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GM-CSF and IL-4 stimulate antibody responses in humanized mice by promoting T, B, and dendritic cell maturation.

ABSTRACT: Engraftment of human hematopoietic stem cells into immunodeficient mice that lack T cells, B cells, and NK cells results in reconstitution of human blood lineage cells, especially B cells, in the recipient mice. However, these humanized mice do not make any significant level of IgG Ab in response to Ag stimulation. In this study, we show that in humanized mice, B cells are immature, and there is a complete deficiency of CD209(+) (DC-SIGN) human dendritic cells. These defects can be corrected by expression of human GM-CSF and IL-4 in humanized mice. As a result, these cytokine-treated humanized mice produced significant levels of Ag-specific IgG after immunization, including the production of neutralizing Abs specific for H5N1 avian influenza virus. A significant level of Ag-specific CD4 T cell response was also induced. Thus, we have identified defects in humanized mice and devised approaches to correct these defects such that the platform can be used for studying Ab responses and to generate novel human Abs against virulent pathogens and other clinically relevant targets.

SUBMITTER: Chen Q

PROVIDER: S-EPMC4106296 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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GM-CSF and IL-4 stimulate antibody responses in humanized mice by promoting T, B, and dendritic cell maturation.

Chen Qingfeng Q He Fang F Kwang Jimmy J Chan Jerry K Y JK Chen Jianzhu J

Journal of immunology (Baltimore, Md. : 1950) 20121022 11

Engraftment of human hematopoietic stem cells into immunodeficient mice that lack T cells, B cells, and NK cells results in reconstitution of human blood lineage cells, especially B cells, in the recipient mice. However, these humanized mice do not make any significant level of IgG Ab in response to Ag stimulation. In this study, we show that in humanized mice, B cells are immature, and there is a complete deficiency of CD209(+) (DC-SIGN) human dendritic cells. These defects can be corrected by ...[more]

PMID: 23089398

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Project description:Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function in vitro. Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.

Project description:Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14-CD1c+ conventional DCs (cDCs) and CD14-CD141+ cDCs, in addition to CD14+ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123+ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c+ myeloid cells, CD141+ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3+ cells within splenic CD4+ T cells were significantly increased in the presence of GM-CSF. Foxp3+ T cells could be subdivided into two subpopulations, CD45RA-Foxp3hi and CD45RA-Foxp3lo T cells. Whereas CD45RA-Foxp3hi T cells were increased only after treatment with GM-CSF alone, CD45RA-Foxp3lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3+ T cells. The correlation analysis demonstrated that the development of these Foxp3+ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.

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GM-CSF and IL-4 stimulate antibody responses in humanized mice by promoting T, B, and dendritic cell maturation.

Publications

GM-CSF and IL-4 stimulate antibody responses in humanized mice by promoting T, B, and dendritic cell maturation.

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