n = 61). NK alloreactive donors possessed HLA class I, killer-cell immunoglobulin-like receptor (KIR) ligand(s) which were missing in the recipients, KIR gene(s) for missing self recognition on recipient targets, and alloreactive NK clones against recipient targets. Transplantation from NK-alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in CR (3% versus 47%) (P > .003), better event-free survival in patients transplanted in relapse (34% versus 6%, P = .04) and in remission (67% versus 18%, P = .02), and reduced risk of relapse or death (relative risk versus non-NK-alloreactive donor, 0.48; 95% CI, 0.29-0.78; P > .001). In all patients we tested the "missing ligand" model which pools KIR ligand mismatched transplants and KIR ligand-matched transplants from donors possessing KIR(s) for which neither donor nor recipient have HLA ligand(s). Only transplantation from NK-alloreactive donors is associated with a survival advantage.

Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation.

Aspergillus and cytomegalovirus are major causes of morbidity/mortality after haploidentical hematopoietic transplantation. The high degree of mismatching makes cell immunotherapy impossible as it would result in lethal graft-versus-host disease (GvHD). We generated large numbers of donor T-cell clones specific for Aspergillus or cytomegalovirus antigens. We identified clones potentially responsible for causing GvHD by screening them for cross-reactivity against recipient mononuclear cells. Non-recipient reactive, pathogen-specific clones were infused soon after transplantation. They were CD4+ and produced high levels of interferon-gamma and low levels of interleukin-10. In 46 control transplant recipients who did not receive adoptive therapy, spontaneous pathogen-specific T cells occurred in low frequency 9 to 12 months after transplantation and displayed a non-protective low interferon-gamma/high interleukin-10 production phenotype. In the 35 recipients who received adoptive therapy, one single infusion of donor alloantigen-deleted, pathogen-specific clones in the dose range of 10(5) to 10(6) cells/kg body weight did not cause GvHD and induced high-frequency T-cell responses to pathogens, which exhibited a protective high interferon-gamma/low interleukin-10 production phenotype within 3 weeks of infusion. Frequencies of pathogen-specific T cells remained stable over time, and were associated with control of Aspergillus and cytomegalovirus antigenemia and infectious mortality. This study opens new perspectives for reducing infectious mortality after haploidentical transplantations.

Alloreactive natural killer cells in mismatched hematopoietic stem cell transplantation.

Natural killer (NK) cell-mediated, donor-vs.-recipient alloresponses occur following transplantation of human leukocyte antigen (HLA) haplotype-mismatched hematopoietic stem cells (HSCs). NK cell alloreactivity reduced the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-vs.-host disease (GvHD). NK cells are primed to kill by several activating receptors. NK killing of autologous cells is prevented because NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) MHC class I alleles. As KIRs are clonally distributed, the NK population in any individual is constituted of a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and in some cases, functional assessment of donor NK clones will identify haploidentical donors who are able to mount donor-vs.-recipient NK alloreactions.

Tuber aestivum and Tuber uncinatum: two morphotypes or two species?

Tuber spp. are ectomycorrhizal fungi that establish symbioses with shrubs and trees. Because of their different smell and taste, Tuber uncinatum and Tuber aestivum are two truffle morphotypes with a different market value, but whether or not T. uncinatum and T. aestivum are different taxa is still an open debate among mycologists. In order to identify molecular keys characterizing both T. aestivum and T. uncinatum morphotypes, ITS/RFLPs analyses were carried out on a large collection of samples from all over Italy and from other European countries, followed by a study of the phylogenesis of ITS, beta-tubulin and EF 1-alpha genes, on representative samples. The present study provides compelling evidence that: (i) T. uncinatum and T. aestivum belong to the same species, (ii) neither morphotype presents a specific molecular fingerprint, but they may even share identical alleles at any of the loci analysed; (iii) T. aestivum is most likely under a selfing reproductive mode. Our findings suggest that ecological, rather than genetic causes may account for differences in sporal morphology, taste and smell between T. aestivum and T. uncinatum truffles.