Gianluca Ubezio

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.

Granulocytes and monocytes apheresis induces upregulation of TGFβ1 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 TGFβ1 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, TGFβ1 plasma levels, and the percentage of TGFβ1 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 TGFβ1 plasma levels and the percentage of TGFβ1 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.

Transient transforming growth factor β1 modulation in monocytes and natural killer cells following plasma or plasma-platelet apheresis donation procedures.

Dear Sir, Our group has long been concerned with the immunomodulatory role of soluble HLA class I molecules (sHLA-I) in all clinically available blood products and derivatives. Recently, we have shown that whole and/or re-folded sHLA-I molecules bind to apheresis circuit surfaces fabricated from synthetic polymers during therapeutic and donation centrifugation-based apheresis procedures1–3. CD8+ T lymphocytes and neutrophils, rolling into the circuits during the procedure, bind sHLA-I molecules adsorbed to the circuit’s polymers and thereby become sensitive to the biological effects of sHLA-I, such as transcriptional/post-transcriptional modulation of transforming growth factor β1 (TGFβ1)1–3. As monocytes and natural killer (NK) cells can reportedly bind sHLA-I molecules with immunoglobulin-like transcripts (ILTs) and CD8 membrane molecules, respectively4,5, these leucocytes, too, may be exposed to similar immunosuppressant activities during apheresis procedures. Integrating the previous studies focused on sHLA-I mediated immunomodulation following plasma-platelet apheresis donation procedures1,3, the present study concentrated on the effects on donors’ NK cells and monocytes. To this aim events leucocytes from 20 donors (16 male) were analysed before, immediately after, and 7 and 14 days following three closely timed plasma and platelet donation procedures. Moreover, to exclude that a possible transcriptional/post-transcriptional modulation of TGFβ1 could be based on technical differences between the cell separators and their related methods of collection, in this study we compared different plasma and/or platelet donation procedures and Haemonetics PCS2, model 6002, Haemonetics MCS Plus version C (Haemonetics Corp., Baintree, MA, USA) and Trima Accel version 4.0 separators (Gambro BTC, Zaventem, Belgium)1,3. Apheresis donor procedures were performed in accordance with the Italian law, in healthy donors fulfilling the criteria laid down by the Italian blood donation guidelines and SIMTI recommendations for apheresis donation. Informed consent was obtained from all the donors. Samples to perform a complete pre-procedure laboratory profile were obtained from each donor. In addition to biochemical and serological tests required by law, C-reactive protein, erythrocyte sedimentation rate, protein electrophoresis, fibrinogen and ferritin were also evaluated in order to further exclude any sub-clinical inflammatory disorder possibly causing leucocyte activation. Follow-up assessments were performed every 6 months up to 2 years, by physical examination, history and the same biochemical/serological tests done during the enrolment, as previously described1,3. Monoclonal antibodies and other reagents for research use were purchased from several renowned companies. Flow cytometer assays for CD14, CD56 and TGFβ1 were performed with standard methods. Immunofluorescence tests of the return line of the apheretic circuits were performed as described1. A matched pair Wilcoxon’s sign rank test was used to perform the comparisons between results at different times. Results of sHLA-I staining in apheresis circuits revealed a homogeneous layer consisting of sHLA-I molecules binding to the plastic surfaces during the extracorporeal blood/circuit interaction. Regardless of the type of donation procedure, there was a significant up-regulation of intracytoplasmic TGFβ1 molecules, consistently observed in both monocytes and NK cells from healthy subjects. Specifically, flow-cytometer analysis revealed a clear but not significant up-regulation of TGFβ1 molecules 7 days after each plasma-platelet apheresis donation procedure. An evident reversion to baseline levels was detectable 14 days after each procedure. Matching the above data, the real-time polymerase chain reaction analyses in monocytes showed an analogous increase of TGFβ1-mRNA 7 days after all the three procedures, followed by a likewise evident and significant decrease 14 days following apheresis. On the whole, these findings indicate that sHLA-I molecules are adsorbed onto plastic apheresis circuits during donation procedures as well, and interact there with immunoglobulin-like transcripts and CD8 surface receptors of donors’ monocytes and NK cells (Figure 1). Nevertheless, unlike what was reported for therapeutic apheresis2, a very ephemeral transcriptional/post-transcriptional TGFβ1 modulation was detectable both in monocytes and NK cells following plasma and platelet donation procedures. Figure 1 Pre/post-transcriptional modulation of TGFβ1 molecule. Variations in the percentages of intracytoplasmic TGFβ1 staining in monocytes and NK cells analysed by flow-cytometry are shown. In our opinion, a difference in leucocyte activation status is a possible explanation for our findings: therapeutic apheresis is performed in patients suffering from immune-mediated diseases, in which immunocompetent cells circulate mostly in a persistently activated state and are, therefore, much more prone to a possible protracted sHLA-I-mediated immunomodulation. Conversely, donor apheresis is performed in healthy subjects whose leucocytes mostly circulate in a not activated state, and only after interaction with the plastic circuitry of the apheresis equipment during the procedure can transiently become sHLA-I-sensitive. In accordance with such short-lived immunomodulation, none of the subjects enrolled experienced any adverse reaction during the procedures or showed any possible immunosuppressive effect on viral, bacterial or neoplastic disease during 24 months of monitoring following the donations. Lastly, in the donors studied, NK cells and monocytes showed minor, but not statistically significant changes compared to basal conditions following each type of apheresis procedure (data not shown). On the whole, our findings indicate that durable sHLA-I-mediated immunomodulation does not occur in healthy donors undergoing plasma-platelet apheresis donation, in contrast to what has been reported for therapeutic procedures. This fact might corroborate a conceivable “immunological” harmlessness of donor apheresis following a correct assessment of the donors’ healthy status, which is desirable in order to avoid, among other complications, any degree of sHLA-I-mediated immunomodulation.

Donor neutrophil activation and transforming growth factor-β1 modulation induced by donor apheresis procedures.

Dear Sir, Some concerns have been raised regarding possible immunosuppressive features in plasmapheresis donors, including alterations of neutrophil function1. On the whole, no report has given clear evidence of immunosuppression at a clinical level and in general no long-term harmful effects have been observed in donors who have undergone cytapheresis2. Our group has long been concerned with the immunomodulatory role of soluble human leucocyte antigen class I molecules (sHLA-I) in transfusion medicine3. We recently showed that CD8+ T lymphocytes and neutrophils rolling and floating in the circuit during therapeutic apheresis are susceptible to the bioactivity of sHLA-I molecules bound to the synthetic polymers of the circuit’s surface during procedure4. The circuits for donor apheresis procedures are made from exactly the same biocompatible plastic as that used in circuits for therapeutic apheresis and so it is conceivable that also during donation a boundary layer could form as a result of adsorption of plasma proteins to plastic membranes made of various synthetic polymers. Accordingly, also during plasma and platelet donation procedures, neutrophils could be activated by rolling in the circuits1 and bound to sHLA-I molecules adsorbed to the circuits’ polymers with Ig-like-transcripts, thereafter becoming sensitive to the biological effects of sHLA-I, such as transcriptional/post-transcriptional modulation of transforming growth factor-β1 (TGFβ1)5. Indeed, in contrast to the situation in patients suffering from immune-mediated diseases, in whom immune-competent cells circulate mostly in an enduringly activated status, the leucocytes of healthy donors are only transiently activated by contact with circuits during the donor procedure and should, therefore, show only an ephemeral sensitivity to sHLA-I-mediated immunomodulation. In order to evaluate a possible ephemeral sHLA-I-mediated immunomodulation in productive aphaeresis, neutrophils from 20 donors (16 male) were analysed before, immediately after, and 7 and 14 days following three closely timed plasma and platelet donation procedures. Apheresis donor procedures were performed in accordance with the Italian law, in healthy donors fulfilling the criteria laid down by the Italian blood donation guidelines and SIMTI recommendations for apheresis donation. Informed consent was obtained from all the donors. Samples to perform a complete pre-procedure laboratory assessment were obtained from each donor. In addition to biochemical and serological tests required by law, C-reactive protein, erythrocyte sedimentation rate, protein electrophoresis, fibrinogen and ferritin were also evaluated in order to further exclude any sub-clinical inflammatory disorder possibly causing leucocyte activation. Follow-up assessments were performed every 6 months up to 2 years, by physical examination, history taking and the same biochemical/serological tests performed during the enrolment. Monoclonal antibodies and other reagents for research use were purchased from several renowned companies. Immunofluorescence tests of the return line of the apheretic circuits were performed as described elsewhere4. Flow cytometry assays for CD66b and TGFβ were conducted using standard methods. The expression of CD11b membrane molecules was evaluated as an indicator of neutrophil activation. All data were subjected to appropriate statistical analyses. Results of sHLA-I staining in apheresis circuits revealed a homogeneous layer consisting of sHLA-I molecules binding the plastic vessel surfaces during the extracorporeal blood/circuit interaction. Donor neutrophils were found to be transiently activated by the interaction with plastic circuits during the procedure regardless of the type of donation procedure. More to the point, the comparison between the findings at different times showed a statistically significant increase in the expression of CD11b molecules immediately after the procedures (p<0.001). The above-mentioned activation markers significantly decreased after another 7 days (p<0.001), confirming the transient nature of the leucocyte activation (Figure 1). Once again, regardless of the donation procedure, a significant up-regulation of intracytoplasmic TGFβ1 molecules was consistently observed in neutrophils from healthy subjects. Specifically, flow-cytometry analysis revealed a sharp and significant (p<0.001) up-regulation of TGFβ1 molecules at the end of each apheresis procedure. An evident reversion to baseline levels was detectable 7 days after each procedure. Matching the above data, real-time polymerase chain reaction analyses in donor neutrophils showed a sharp increase of TGFβ1-mRNA immediately after all the three procedures, followed by a similarly evident and significant decrease 7 days after the apheresis (Figure 2). Figure 1 Neutrophil activation. Figure 2 Pre/post-transcriptional modulation of TGFB1 molecule. On the whole these findings indicate that sHLA-I molecules adsorb to plastic apheresis circuits during donation procedures and interact with Ig-like-transcripts surface receptors of donors’ neutrophils. Moreover, neutrophils activated by donation procedures1 become sensitive to the biological effects of sHLA-I, such as transcriptional/post-transcriptional modulation of TGFβ15. Nevertheless, unlike what was reported with regards to therapeutic apheresis, the transcriptional/post-transcriptional TGFβ1 modulation detectable in neutrophils following plasma and platelet donation procedures was very ephemeral. In our opinion, the difference in leucocyte activation status is a possible explanation of our findings: therapeutic apheresis is performed in patients suffering from immune-mediated diseases, in whom immune-competent cells circulate mostly in an enduringly activated status4 and are, therefore, much more prone to a possible protracted sHLA-I-mediated immunomodulation. Conversely, donor apheresis is performed in healthy subjects whose leucocytes mostly circulate in a not activated status, and only after interaction with the plastic circuits of the apheresis equipment during the procedure do they become transiently sensitive to sHLA-I. In accordance with such short-lived immunomodulation, no subject enrolled experienced any adverse reaction during the procedures or showed any possible immunosuppression or viral, bacterial or neoplastic disease during 24 months of monitoring following the donations. Finally, at every time-point of the study, the relative proportions and absolute numbers of the neutrophils were evaluated by flow cytometry. These parameters showed no modifications with respect to basal conditions in all the donors examined and in each type of apheresis procedure (data not shown). Overall, our findings might contribute to confirm the conceivable “immunological” harmlessness of donor apheresis, highlighting the importance of a correct assessment of the donors’ healthy status in order to avoid, among other complications, any kind of prolonged sHLA-I-mediated immunomodulation following platelet or plasma donor apheresis.

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.

Transient inhibition of neutrophil migration following plasma or plasma-platelet apheresis donation procedures.

Dear Sir, Our group has long been interested in the immunomodulatory role of soluble HLA class I molecules (sHLA-I) in all clinically available blood products and derivatives and recently we evaluated possible immunomodulatory effects following plasma-platelet apheresis donation procedures. We found that, following centrifugation-based apheresis, whole and/or re-folded sHLA-I bind to the apheresis circuit surfaces fabricated from synthetic polymers during therapeutic and donation procedures1–3. Monocytes and neutrophils, or CD8+ T lymphocytes and natural killer cells could bind sHLA-I molecules with immunoglobulin-like transcripts and CD8 membrane molecules, respectively. Accordingly, we showed that all these leucocytes, rolling into the circuits during the procedure, could bind sHLA-I molecules adsorbed to the circuits’ polymers and thus become sensitive to the biological effects of sHLA-I, such as transcriptional/post-transcriptional modulation transforming growth factor-beta 1 (TGFβ1)1–3. Finally, we have previously demonstrated that high dilutions of supernatants from stored red blood cells inhibit neutrophil migration. Such inhibitory activity was demonstrated to be due to the TGFβ1 contained in the supernatants, which is capable of desensitizing neutrophils to chemotactic stimulation4. On this basis, it could be hypothesised that TGFβ1 modulation induced by plasma-platelet apheresis donation procedures may also inhibit neutrophil migration. To determine whether this is the case, samples of plasma were collected from three male donors before, and immediately, 7 and 14 days after the plasma and platelet donation procedure. Moreover, to highlight a potential unique mechanism in neutrophil locomotion inhibition following apheresis donation, we compared different plasma and/or platelet donation procedures and separators (Haemonetics PCS2, model 6002, Haemonetics MCS Plus version C [Haemonetics Corp., Baintree, MA, USA] and Trima Accel version 4.0 [Gambro BTC, Zaventem, Belgium])1,3. Apheresis donor procedures were performed in accordance with the Italian law, in healthy donors fulfilling the criteria laid down by the Italian blood donation guidelines and Italian Society of Transfusion Medicine and Immunohaematology (SIMTI) recommendations for apheresis donation. Informed consent was obtained from all the donors. Samples were obtained from each donor to perform a complete pre-procedure laboratory profile. In addition to biochemical and serological tests required by law, C-reactive protein, erythrocyte sedimentation rate, protein electrophoresis, fibrinogen and ferritin were also evaluated in order to further exclude any sub-clinical inflammatory disorder possibly causing leucocyte activation. Follow-up assessments were performed every 6 months up to 2 years, by physical examination, anamnestic evaluation and the same biochemical/serological tests done during the enrolment, as previously described1,3. Monoclonal antibodies and other reagents for research use were purchased from several renowned companies. The modified Boyden chamber migration assay was conducted using human neutrophils from different healthy volunteers isolated from heparinised venous blood and the cells were pre-incubated with chosen dilutions of plasma from donors (plasma dilution range from 1:1×104 to 1:1×105, data not shown). Cells were washed and neutrophil migration toward control medium or human recombinant CXCL8 was assessed in a 48-well micro-chemotaxis chamber as previously described4,5. Data are expressed as net migration. The previous studies focused on sHLA-I-mediated immunomodulation following plasma-platelet apheresis donation procedures1,3, regardless of the type of donation procedure. The comparison between the different timings showed a noteworthy inhibition of neutrophil locomotion induced by plasma pre-treatment (also in comparison with matched non-pretreated controls, data not shown) which was consistently observed immediately the after plasma-platelet apheresis donation procedure. An evident and stable reversion to baseline levels was detectable 7 and 14 days after the procedure (Figure 1). Figure 1 Immediately after plasma-platelet apheresis, the locomotion of neutrophils from healthy subjects was constantly induced by pre-treatment donor’s plasma regardless of the type of donation procedure. An evident and stable reversion to baseline levels ... In accordance with such short-lived immunomodulation, no enrolled subject experienced any adverse reaction during the procedures or showed any possible immunosuppression or viral, bacterial or neoplastic disease during 24 months of monitoring following the donations. On the whole, our findings indicate that durable inhibition of neutrophil locomotion does not occur in healthy donors undergoing plasma-platelet apheresis donation. Completing previous studies focused on sHLA-I mediated immunomodulation following plasma-platelet apheresis donation procedures, this fact might further corroborate a conceivable “immunological” harmlessness of such procedures.