Processing methods and storage duration impact extracellular vesicle counts in red blood cell units.

Extracellular vesicles (EVs) are active components of red blood cell (RBC) concentrates and may be associated with beneficial and adverse effects of transfusion. Elucidating controllable factors associated with EV release in RBC products is thus important to better manage the quality and properties of RBC units. Erythrocyte-derived EVs (EEVs) and platelet-derived EVs (PEVs) were counted in 1226 RBC units (administered to 280 patients) using a standardized cytometry-based method. EV size and CD47 and annexin V expression were also measured. The effects of donor characteristics, processing methods, and storage duration on EV counts were analyzed by using standard comparison tests, and analysis of covariance was used to determine factors independently associated with EV counts. PEV as well as EEV counts were higher in whole-blood-filtered RBC units compared with RBC-filtered units; PEV counts were associated with filter type (higher with filters associated with higher residual platelets), and CD47 expression was higher on EEVs in RBC units stored longer. Multivariate analysis showed that EEV counts were strongly associated with filter type (P < .0001), preparation, and storage time (+25.4 EEV/µL per day [P = .01] and +42.4 EEV/µL per day [P < .0001], respectively). The only independent factor associated with PEV counts was the residual platelet count in the unit (+67.1 PEV/µL; P < .0001). Overall, processing methods have an impact on EV counts and characteristics, leading to large variations in EV quantities transfused into patients. RBC unit processing methods might be standardized to control the EV content of RBC units if any impacts on patient outcomes can be confirmed. The IMIB (Impact of Microparticles in Blood) study is ancillary to the French ABLE (Age of Transfused Blood in Critically Ill Adults) trial (ISRCTN44878718).

How to quantify microparticles in RBCs? A validated flow cytometry method allows the detection of an increase in microparticles during storage.

BACKGROUND: The procoagulant and proinflammatory microparticles (MPs) released during storage of packed red blood cells (pRBCs) can potentially modify transfusion benefits. A robust method to quantify MPs in pRBCs is needed to evaluate their impact in clinical trials. STUDY DESIGN AND METHODS: The objective was to validate the preanalytic conditions required to prepare pRBC supernatant as well as a method to quantify and evaluate MP variations over 42 days of pRBC storage.A flow cytometry method with size-calibrated beads was developed and fully validated. Quantification of MPs in pRBCs (n = 109) was assessed during short-term (7 days) and long-term (42 days) storage at 4°C, during short-term storage (8 hours) at room temperature, and after 2 years frozen. RESULTS: Repeatability, reproducibility, and linearity of the quantification method were validated, and variations during conservation are presented. There was high variability in RBC (erythrocyte) MP (ERMP) and platelet MP (PMP) levels between RBC units, depending on the filter used for leukocyte reduction. During the 42 days of storage at 4°C, significant increases in ERMPs and PMPs occurred (from 58 to 138 ERMPs/µL from Day 2 to Day 42; p = 0.0002; and from 326 to 771 PMPs/µL from Day 2 to Day 42; p = 0.00026). CONCLUSION: We use a robust method to confirm that ERMPs and PMPs are present to various degrees in pRBCs and that storage for 42 days significantly increases their generation. This method is robust enough to allow MP quantification in pRBCs and is adapted to evaluate the clinical impact of transfused MPs in prospective clinical trials.

Rapalogs Efficacy Relies on the Modulation of Antitumor T-cell Immunity.

The rapalogs everolimus and temsirolimus that inhibit mTOR signaling are used as antiproliferative drugs in several cancers. Here we investigated the influence of rapalogs-mediated immune modulation on their antitumor efficacy. Studies in metastatic renal cell carcinoma patients showed that everolimus promoted high expansion of FoxP3 (+)Helios(+)Ki67(+) regulatory CD4 T cells (Tregs). In these patients, rapalogs strongly enhanced the suppressive functions of Tregs, mainly in a contact-dependent manner. Paradoxically, a concurrent activation of spontaneous tumor-specific Th1 immunity also occurred. Furthermore, a high rate of Eomes(+)CD8(+) T cells was detected in patients after a long-term mTOR inhibition. We found that early changes in the Tregs/antitumor Th1 balance can differentially shape the treatment efficacy. Patients presenting a shift toward decreased Tregs levels and high expansion of antitumor Th1 cells showed better clinical responses. Studies conducted in tumor-bearing mice confirmed the deleterious effect of rapalogs-induced Tregs via a mechanism involving the inhibition of antitumor T-cell immunity. Consequently, the combination of temsirolimus plus CCR4 antagonist, a receptor highly expressed on rapalogs-exposed Tregs, was more effective than monotherapy. Altogether, our results describe for the first time a dual impact of host adaptive antitumor T-cell immunity on the clinical effectiveness of rapalogs and prompt their association with immunotherapies. Cancer Res; 76(14); 4100-12. ©2016 AACR.

CD20 alternative splicing isoform generates immunogenic CD4 helper T epitopes.

Cancer-specific splice variants gain significant interest as they generate neo-antigens that could be targeted by immune cells. CD20, a membrane antigen broadly expressed in mature B cells and in B cell lymphomas, is subject to an alternative splicing named D393-CD20 leading to loss of membrane expression of the spliced isoform. D393-CD20 expression is detectable in transformed B cells and upregulated in various lymphoma B cells. In this study, we show that D393-CD20 is translated in malignant B cells and that D393-CD20 specific CD4 T cells producing IFN-γ are present in B-cell lymphoma patients. Then, we have investigated whether the 20mer D393-CD20 peptide spanning the splicing site might be targeted by the immune system and we have shown that D393-CD20-specific CD4 Th1 clones could directly recognize malignant B cell lines and kill autologous lymphoma B cells indicating that D393-CD20-derived epitopes are naturally processed and presented on tumor cells. Finally, D393-CD20 peptide-based vaccination induced specific CD8 and CD4 T cell responses in HLA-humanized transgenic mice suggesting the presentation of D393-CD20 derived peptides on both HLA Class-I and -II. These findings support further investigations on the potential use of D393-CD20 directed specific immunotherapy in B cell malignancies.

New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.

BACKGROUND: CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors. RESULTS: Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL. CONCLUSION: The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies.

Erratum to: New CD20 alternative splice variants: molecular identification and differential expression within hematological B cell malignancies.

[This corrects the article DOI: 10.1186/s40164-016-0036-3.].

Lack of expression of an alternative CD20 transcript variant in circulating B cells from patients with pemphigus.

We have identified a spliced mRNA transcript of CD20 (named D393-CD20) which was associated with resistance to RTX in primary B cell from patients with lymphoma and leukaemia. In the present work, we wished to investigate whether D393-CD20 variant was expressed by B cells from patients with pemphigus. Ten patients with bullous pemphigoid and twenty-five patients with pemphigus were included. All patients were responder to conventional immunosuppressive agents or rituximab (n = 11). Efficacy of B-cell activation by pokeweed mitogen was assessed by CD86 expression using a FACS Canto II flow cytometer. mRNA CD20 expression study was then performed using RT-PCR assay allowing first to discriminate wild-type (wt)-CD20 and D393-CD20 transcript. Although wt-CD20 expression was always detected, we were unable to detect D393-CD20, even after B-cell activation or RTX treatment. Our results suggest that D393-CD20 transcript may be a molecular marker of B-cell malignancies rather than autoimmune disease like pemphigus. Further study of RTX non-responders or non-escaping PV patients is thus still required to appreciate whether D393-CD20 expression may be detected under the pressure of RTX therapy.