Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001

Abstract

Monoclonal antibodies that bind to human leukocyte antigen (HLA) are useful tools for HLA-typing, tracking donor-recipient chimerisms after bone marrow transplants, and characterizing specific major histocompatibility complexes (MHC) on cell surfaces. Unfortunately, equivalent reagents are not available for rhesus macaques, which are commonly used animal as models in organ transplant and infectious disease research. To address this deficiency, we isolated an antibody that recognizes the common Indian rhesus macaque MHC class I molecule, Mamu-A1*001. We induced Mamu-A1*001-binding antibodies by alloimmunizing a female Mamu-A1*001-negative rhesus macaque with peripheral blood mononuclear cells (PBMC) from a male Mamu-A1*001-positive donor. A Fab phage display library was constructed with PBMC from the alloimmunized macaque and panned to isolate an antibody that binds to Mamu-A1*001 but not to other common rhesus macaque MHC class I molecules. The isolated antibody distinguishes PBMC from Mamu-A1*001-positive and -negative macaques. Additionally, the Mamu-A1*001-specific antibody binds the cynomolgus macaque MHC class I ortholog Mafa-A1*001:01 but not variants Mafa-A1*001:02/03, indicating a high degree of binding specificity. The Mamu-A1*001-specific antibody will be useful for identifying Mamu-A1*001-positive rhesus macaques, for detecting Mamu-A1*001-positive cells in populations of Mamu-A1*001-negative cells, and for examining disease processes that alter expression of Mamu-A1*001 on cell surfaces. Moreover, the alloimmunization process we describe will be useful for isolating additional MHC allomorph-specific monoclonal antibodies or antibodies against other polymorphic host proteins which are difficult to isolate with traditional technologies.

Evidence for persistence of the SHIV reservoir early after MHC haploidentical hematopoietic stem cell transplantation

“…3h ), and by flow cytometry using a donor vs. recipient discriminatory Mamu-A01 antibody, which was applicable in both R.702 and R.703 (Fig. 3i–k ) 34 . Both animals demonstrated rapid engraftment of donor neutrophils, Fig.…”

“…Donor chimerism was monitored by molecular chimerism analysis-based divergent microsatellite markers as previously described 30 , 31 , as well as by flow cytometry using a discriminatory anti-Mamu A01 antibody 34 . Flow cytometric analysis was performed with FlowJo (Tree Star) using the following analysis pipeline: (1) Doublets were excluded on the basis of FSC-H vs. FSC-A and SSC-H vs. SSC-A gating, (2) Neutrophils cells were identified as high SSC, CD11b + CD3- cells, (3) T cells as CD3 + CD20- CD11b- cells present in the lymphocyte gate (then sub-gated for CD4 or CD8 positivity), and (4) B cells as CD3- CD20 + lymphocytes.…”

“…Flow cytometric analysis was performed with FlowJo (Tree Star) using the following analysis pipeline: (1) Doublets were excluded on the basis of FSC-H vs. FSC-A and SSC-H vs. SSC-A gating, (2) Neutrophils cells were identified as high SSC, CD11b + CD3- cells, (3) T cells as CD3 + CD20- CD11b- cells present in the lymphocyte gate (then sub-gated for CD4 or CD8 positivity), and (4) B cells as CD3- CD20 + lymphocytes. The following antibody clones were used for this analysis: anti-Mamu A01 clone P12 34 (Anti-Mamu A01-PE, NHP Reagent Resources,1:50 dilution), anti-CD11b clones ICRF44 and D12 (BD Biosciences, catalog numbers 557918 and 340936, 1:100 dilution), anti-CD3 clone SP34–2 (BD Biosciences, catalog number 557757, 1:50 dilution), anti-CD4 clone OK-T4 (Biolegend, catalog number 317442, 1:100), anti-CD8 clone RPA-T8 (BD Biosciences, catalog number 563795, 1:50 dilution), anti-CD20 clone 2H7 (ThermoFisher, catalog number 45-0209-42, 1:50 dilution).…”

“…7a ): (1) Doublets were excluded on the basis of FSC-H vs. FSC-A and SSC-H vs. SSC-A gating; (2) live cells were selected as 7AAD- cells; (3) CD4 + T cells were sorted as CD3 + CD4 + CD8- CD20- CD14- cells present in the lymphocyte gate, then sub-gated as A01 + donor vs. A01- recipient T cells; (4) CD4 + non-T cells were sorted as CD3- Lin + (positive in the CD20/CD14 channel), then were sub-gated as CD4 + A01 + donor vs. CD4 + A01- recipient non-T cells. The following antibody clones were used: anti-Mamu A01 clone P12 34 (Anti-Mamu A01-PE, NHP Reagent Resources,1:50 dilution), anti-CD3 clone SP34-2 (BD Biosciences, catalog number 557757, 1:50 dilution), anti-CD4 clone OK-T4 (Biolegend, catalog number 317442, 1:100), anti-CD8 clone RPA-T8 (BD Biosciences, catalog number 563795, 1:50 dilution), anti-CD14 clone M5E2 (BD Biosciences, catalog number 557742, 1:100 dilution), and anti-CD20 clone 2H7 (BD Biosciences, catalog number 560735, 1:50 dilution). In samples where enough cells were available post-sort, we performed a quality control analysis to assess the purity of the A01 + and A01- cells, with most samples tested showing >90% purity (Supplementary Table 1 ).…”

Identification of a lymphocyte minor histocompatibility antigen in Mauritian cynomolgus macaques

“…Activated PBMCs were harvested the following morning, washed twice with phosphate-buffered saline (PBS), resuspended in PBS, and loaded into tuberculin syringes. Each macaque was then immunized with up to 4x10 7 activated PBMCs as described previously (59).…”