Granulocytes and monocytes apheresis induces upregulation of TGFß1 in patients with active ulcerative colitis: A possible involvement of soluble HLA-I.

Granulocyte and monocyte apheresis has been used in different immune-mediated disorders, mainly inflammatory bowel diseases. The removal of activated leukocytes and several additional immunomodulatory mechanisms have been so far suggested to explain the anti-inflammatory effects of the treatment. Recent data indicate that, during centrifugation based apheresis, sHLA-I adsorbed to plastic circuits is able to induce TGFß1 production in activated leukocytes. On these bases, the present study was aimed at analyzing if this model could be applied to a noncentrifugation based apheresis, such as granulocyte and monocyte apheresis. Ten patients with ulcerative colitis were enrolled. Every patient received 5 weekly apheresis treatments. Cellulose acetate beads removed from the column post-GMA were stained by fluorescent anticlass I mAb and examined by fluorescent microscope. Moreover, sFasL plasma concentration, TGFß1 plasma levels, and the percentage of TGFß1 positive neutrophils were evaluated before and immediately after each single apheresis. Immunofluorescent images revealed a homogeneous layer of a sHLA-I adsorbed to the surface of the beads recovered following the procedure. sFasL plasma concentration progressively increased both following the procedures and during inter-procedure periods. Consistently, also TGFß1 plasma levels and the percentage of TGFß1 positive neutrophils increased during the procedures with a meaningful relationship with sFasL plasma levels. Taken together, these findings suggest that the immunosuppressive effects attributed to granulocyte and monocyte apheresis might depend, at least in part, on the sensitivity of activated leucocytes to the bioactivity of sHLA-I molecules. J. Clin. Apheresis 32:49-55, 2017. © 2016 Wiley Periodicals, Inc.

Immunomodulation due to plasma or plasma-platelet apheresis donation: Events occurring during donation procedures.

BACKGROUND: It has been recently shown that during therapeutic apheresis procedure, a large amount of soluble HLA class I molecules settles onto plastic apheresis circuits, inducing sustained TGFß1 pre/post-transcriptional modulation in activated patients' leukocytes. Reportedly, donors' leukocytes may be exposed to similar immunosuppressing activities during donor apheresis procedures. On this basis, it could be hypothesized that such events can cause immune modulation. It is uncertain which blood cell population is most impacted by these events. This study is focused on the effects on the T lymphocytes. STUDY DESIGN AND METHODS: To assess if such events occur, lymphocytes from 20 apheresis donors collected before and after three closely timed plasma and platelet donation procedures were analyzed for sHLA-I mediated immunomodulation. RESULTS: The results confirmed that sHLA-I molecules bind to the apheresis circuit surfaces. Circuits can also transiently activate donors' CD8(+) T lymphocytes, to which sHLA-I molecules can bind, thus modulating short-lasting immune effects, such as transcriptional and post-transcriptional TGFß1 modulation and soluble Fas ligand release. However, no significant change in relative proportions, absolute number and cell viability of lymphocyte subpopulations was found and no ex vivo immune effect was detectable longer than 14 days after procedure in any cell type in all donors. CONCLUSION: Short-lived sHLA-I mediated immunomodulation was demonstrable in lymphocytes from every donor as a consequence of apheresis procedures, but no enrolled subject experienced any adverse reaction or showed any sign of immunosuppression during 24 months of follow-up after the donations.

Identification of a novel cord blood NK cell subpopulation expressing functional programmed death receptor-1.

Background: Natural Killer cells (NKs) represent the innate counterpart of TCRαß lymphocytes and are characterized by a high anti-tumor and an anti-viral cytotoxic activity. Recently, it has been demonstrated that NKs can express PD-1 as an additional inhibitory receptor. Specifically, PD-1 was identified on a subpopulation of terminally differentiated NKs from healthy adults with previous HCMV infection. So far it is unknown whether PD-1 appears during NK-cell development and whether this process is directly or indirectly related to HCMV infection. Methods: In this study, we analyzed the expression and function of PD-1 on Cord Blood derived NKs (CB-NKs) on a large cohort of newborns through multiparametric cytofluorimetric analysis. Results: We identified PD-1 on CB-NKs in more than of half the newborns analyzed. PD-1 was present on CD56dim NKs, and particularly abundant on CD56neg NKs, but only rarely present on CD56bright NKs. Importantly, unlike in adult healthy donors, in CB-NKs PD-1 is co-expressed not only with KIR, but also with NKG2A. PD-1 expression was independent of HCMV mother seropositivity and occurs in the absence of HCMV infection/reactivation during pregnancy. Notably, PD-1 expressed on CB-NKs was functional and mediated negative signals when triggered. Conclusion: To our understanding, this study is the first to report PD-1 expression on CB derived NKs and its features in perinatal conditions. These data may prove important in selecting the most suitable CB derived NK cell population for the development of different immunotherapeutic treatments.

Characterizing Features of Human Circulating B Cells Carrying CLL-Like Stereotyped Immunoglobulin Rearrangements.

Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5+ B cells, nor in specific B-cell subpopulations or the CD5+ cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin.

sHLA-I Contamination, a novel mechanism to explain ex vivo/in vitro modulation of IL-10 synthesis and release in CD8(+) T lymphocytes and in neutrophils following intravenous immunoglobulin infusion.

BACKGROUND: Numerous mechanisms have been proposed to explain the beneficial action of intravenous immune globulin (IVIG) in autoimmune and systemic inflammatory disorders; among others, they could decrease pro-inflammatory cytokine levels and also induce anti-inflammatory cytokines. MATERIALS AND METHODS: Ex vivo analysis of cells from ten IVIG recipients showed significant increase of IL-10 mRNA and intra-cellular IL-10 molecules in both leukotypes. RESULTS: In vitro comparable results were obtained incubating CD8(+) T lymphocytes and neutrophils from healthy donors with IVIG. sHLA-I and/or sFasL immunodepletion abolished IL-10 modulation. Co-culture with contaminant-free IgM or MabThera did not exert any mRNA modulation. Finally, IgM or MabThera plus purified sHLA-I molecules enhanced IL-10-mRNA in both leukotypes to levels comparable to those obtained with IVIG incubation. CONCLUSION: As IVIG infusion involves administration of soluble contaminants, these data consent to speculate that IVIG might modulate IL-10 via the immunomodulatory activities of sHLA-I contaminant molecules inducing transcriptional and post-transcriptional modulation of IL-10 in CD8(+) T lymphocytes and neutrophils.

sHLA-I contaminating molecules as novel mechanism of ex vivo/in vitro transcriptional and posttranscriptional modulation of transforming growth factor-beta in CD8+ T lymphocytes and neutrophils after intravenous immunoglobulin treatment.

BACKGROUND: Numerous mechanisms have been proposed to explain the beneficial action of intravenous immune globulin (IVIG) in autoimmune and systemic inflammatory disorders. Among others' data, an in vitro increase of intracellular TGF-beta expression when culturing CD4+ T lymphocytes in the presence of IVIG has been reported. As IVIG infusion involves administration of soluble contaminants likewise all hemoderivative preparations, we hypothesized that, besides several other immunomodulatory proposed mechanisms, the clinical effects of IVIG therapy might be, at least partly, due to contaminating soluble HLA Class I (sHLA-I) molecules capable to exert pleiotropic immunomodulatory effects among which TGF-beta(1) modulation. STUDY DESIGN AND METHODS: Ex vivo and in vitro transcriptional and posttranscriptional modulation of TGF-beta(1) in CD8+ T lymphocytes and neutrophils after IVIG infusion was analyzed. RESULTS: Ex vivo analysis of cells drawn from 10 enrolled IVIG recipients pointed out a significant increase of TGF-beta(1) mRNA and intracellular TGF-beta(1) molecules in both leukotypes. In vitro comparable results were obtained incubating CD8+ T lymphocytes and neutrophils from healthy donors with IVIG. The immunodepletion of sHLA-I and/or soluble Fas ligand (sFasL) abolished TGF-beta(1) modulation in both leukotypes. Coculture with human immunoglobulin (Ig)M monoclonal antibody or chimeric IgG (MabThera, Roche), whose manufacturing excludes "contamination," did not exert any mRNA modulation. Finally, IgM or MabThera plus purified sHLA-I molecules enhanced TGF-beta(1) mRNA in both white blood cells to levels comparable to those obtained with IVIG incubation. CONCLUSION: On the whole, these data lead us to speculate that the ability of IVIG administration to modulate TGF-beta(1) might be related to the immunomodulatory activities of sHLA-I and sFasL molecules on activated CD8+ T lymphocytes and neutrophils.

"Normal values" for the lymphocyte cytokinesis-block micronucleus cytome parameters: Repeatability and reproducibility in a healthy reference population.

The micronucleus test in peripheral blood lymphocytes is the most widely validated technique to evaluate the DNA damage and chromosomal instability in human populations. The test is largely applied in monitoring environmental and occupational exposure to genotoxic agents. It was also proposed as a biomarker of risk/susceptibility for cancer and other degenerative diseases. The availability of "normal values" in healthy populations is a main requisite for the assay application in human biomonitoring. Age and gender-related ranges of micronucleated binucleated cells (MNBN) baseline values were established in a group of 103 healthy platelet donors (50 males and 53 females) not recently exposed to genotoxic agents and characterized for demographic, lifestyle and dietary factors. Repeatability of the test by the same scorer was evaluated. Reproducibility was estimated through analysis of repeated blood samples. High correlation between the results of the three blood samplings in two separate scoring sessions of MNBN/1000BN (R2 values were 0.83, 0.74 and 0.68; P < 0.0001) and PI values (R2 values were 0.69, 0.62 and 0.65; P < 0.0001) was detected. High consistency among the values obtained in three different samplings in the same individual was observed (Intraclass Correlation Coefficient (ICC) = 0.905, (95% CI = 0.868-0.933, P < 0.0001) The range of "normal" values predicted on the basis of the results of the present study appears to be sufficiently narrow to warrant application of the assay in the comparison of data obtained from groups of exposed or susceptible subjects, supporting its use in preventive programs. The large inter-individual variability predicted by the model used in the present study hampers a clinical application of the assay at individual level. The method applied in the present study represents a generally applicable model to derive "normal values" in any population, as an essential step before starting a biomonitoring study.

Blood transfusions with high levels of contaminating soluble HLA-I correlate with levels of soluble CD8 in recipients' plasma; a new control factor in soluble HLA-I-mediated transfusion-modulated immunomodulation?

BACKGROUND: The cause of transfusion-related immunomodulation (TRIM) has proved tantalisingly elusive. An ever-growing body of evidence indicates that the infusion of large amounts of soluble and cell-associated antigens into a recipient can somehow induce TRIM. One soluble molecule that has been implicated in TRIM is soluble human leucocyte antigen I (sHLA-I). However, patients infused with large amounts of sHLA-I do not always and unambiguously experience TRIM. As soluble CD8 (sCD8) molecules have been shown to capable of binding membrane and soluble HLA-I molecules, we focused on sCD8 as a possible modulator of sHLA-I-mediated TRIM. MATERIAL AND METHODS: To this aim we compared the up-regulation of circulating sCD8 in plasma from patients suffering from the same pathology, but chronically transfused with two different blood derivatives: pre- and post-storage leucodepleted red blood cells which contain low and high levels of contaminating sHLA-I, respectively. RESULTS: Significantly larger amounts of sCD8 circulating molecules were detectable in the plasma of patients transfused with post-storage leucodepleted red blood cells whose supernatants contained significantly larger amounts of sHLA-I contaminating molecules. CONCLUSION: With the limitation of indirect evidence, this report introduces a new facet of the bioactivity of sCD8 as a possible modulator of sHLA-I-mediated TRIM.

Bio-modulators in platelet-rich plasma: a comparison of the amounts in products from healthy donors and patients produced with three different techniques.

BACKGROUND: Platelet-rich plasma consists of platelets concentrated in a small volume of plasma and constitutes a reservoir of bio-modulators potentially useful in tissue repair. The amounts of bio-modulators detectable in platelet-rich plasma prepared with various commercial or "in house" methods have been reported, but virtually all the analyses described have been performed on platelet-rich plasma derived from healthy donors. Since leucocyte contamination is technically unavoidable, we investigated whether platelet-rich plasma prepared from patients could contain different amounts of bio-modulators because of a possible activated status of the leucocytes. MATERIALS AND METHODS: We evaluated platelet-rich plasma prepared with three different techniques (the commercial Vivostat and Biomet recover GPS II systems and an "in house" method) starting from whole blood from healthy donors and patients. Specifically, we compared the levels of sHLA-I, sFasL, platelet-derived growth factor, transforming growth factors-beta and vascular endothelial growth factor in the platelet-rich plasma releasates according to the method of preparation and to the immune system activation status of the subjects. RESULTS: With the exception of sHLA-I levels, no differences were found in the surrogate indices of lymphocyte activation between healthy donors and patients. No significant differences were found in sHLA-I, sFasL, platelet-derived growth factor, transforming growth factors-beta and vascular endothelial growth factor levels detectable in platelet-rich plasma produced with the three different methods in either healthy donors or patients. DISCUSSION: On the whole our findings indicate that the overall content of bio-modulators in autologous platelet-rich plasma is not influenced by T-lymphocyte activation status, at least in patients with uncomplicated femoral fractures. The amounts of sFasL and sHLA-I detected in all the platelet-rich plasma releasates studied were very small, far below the amounts detectable in all clinically available blood derivatives and absolutely insufficient to induce sHLA-I and/or sFasL mediated immunomodulation.

Contact with the bone marrow microenvironment readdresses the fate of transplanted hematopoietic stem cells.

OBJECTIVE: Despite enormous advancements in our comprehension of molecular mechanisms governing hematopoietic stem cells (HSCs) engraftment in the bone marrow, current clinical protocols of intravenous (IV) transplantation suffer from a relatively low seeding efficiency. To solve this problem, intrabone (IB) injection of HSCs has been proposed. However, the mechanisms underlying the benefit provided by this procedure remain unknown. This study aims to evaluate the effect of IB on HSCs trafficking and homing features in the living rat. MATERIALS AND METHODS: A total of 35 Lewis rats underwent IB or IV administration of HSCs harvested from syngeneic animals and purified according to CD90 expression. These cells were labeled with 37 MBq 99mTc-exametazime and injected either IV or IB. Cell trafficking and distribution in heart, lung, spleen, liver, and forelimb was evaluated by dynamic radionuclide imaging. Logan graphical approach was used to estimate tissue recruitment of HSCs. RESULTS: More than 90% of cells escaped from the injected bone to the bloodstream in <15 seconds. However, this short contact profoundly modified HSCs kinetics, reducing their lung sequestration and shortening their blood persistence with respect to IV. More importantly, IB passage resulted in reduced lung uptake and in a fourfold increase in homing of remote bone marrow sites. CD90(+) cells transplantation restored hematopoiesis in eight further rats previously exposed to lethal irradiation. CONCLUSION: The first-entry contact with the hematopoietic microenvironment immediately readdresses the fate of transplanted HSCs, providing them with "the final destination stamp" to define their bone marrow homing.

Immunomodulatory effects of blood transfusions: the synergic role of soluble HLA Class I free heavy-chain molecules detectable in blood components.

BACKGROUND: Over the past decades, the weight of the published literature demonstrates that blood transfusions can induce clinically significant immunosuppression in recipients. Several studies showed significant improved clinical outcomes in the patients receiving leukoreduced transfusions, compared with control patients who received nonleukoreduced transfusions. Moreover, the immunosuppressive potential of blood products grows with the time of their storage and becomes highest in nonleukoreduced blood products stored for a long time. STUDY DESIGN AND METHODS: The interest was previously focused on the determination of immunomodulatory soluble molecules such as soluble HLA Class I (sHLA-I) and soluble Fas ligand (sFasL) in different blood components and on the evaluation of their immunomodulatory activities. On this basis, whether soluble beta2-microglobulin free HLA Class I heavy chains (sHLA-beta2fHC) could be detected and immunochemically characterized in different blood components was evaluated. Immunomodulatory activity of detectable sHLA-beta2fHC molecules was evaluated by apoptosis inducing capacity in interleukin-2-activated antigen-specific cytotoxic T lymphocytes (CTL). RESULTS: Double-determinant immunoenzymatic assay indicates that sHLA-beta2fHC levels in red blood cells stored for up to 30 days and in random-donor platelets are significantly (p < 0.001) higher than in other blood components, and the immunochemical characterization suggests that the major source of sHLA-beta2fHC molecules might be the residual white cells that undergo membrane damage during storage. Finally, allogeneic CD8+ CTL apoptosis induction confirmed biofunctionality of sHLA-beta2fHC molecules. CONCLUSION: These data are comparable with those previously reported dealing with contaminant soluble molecules in allogeneic and autologous blood components, suggesting that sHLA-beta2fHC molecules could contribute to the immunosuppressive effects of blood transfusions.

Fas-mediated T-cell apoptosis is impaired in patients with chronic inflammatory demyelinating polyneuropathy.

The Fas death receptor is expressed by activated lymphocytes and is involved in switching-off the immune response. Its inherited defects cause auto-immune lymphoproliferative syndrome. Impaired Fas function may also play a role in other auto-immune diseases, such as multiple sclerosis and type 1 diabetes mellitus. The aim of this work was to evaluate Fas function in T cells from patients with chronic inflammatory demyelinating polyneuropathy (CIDP). We evaluated Fas-induced apoptosis in T-cell lines from 27 patients with CIDP, 12 patients with acute inflammatory demyelinating polyneuropathy (AIDP), and 110 controls. CIDP patients displayed lower Fas function than both AIDP patients and controls, whereas no statistically significant difference was found between AIDP patients and controls. Moreover, Fas function was lower in CIDP patients with progressive course than in those with relapsing-remitting course and lower in CIDP patients with axonal damage than in those with pure demyelination. These data suggest that defective Fas function favours CIDP development and aggressive evolution.