The “in situ” expression of Human Leukocyte Antigen Class I antigens is not altered by cryopreservation in human arterial allografts

Abstract

This study was aimed to establish whether the cryopreservation procedure we currently use in clinics can modify arterial homograft antigenicity. To this purpose, we performed an immunohistochemical study on fresh and cryopreserved human arterial homografts to visualize the expression of HLA class I heavy and light chains "in situ" by using the HC-10 and Namb-1 monoclonal antibodies. Human femoral arteries and thoracic aortas were harvested from 18 heart-beating donors and sampled before and after cryopreservation. Arterial segments were frozen in liquid nitrogen vapors in a controlled rate freezing system. After thawing, samples were processed for routine immunohistochemistry. To standardize immunostaining, flow-cytometry indirect immunofluorescence analysis was performed on HUVEC; immunohistochemistry of human ovarian cortical vessels was performed as an additional positive control. Negative controls were performed by omitting tissue incubation with primary antibodies. HLA-class I antigens were markedly expressed by endothelial cells lining surface intima and adventitial vasa vasorum; a moderate expression was found in medial smooth muscle cells. Except for the surface unreactivity caused by loss of endothelium, results from cryopreserved arterial allografts were strictly comparable to those observed in fresh, unfrozen tissues. These results support the view that cryopreserved arterial allografts are immunogenic as their fresh counterparts; apart from smooth muscle cells which retained a moderate expression of HLA class I antigens following cryopreservation, our study suggests that the highly HC-10 positive endothelial cells we found to line the rich adventitial network of vasa vasorum are expected to be one of the major targets of the serological response in the recipient.

Thoracic Aortas from Multiorgan Donors Are Suitable for Obtaining Resident Angiogenic Mesenchymal Stromal Cells

“…CD34 ϩ vascular channels, which adjacent sections showed to be vWF Ϫ , could also be found embedded within the media. A similar vascular organization composed of cells highly expressing human leukocyte antigen class I antigen [16] was also found in another study and may therefore suggest that distinct patterns of CD34 ϩ cell aggregation exist in elastic and muscular arteries.…”

“…To detect surface antigens, aliquots of cells were stained by MoAbs as previously described [16]. Cells taken at passages 3-5 were washed twice with phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) and stained for 20 minutes at room temperature using the following MoAbs: CD133-phycoerythrin (PE) (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.…”

“…Reverse transcription reactions were performed in a 20-l volume with 2 g of total RNA using M-MLV Reverse Transcriptase (Invitrogen) following the manufacturer's protocol. Oligo(dT) [12][13][14][15][16][17][18] primers (Invitrogen) were used for first strand synthesis (details are provided in the supplemental online material). The expression of the following mRNAs was investigated: OCT4, BMI-1, BCRP-1, Interleukin-6 (IL6), c-kit, CD133, KDR, and ␤2 microglobulin (control).…”

Strong Humoral Anti-HLA Immune Response Upon Arbitrarily Chosen Allogeneic Arterial Vessel Grafts

“…In their later publication dealing with the same cohort of patients, however, the authors referred to HLA-matching of venous allografts as impossible due to their generally limited numbers [26]. The final refusal to the approach of reducing the vessel's antigenicity through cryopreservation was published in 2007 by Pasquinelli et al [27]. The authors convincingly described and reviewed the up-to-that-date existing publications that in follow-up investigations both humoral and cellular immune responses basically arise within 30-60 days after surgery although cryopreservation alters the number and viability of endothelial and smooth muscle cells.…”

“…In this context the authors pointed to the relevance of HLA class I antigens which were clearly expressed in human aortic and femoral arteries after their adequate cryopreservation using e.g. controlled-rate freezing machines [27,28].…”