A SMART method for isolating monoclonal antibodies from individual rhesus macaque memory B cells
Weinfurter JT, Bennet SN, and Reynolds MR
Journal of Immunological Methods 2024
In this paper, we introduce a new method for isolating monoclonal antibodies from individual memory B cells. Traditional approaches for isolating antibodies from B cells use 5′ primers in nested polymerase chain reactions (PCRs) that bind to the immunoglobulin (Ig) leader sequences, which are immediately upstream of the heavy and light chain variable genes. However, the extensive diversity within rhesus macaque leader sequences necessitates using numerous primers to amplify the Ig variable genes, reducing PCR efficiency. To address this problem, we developed a switching mechanism at the 5′ ends of the RNA transcript (SMART)-based method for amplifying IgV genes from single rhesus macaque B cells to efficiently capture Ig heavy and light chain pairs. This approach has several advantages. First, it generates full-length cDNAs from individual B cells using SMART 5′ and 3′ rapid amplification of cDNA ends (RACE) reactions. Second, it adds primer binding sites to the 5′ and 3′ ends of cDNA during synthesis, enabling PCR amplification of low-abundance antibody templates. Third, it simplifies nested PCR reactions by using universal 5′ primers, eliminating the need for complex primer sets targeting the Ig leader sequences. In our hands, this method efficiently amplifies the heavy and light chain genes from individual rhesus macaque memory B cells. We anticipate this method will also be valuable for isolating antibodies from other animal species, especially those with poorly characterized immunoglobulin genes.
Generation of SIV-resistant T cells and macrophages from nonhuman primate induced pluripotent stem cells with edited CCR5 locus
Stem Cell Reports 2022
Working with the Slukvin lab, we used CRISPR-Cas9 technology to edit the CCR5 gene in induced pluripotent stem cells (iPSCs) derived from T cells (T-iPSCs) or fibroblasts (fib-iPSCs) of Mauritian cynomolgus macaques (MCM). Notably, we observed that T cells and macrophages from CCR5-edited MCM iPSCs could not support the replication of CCR5-tropic simian immunodeficiency viruses (SIVs). Overall, these findings lay the groundwork for investigating gene-edited iPSC-based therapies for AIDS in nonhuman primates.
§co-senior authors PubMed Link
Transplantation of T-cell receptor α/β-depleted allogeneic bone marrow in nonhuman primates
In partnership with the Slukvin lab, we investigated whether removing T cells from donor bone marrows before transplant could reduce graft-versus-host disease (GvHD) in Mauritian cynomolgus macaques (MCMs) after transplantation. We transplanted T-cell-depleted bone marrow cells into major histocompatibility complex (MHC)-matched MCMs that had undergone total body irradiation conditioning. MCMs treated with immunosuppressants cyclophosphamide and tacrolimus post-transplant showed mixed chimerisms without GvHD, and one animal displayed long-term engraftment. These findings indicate that donor bone marrow cells can engraft without causing severe GvHD in MHC-matched MCMs with intense conditioning and anti-GvHD measures. The model may be valuable for studying how GvH responses eliminate HIV-infected cells.
§co-senior authors PubMed Link
Identifying a Minor Histocompatibility Antigen in Mauritian Cynomolgus Macaques Encoded by APOBEC3C
Front. Immunol. 2020
This study employed alloimmunization, whole exome sequencing, and bioinformatics to identify a minor histocompatibility antigen (mHAg) in Mauritian cynomolgus macaques (MCMs). The mHAg, named SW10, is 10 amino acids long and lies within the cytidine deaminase APOBEC3C. We determined that it is bound by the major histocompatibility complex class I (MHC-I) molecule Mafa-A1∗063, expressed by almost 90% of MCMs. Notably, CD8+ T cells specific for APOBEC3C SW10 recognized immortalized B cells but not fibroblasts, suggesting that the mHAg is expressed by a limited number of tissues. These findings established a method for mapping mHAgs in a non-transplant setting and identified APOBEC3C SW10 as a potential model antigen for testing mHAg-targeted therapies in NHPs.
Isolation of a monoclonal antibody from a phage display library binding the rhesus macaque MHC class I allomorph Mamu-A1*001
This study isolated an antibody recognizing the common Indian rhesus macaque (RM) major histocompatibility complex class I (MHC-I) molecule, Mamu-A1*001. We induced Mamu-A1*001-binding antibodies by alloimmunizing a Mamu-A1*001-negative RM with peripheral blood mononuclear cells (PBMC) from a Mamu-A1*001-positive donor. Using a Fab phage display library that we constructed from PBMC from the alloimmunized macaque, we isolated an antibody, named P12, that binds specifically to Mamu-A1*001 but not to other common RM MHC-I molecules. P12 can distinguish PBMC from Mamu-A1*001-positive and -negative macaques. Additionally, P12 binds the cynomolgus macaque MHC-I ortholog Mafa-A1*001:01 but not variants Mafa-A1*001:02/03, demonstrating high binding specificity. We anticipate that P12 will be useful for detecting chimerism after bone marrow transplants between Mamu-A1*001 mismatched donor-recipient pairs and studying diseases affecting the cell surface expression of Mamu-A1*001. The alloimmunization strategy described in this study can also facilitate the isolation of additional MHC-specific monoclonal antibodies or antibodies targeting other polymorphic host proteins, which are challenging to isolate with traditional methods.