Enhanced immunogenic potential of cancer immunotherapy antibodies in human IgG1 transgenic mice.

ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.

Accumulation of Multipotent Hematopoietic Progenitors in Peripheral Lymphoid Organs of Mice Over-expressing Interleukin-7 and Flt3-Ligand.

Interleukin-7 (IL-7) and Flt3-ligand (FL) are two cytokines important for the generation of B cells, as manifested by the impaired B cell development in mice deficient for either cytokine or their respective receptors and by the complete block in B cell differentiation in the absence of both cytokines. IL-7 is an important survival and proliferation factor for B cell progenitors, whereas FL acts on several early developmental stages, prior to B cell commitment. We have generated mice constitutively over-expressing both IL-7 and FL. These double transgenic mice develop splenomegaly and lymphadenopathy characterized by tremendously enlarged lymph nodes even in young animals. Lymphoid, myeloid and dendritic cell numbers are increased compared to mice over-expressing either of the two cytokines alone and the effect on their expansion is synergistic, rather than additive. B cell progenitors, early progenitors with myeloid and lymphoid potential (EPLM), common lymphoid progenitors (CLP) and lineage-, Sca1+, kit+ (LSK) cells are all increased not only in the bone marrow but also in peripheral blood, spleen and even lymph nodes. When transplanted into irradiated wild-type mice, lymph node cells show long-term multilineage reconstitution, further confirming the presence of functional hematopoietic progenitors therein. Our double transgenic mouse model shows that sustained and combined over-expression of IL-7 and FL leads to a massive expansion of most bone marrow hematopoietic progenitors and to their associated presence in peripheral lymphoid organs where they reside and potentially differentiate further, thus leading to the synergistic increase in mature lymphoid and myeloid cell numbers. The present study provides further in vivo evidence for the concerted action of IL-7 and FL on lymphopoiesis and suggests that extramedullary niches, including those in lymph nodes, can support the survival and maintenance of hematopoietic progenitors that under physiological conditions develop exclusively in the bone marrow.

Prophylactic and Therapeutic Effects of Interleukin-2 (IL-2)/Anti-IL-2 Complexes in Systemic Lupus Erythematosus-Like Chronic Graft-Versus-Host Disease.

Murine chronic graft-versus-host-disease (cGvHD) induced by injection of parental lymphocytes into F1 hybrids results in a disease similar to systemic lupus erythematosus. Here, we have used DBA/2 T cell injection into (C57BL/6 × DBA/2)F1 (BDF1) mice as a model system to test the prophylactic and therapeutic effects of interleukin-2 (IL-2)/anti-IL-2 immune complexes on the course of cGvHD. Our findings demonstrate that pretreatment with Treg inducing JES6/IL-2 complexes render BDF1 mice largely resistant to induction of cGvHD, whereas pretreatment with CD8+ T cell/NK cell inducing S4B6/IL-2 complexes results in a more severe cGvHD. In contrast, treatment with JES6/IL-2 complexes 4 weeks after induction had no beneficial effect on disease symptoms. However, similar treatment with S4B6/IL-2 complexes led to a significant amelioration of the disease. This therapeutic effect seems to be mediated by donor CD8+ T cells. The fact that a much stronger cGvHD is induced in BDF1 mice depleted of donor CD8+ T cells strongly supports this conclusion. The contrasting effects of the two different IL-2 complexes are likely due to different mechanisms.

Two Distinct Pathways in Mice Generate Antinuclear Antigen-Reactive B Cell Repertoires.

The escape of anti-self B cells from tolerance mechanisms like clonal deletion, receptor editing, and anergy results in the production of autoantibodies, which is a hallmark of many autoimmune disorders. In this study, we demonstrate that both germline sequences and somatic mutations contribute to autospecificity of B cell clones. For this issue, we investigated the development of antinuclear autoantibodies (ANAs) and their repertoire in two different mouse models. First, in aging mice that were shown to gain several autoimmune features over time including ANAs. Second, in mice undergoing a chronic graft-versus-host disease (GVHD), thereby developing systemic lupus erythematosus-like symptoms. Detailed repertoire analysis revealed that somatic hypermutations (SHM) were present in all Vh and practically all Vl regions of ANAs generated in these two models. The ANA B cell repertoire in aging mice was restricted, dominated by clonally related Vh1-26/Vk4-74 antibodies. In the collection of GVHD-derived ANAs, the repertoire was less restricted, but the usage of the Vh1-26/Vk4-74 combination was still apparent. Germline conversion showed that the SHM in the 4-74 light chain are deterministic for autoreactivity. Detailed analysis revealed that antinuclear reactivity of these antibodies could be induced by a single amino acid substitution in the CDR1 of the Vk4-74. In both aging B6 and young GVHD mice, conversion of the somatic mutations in the Vh and Vl regions of non Vh1-26/Vk4-74 using antibodies showed that B cells with a germline-encoded V gene could also contribute to the ANA-reactive B cell repertoire. These findings indicate that two distinct pathways generate ANA-producing B cells in both model systems. In one pathway, they are generated by Vh1-26/Vk4-74 expressing B cells in the course of immune responses to an antigen that is neither a nuclear antigen nor any other self-antigen. In the other pathway, ANA-producing B cells are derived from progenitors in the bone marrow that express B cell receptors (BCRs), which bind to nuclear antigens and that escape tolerance induction, possibly as a result of crosslinking of their BCRs by multivalent determinants of nuclear antigens.

FGF2 induces RANKL gene expression as well as IL1ß regulated MHC class II in human bone marrow-derived mesenchymal progenitor stromal cells.

OBJECTIVE: Human bone marrow mesenchymal stromal cells (hBM-MSC) are being applied in tissue regeneration and treatment of autoimmune diseases (AD). Their cellular and immunophenotype depend on isolation and culture conditions which may influence their therapeutic application and reflect their in vivo biological functions. We have further characterised the phenotype induced by fibroblast growth factor 2 (FGF2) on healthy donor hBM-MSC focusing on the osteoimmunological markers osteoprotegerin (OPG), receptor activator of nuclear factor kB (RANK), RANK ligand (RANKL) and HLA-DR and their regulation of expression by the inflammatory cytokines IL1ß and IFNγ. METHODS: RANK, RANKL, OPG and HLA-DR expression in hBM-MSC expanded under specific culture conditions, were measured by RT-PCR and flow cytometry. MAPKs induction by FGF2, IL1ß and IFNγ in hBM-MSC was analysed by immunoblotting and RT-PCR. RESULTS: In hBM-MSC, OPG expression is constitutive and FGF2 independent. RANKL expression depends on FGF2 and ERK1/2 activation. IL1ß and IFNγ activate ERK1/2 but fail to induce RANKL. Only IL1ß induces P38MAPK. The previously described HLA-DR induced by FGF2 through ERK1/2 on hBM-MSC, is suppressed by IL1ß through inhibition of CIITA transcription. HLA-DR induced by IFNγ is not affected by IL1ß in hBM-MSC, but is suppressed in articular chondrocytes and lung fibroblasts. CONCLUSIONS: RANKL expression and IL1ß regulated MHC-class II, both induced via activation of the ERK1/2 signalling pathway, are specific for progenitor hBM-MSC expanded in the presence of FGF2. HLA-DR regulated by IL1ß and ERK1/2 is observed on hBM-MSC during early expansion without FGF2 suggesting previous in vivo acquisition. Stromal progenitor cells with this phenotype could have an osteoimmunological role during bone regeneration.