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. 2024 Nov;105(11):800-806.
doi: 10.1002/cyto.a.24899. Epub 2024 Sep 18.

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

M Esad Gunes  1 , Daniel H Wolbrom  1 , Emilie Ditlev Nygaard  1 , Elin Manell  1   2 , Philip Jordache  1 , Susan Qudus  1 , Alexander Cadelina  1 , Joshua Weiner  1   3 , Greg Nowak  1   4
Affiliations

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

M Esad Gunes et al. Cytometry A. 2024 Nov.
No abstract available

Keywords: baboon; bone marrow transplantation; immune reconstitution; mixed chimerism; porcine; recent thymic emigrants; thymus transplantation; xenotransplantation.

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Figures

Figure 1.
Figure 1.. Gating strategy for the panel.
The sample was created by mixing baboon and pig blood at an 8:2 ratio. A) Leukocytes were gated, and the live and singlets were selected. B) To select pig cells within the sample, the singlets were plotted with pCD45 and pMHC class I (pSLA class I), and all pCD45+pMHC class I+ cells were selected as pig cells for further downstream analysis. The pCD45+pMHC class I+ cells were plotted with pSWC3a and pCD3. pSWC3a+ cells were selected as pig myeloid cells, and pCD3+ cells were selected as pig T cells. pCD3+ cells were plotted with pCD4 and pCD8 and pCD4+pCD8- cells were gated as pig Thelper, pCD4-pCD8+ as pig Tcytotoxic, and pCD4+pCD8+ as pig double positive T cells. pCD4-pCD8- cells were not gated; however, most represent pig γδ T-cells. C) Singlets were plotted with pCD45+ and bCD45+ to analyze baboon cells. bCD45+ cells were selected as baboon cells. bCD45+ cells were separated into bCD11b+ myeloid cells and bCD45+bCD11b- lymphoid cells. D) bCD45+bCD11b- cells were plotted using bCD3 and bCD45RA and bCD3 and bCD69 to see an overview of CD45RA and CD69 expression of the entire lymphoid series. E) bCD45+bCD11b- lymphoid cells were plotted using bCD3 and bCD20 to gate bCD20+ B cells. bCD45+bCD11b-bCD20- cells were selected for further downstream analysis as non-B-cell lymphoid cells. F) bCD45+bCD11b-bCD20- cells were plotted against bCD3 and bCD56. bCD3+bCD56- cells were selected as T-cells for further downstream analysis. bCD3+bCD56+ cells were selected as a group of NKT cells, and bCD3-bCD56+ cells were selected as NK cells. NK and NKT cells were plotted against bCD8 and SSC-A to identify bCD8hi+, bCD8lo+, and bCD8- NK and NKT cells. G) bCD45+bCD11b-bCD20-bCD56-bCD3+ T-cells were plotted using bCD4 and bCD8 to gate bCD4+ Thelper and bCD8+ Tcytotoxic cells. bCD4+ Thelper cells were further plotted using bCD25 and bCD127 to select bCD4+bCD25+bCD127- Treg cells. H) bCD45+bCD11b-bCD20-bCD56-bCD3 T-cells, bCD45+bCD11b-bCD20-bCD56-bCD3+bCD4+ Thelper cells and bCD45+bCD11b-bCD20-bCD56-bCD3+bCD8+ Tcytotoxic cells were gated using bCCR7 and bCD45RA, to gate bCCR7-bCD45RA- TEM, bCCR7+bCD45RA- TCM and bCCR7-bCD45RA+ TEMRA cell populations. bCCR7+bCD45RA+ cells were further plotted using bCD28 and bCD95 to gate bCD28+bCD95- as Tnaïve cells. bCCR7+bCD45RA+bCD28+bCD95- Tnaïve cells were lastly plotted against bCD31 and SSC-A to select bCCR7+bCD45RA+bCD28+bCD95-bCD31+ recent thymic emigrants.
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