NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination

<div><p>Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs) are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD), which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2r<b>γ</b>^null (NOG) mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg). After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2) cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP) or keyhole limpet hemocyanin (KLH) successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.</p></div

Generation of NOG-hIL-4-Tg mice.

<p><b>A,</b> Genotyping of NOG-hIL-4-Tg mice. Human IL-4-specific bands (561 bp) were detected along with the internal control (151 bp). PC: positive control. These results confirmed the gene transfer. NC: negative control. Conventional NOG mice were used. <b>B,</b> ELISA of NOG-IL-4-Tg plasma corresponding to the mice submitted for genotyping. The mice producing more than 100 pg/ml hIL-4 (higher than the broken line) were selected and used for the assays. A representative assay is shown. <b>C,</b> Comparison of hIL-4 protein levels in HD plasma (n = 12) and NOG-hIL-4-Tg mice (n = 57). The broken line shows the 100 pg/ml level. The high hIL-4 (n = 47) and low hIL-4 groups (n = 10) are shown. <b>D</b>, Tissue-specific expression of hIL-4 mRNA. Representative data of 1 NOG and 2 NOG-hIL-4-Tg mice are shown. Human IL-4 specific bands (449 bp) were detected along with β-actin (569 bp).</p

Comparison of engrafted human lymphocytes in NOG-hIL-4-Tg and conventional NOG mice.

<p><b>A,</b> Cellularity in HD PBMC-transferred NOG or NOG-hIL-4-Tg mice. NOG-hIL-4-Tg and NOG mice were transplanted with PBMCs (5x10⁶) from the same HD. After 4 weeks, the ratios of each T or B cell subset in BM or spleen obtained from PBMC-NOG (n = 6) and PBMC-NOG-hIL-4-Tg mice (n = 3) were analyzed by FCM. HD PBMCs (n = 20) are shown as a control. CD4-N; naïve CD4+ T cells, CD8-N; naïve CD8+ T cells, CD4-M; memory CD4+ T cells, CD8-M; memory CD8+ T cells, B-T; transitional B cells, B-N; naïve B cells, B-M; memory B cells, B-P; plasmablast/plasma cells. The mean ± SD is shown with the percentage score above each bar. <b>B.</b> Human naïve CD4+ T cells were purified and transferred to both NOG mice (n = 5) and NOG-hIL-4-Tg mice (n = 8). The left 4 panels show the typical expression profiles of CD45RA, CD45RO, CD3 and CD4 among purified naïve CD4+ T cells. MNCs were obtained from PBMC-NOG or PBMC-NOG-hIL-4-Tg mice, and the human CD4+ T cells were analyzed by FCM as shown in the 4 right panels. The lower panels were all gated on the upper gates surrounded by the lines. <b>C,</b> Isolated human CD4+ T cells were stimulated with PMA and ionomycin, and the expression levels of human IL-4 and IFN-γ were analyzed by FCM. Typical patterns are shown.</p

The list of classical HLA types of HDs.

<p>The list of classical HLA types of HDs.</p

Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs

<div><p>The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen <i>(SLA)-1</i>*<i>0401</i> after the stimulation of peripheral blood mononuclear cells (PBMCs) by <i>Staphylococcus aureus</i> superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only <i>SLA-1</i>*<i>0401</i>, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from <i>SLA-1</i>*<i>0401</i> homozygous pigs were stimulated, the <i>SLA-1</i>*<i>0401</i> mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the <i>SLA-1</i>*<i>0401</i> mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.</p></div

Tertiary structure of X2F6 mAb and the predicted antibody epitope.

<p>(A) Amino acid sequences of heavy and light chains of the X2F6 variable region. The database sequence PDB ID 3V7A is shown as the control sequence. (B) The predicted tertiary structure of the X2F6 mAb. (C) The tertiary structures of the YLL set in SLA-1*0501, which reacts with X2F6 with high reactivity (left panel), and the DVF set in SLA-1*1104, which cannot react with X2F6 (right panel), are shown. Pink (hydrophobic) and green (hydrophilic) colors represent the amino acid character. The structure is largely different, and the binding affinity is predicted to be different.</p

Class I SLA-related mRNA expression after TSST-1 stimulation.

<p>The PBMCs of two pigs with the Hp-16.0 haplotype (individuals #965 and #1938) were examined for classical class I SLA (A) and related mRNA (B) expression after stimulation. Closed squares with a solid line show TSST-1-stimulated PBMCs, open squares with a broken line show IFN-γ stimulation, and closed squares with a dotted line show the negative control.</p

Primer sequences.

<p>Primer sequences.</p

Specificity of the X2F6 mAb.

<p>(A) <i>SLA-1</i>*<i>0401</i>, <i>SLA-2</i>*<i>0901</i>, and <i>SLA-3</i>*<i>0602</i>, which are the classical class-I SLA alleles of Haplotype Hp-16.0, and <i>SLA-6</i>*<i>0101</i>, which is a non-classical class-I SLA allele of Hp-16.0, were transfected into HEK293 parent cells, and the reactivity of X2F6 (right panels) was examined by flow cytometry (FCM). Propidium iodide (PI) positive-dead cells were avoided for the gating. PT-85A, the pan-specific MHC class-1 antibody, was used for the positive control (middle panels). (B) The species specificity was examined using swine (Hp-16.0), human, and common marmoset PBMCs. Lymphoid gate was used for the analysis. The percentages shown above the panels are the double-positive cell percentages.</p

Amino acid alignment of each classical class I allele in five SLA class I haplotypes.

<p>The amino acid sequence alignment of the alleles of each SLA locus is shown. For the haplotypes with a specific set of amino acids (Y102, L103, L109; the YLL set), in which each allele reacted with X2F6, the number of YLL sets determined the level of reactivity. The MFI for X2F6 reactivity was highest in the Hp-10.0 PBMCs that possessed three YLL sets in the SLA-1, SLA-2 and SLA-3 loci.</p