ALBERT

Description: Transfusion or transplantation of T lymphocytes into an allogeneic recipient can evoke potent immune responses including, in immunocompromised patients, graft-versus-host disease (GVHD). As our previous studies demonstrated attenuated immunorecognition of red blood cells covalently modified with methoxy(polyethylene glycol) (mPEG), we hypothesized that T-cell activation by foreign antigens might similarly be prevented by mPEG modification. Mixed lymphocyte reactions (MLR) using peripheral blood mononuclear cells (PBMC) from HLA class II disparate donors demonstrate that mPEG modification of PBMC effectively inhibits T-cell proliferation (measured by 3H-thymidine incorporation) in a dose-dependent manner. Even slight derivatization (0.4 mmol/L mPEG per 4 × 106 cells) resulted in a ≥75% decrease, while higher concentrations caused ≥96% decrease in proliferation. Loss of PBMC proliferation was not due to either mPEG-induced cytotoxicity, as viability was normal, or cellular anergy, as phytohemagglutinin (PHA)-stimulated mPEG-PBMC demonstrated normal proliferative responses. Addition of exogenous interleukin (IL)-2 also had no proliferative effect, suggesting that the mPEG-modified T cells were not antigen primed. Flow cytometric analysis demonstrates that mPEG-modification dramatically decreases antibody recognition of multiple molecules involved in essential cell:cell interactions, including both T-cell molecules (CD2, CD3, CD4, CD8, CD28, CD11a, CD62L) and antigen-presenting cell (APC) molecules (CD80, CD58, CD62L) likely preventing the initial adhesion and costimulatory events necessary for immune recognition and response.

Description: We previously showed that the covalent modification of the red blood cell (RBC) surface with methoxypoly(ethylene glycol) [mPEG; MW ∼5 kD] could significantly attenuate the immunologic recognition of surface antigens. However, to make these antigenically silent RBC a clinically viable option, the mPEG-modified RBC must maintain normal cellular structure and functions. To this end, mPEG-derivatization was found to have no significant detrimental effects on RBC structure or function at concentrations that effectively blocked antigenic recognition of a variety of RBC antigens. Importantly, RBC lysis, morphology, and hemoglobin oxidation state were unaffected by mPEG-modification. Furthermore, as shown by functional studies of Band 3, a major site of modification, PEG-binding does not affect protein function, as evidenced by normal SO4− flux. Similarly, Na+ and K+ homeostasis were unaffected. The functional aspects of the mPEG-modified RBC were also maintained, as evidenced by normal oxygen binding and cellular deformability. Perhaps most importantly, mPEG-derivatized mouse RBC showed normal in vivo survival (∼50 days) with no sensitization after repeated transfusions. These data further support the hypothesis that the covalent attachment of nonimmunogenic materials (eg, mPEG) to intact RBC may have significant application in transfusion medicine, especially for the chronically transfused and/or allosensitized patient.

Description: Transfusion or transplantation of T lymphocytes into an allogeneic recipient can evoke potent immune responses including, in immunocompromised patients, graft-versus-host disease (GVHD). As our previous studies demonstrated attenuated immunorecognition of red blood cells covalently modified with methoxy(polyethylene glycol) (mPEG), we hypothesized that T-cell activation by foreign antigens might similarly be prevented by mPEG modification. Mixed lymphocyte reactions (MLR) using peripheral blood mononuclear cells (PBMC) from HLA class II disparate donors demonstrate that mPEG modification of PBMC effectively inhibits T-cell proliferation (measured by 3H-thymidine incorporation) in a dose-dependent manner. Even slight derivatization (0.4 mmol/L mPEG per 4 × 106 cells) resulted in a ≥75% decrease, while higher concentrations caused ≥96% decrease in proliferation. Loss of PBMC proliferation was not due to either mPEG-induced cytotoxicity, as viability was normal, or cellular anergy, as phytohemagglutinin (PHA)-stimulated mPEG-PBMC demonstrated normal proliferative responses. Addition of exogenous interleukin (IL)-2 also had no proliferative effect, suggesting that the mPEG-modified T cells were not antigen primed. Flow cytometric analysis demonstrates that mPEG-modification dramatically decreases antibody recognition of multiple molecules involved in essential cell:cell interactions, including both T-cell molecules (CD2, CD3, CD4, CD8, CD28, CD11a, CD62L) and antigen-presenting cell (APC) molecules (CD80, CD58, CD62L) likely preventing the initial adhesion and costimulatory events necessary for immune recognition and response.

Description: BACKGROUND: The Fresenius COM.TEC is a newer automated leukapheresis device with a software feature that predicts the CD34+ cell yield in peripheral blood progenitor cell (PBPC) collections. There is little published information comparing the collection efficacy of PBPC on the Fresenius COM.TEC with other apheresis machines, including the COBE Spectra. AIM: To compare the collection and cell processing parameters of PBPC collected on the Fresenius COM.TEC with those collected on the COBE Spectra. METHODS: A prospective, single institution study of patients undergoing PBPC collection following mobilisation with chemotherapy and G-CSF was performed. Patients were alternately assigned to have the 1st day of PBPC collection performed on either the COBE Spectra (Version 6.0, MNC) or the Fresenius COM.TEC (Version 4.02, Auto MNC), dependent upon the availability of trained operators for the Fresenius COM.TEC. Patients were scheduled to have 3 blood volumes processed during leukapheresis. RESULTS: Overall, 28 patients had PBPC collected on the COBE Spectra and 20 on the Fresenius COM.TEC. All collections except one were commenced when the peripheral blood CD34+ cell count was ≥10 × 106/L. Two patients on the COBE Spectra and 6 patients on the Fresenius COM.TEC had collections terminated after processing of only 2 blood volumes due to operational requirements. The median volume of PBPC collected on the Fresenius COM.TEC (328mL) was significantly higher than that collected on the COBE Spectra (257mL; p=0.015). This impacted upon the requirement for plasma reduction within the processing laboratory. There was no significant difference in the median number of total nucleated cells, total CD34+ cells or total platelets collected on the 2 machines. Collection efficiency of CD34+ cells was also similar on both machines. Regardless of apheresis machine used, there was a high degree of correlation between predicted and actual yield of total CD34+ cells. However an increased level of red cell contamination in PBPC collected on the Fresenius COM.TEC (median total red cells of 280.2 × 109) compared with the COBE Spectra (median total red cells of 154.3 × 109; p = 0.005) was observed. Also, there was significantly less overall recovery of viable CD34+ cells after laboratory processing of PBPC collected on the Fresenius COM.TEC (median of 73% recovery) compared with the COBE Spectra (median of 89% recovery; p= 0.0004). Of note, technical difficulty was experienced in the processing laboratory with plasma reduction of 7 PBPC products collected on the Fresenius COM.TEC, necessitating deviation from standard operating procedure and re-centrifugation at elevated g force (1000g) to ensure adequate sedimentation of nucleated and CD34+ cells. It is unclear whether the difficultly in plasma reduction in the Fresenius products may have contributed to the reduced recovery of progenitor cells post cryopreservation and thawing. CONCLUSION: Although the Fresenius COM.TEC collects CD34+ cells with similar efficiency to that of the COBE Spectra, PBPC are collected in a larger volume with a higher level of red cell contamination. Furthermore, the recovery of CD34+ cells post cryopreservation and thawing is lower for PBPC collected on the Fresenius COM.TEC. Insufficient patients with collections performed on the COM.TEC have been infused to determine any effect on engraftment kinetics. This study underscores the importance of extending studies evaluating the collection of PBPC to investigate the entire production process including possible post processing effects.