{"doi":"10.1002/cyto.a.24899","title":"OMIP‐108: 22‐color flow cytometry panel for detection and monitoring of chimerism and immune reconstitution in porcine‐to‐baboon models of operational xenotransplant tolerance studies","abstract":"It is a challenge to build cross-species flow cytometry panels due to cross-reactivity between antibodies of different species and having to use antibodies that have yet to be thoroughly validated and, in some cases, need to be conjugated in-house. Therefore, we developed a 22-color panel for spectral flow cytometry of pig and baboon cells and optimized it as a one-tube assay with thoroughly validated antibodies with minimal to no cross reactivity. Our primary aim was to build a 22-color panel to detect porcine chimerism rigorously and confidently in baboons after bone marrow (BM) or thymus transplantation (TTx) using porcine pan-leukocyte markers, pCD45 and pMHC class I, and to characterize and track major porcine myeloid and lymphoid lineages using pSWC3a (porcine swine workshop cluster 3a–a porcine pan-myeloid marker), pCD3, pCD4, and pCD8. Our secondary aim was to track the immune reconstitution of major lymphoid lineages in the immunodepleted recipient baboons confidently and rigorously using bCD3, bCD4, bCD8, bCD20, bCD25, bCD45RA, bCD56, bCD69, bCD127, and bCCR7. Our final goal was to track the development of bCD4+ and bCD8+ recent thymic emigrants (RTEs) using bCD45RA, bCCR7, bCD28, bCD95, and bCD31 to verify the functional status of the transplanted porcine thymus in completely thymectomized baboons. In summary, the aim of the presented panel is to simultaneously detect porcine chimerism and monitor immune reconstitution in porcine-to-baboon transplantation models of operational xenotransplant tolerance that utilize porcine (Sus scrofa domesticus) bone marrow and thymus transplantation in baboons (Papio anubis, Papio hamadryas) (Table 1). With the advancement of our knowledge in surgical techniques, immunology, and pharmacology, solid organ transplantation has become a widespread standard treatment modality in 21st-century medicine [1]. However, with all these advancements, further progress and improvements in the field of transplantation continue to be limited by the worldwide shortage of suitable organs for transplantation [2-4]. With the latest progress and achievements in developing genetically modified pigs using CRISPR-Cas9 technology, xenotransplantation has emerged as a potential solution to the organ shortage in the clinic [5]. The main challenge in xenotransplantation has always been the powerful cross-species immune response that eventually leads to graft loss within minutes to hours after xenotransplantation [5]. Even though recent progress has been made in the avoidance of hyperacute and acute rejection with the creation of Gal-KO pigs in the early 2000s, subacute and chronic rejection still pose a significant challenge [5]. As such, current studies in the field of xenotransplantation aim to prolong graft survival by mitigating the immunological response after organ transplantation by creating operational tolerance [5]. Clinical “operational tolerance” is defined as stable and acceptable graft function without immunosuppression with an otherwise functioning immune system of the recipient [6]. Our center investigates the development of operational tolerance through thymic transplantation and creating mixed lymphohematopoietic chimerism in porcine-to-porcine, porcine-to-humanized mice, and porcine-to-baboon transplantation models [5, 7, 8]. Accordingly, with the latest developments in flow cytometry and related technologies, we built a 22-color panel using a 5-Laser Cytek Aurora Spectral Flow Cytometer (Cytek, Bethesda, MD) that can be used interchangeably in most current porcine-to-baboon operational xenotransplant tolerance studies that utilize porcine BM and thymus transplantation. It is a challenge to build cross-species flow cytometry panels due to cross-reactivity between antibodies of different species and having to use antibodies that have yet to be thoroughly validated and, in some cases, need to be conjugated in-house. Regarding baboon antibodies, we have found that most commercially available ant","journal":"Cytometry Part A","year":2024,"id":450651,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":4,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9507,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1082346,"name":"Daniel H. Wolbrom","orcid":null,"position":1,"is_corresponding":false},{"id":1271627,"name":"Emilie Ditlev Nygaard","orcid":null,"position":2,"is_corresponding":false},{"id":1127190,"name":"Elin Manell","orcid":"0000-0002-4491-7609","position":3,"is_corresponding":false},{"id":1127614,"name":"Philip Jordache","orcid":null,"position":4,"is_corresponding":false},{"id":1271628,"name":"Susan Qudus","orcid":null,"position":5,"is_corresponding":false},{"id":1271629,"name":"Alexander Cadelina","orcid":null,"position":6,"is_corresponding":false},{"id":402550,"name":"Joshua Weiner","orcid":"0000-0003-3780-1875","position":7,"is_corresponding":false},{"id":1271157,"name":"Greg Nowak","orcid":"0000-0001-8766-8860","position":8,"is_corresponding":false},{"id":1271156,"name":"M. Esad Gunes","orcid":"0000-0002-7025-3826","position":0,"is_corresponding":true}],"reference_count":32,"raw_metadata":null,"created_at":"2026-07-19T02:02:28.969209Z","pmid":"39291632","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}