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Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

ABSTRACT: Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as ?HAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HAC?, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane ? and ? immunoglobulins (bIg? and bIg?) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIg?/Ig?; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HAC? (cKSL-HAC?/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

SUBMITTER: Sano A

PROVIDER: S-EPMC3813428 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

Sano Akiko A Matsushita Hiroaki H Wu Hua H Jiao Jin-An JA Kasinathan Poothappillai P Sullivan Eddie J EJ Wang Zhongde Z Kuroiwa Yoshimi Y

PloS one 20131024 10

Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human im ...[more]

PMID: 24205120

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Project description:Immunoglobulin (Ig) class switch recombination is associated with the production and splicing of germline IgCH messenger RNA transcripts. Levels of gamma 1 transcripts in mouse spleen sections were assessed by semiquantitative analysis of reverse transcriptase polymerase chain reaction (PCR) products during primary and secondary antibody responses to chicken gamma globulin (CGG). This was correlated with the appearance of CGG-specific B cells and their growth and differentiation to plasma cells. After primary immunization with CGG, gamma 1 switch transcripts appeared after 4 d, peaked at a median of six times starting levels between 10 and 18 d after immunization, and returned to background levels before secondary immunization at 5 wk. By contrast, after secondary challenge with CGG, a sevenfold increase in transcripts occurs during the first d. The level again doubles by day 3, when it is six times that which is seen at the peak of the primary response. After day 4, there was a gradual decline over the next 2-3 wk. Within 12 h of secondary immunization, antigen-specific memory B cells appeared in the outer I zone and by 24 h entered S phase, presumably as a result of cognate interaction with primed T cells. Over the next few hours, they migrated to the edge of the red pulp, where they grew exponentially until the fourth day, when they synchronously differentiated to become plasma cells. The same pattern was seen for the migration, growth, and differentiation of virgin hapten-specific B cells when CGG-primed mice were challenged with hapten protein. The continued production of transcripts after day 3 indicates that switching also occurs in germinal centers, but in a relatively small proportion of their B cells. The impressive early production of switch transcripts during T cell-dependent antibody responses occurs in cells that are about to undergo massive clonal expansion. It is argued that Ig class switching at this time, which is associated with cognate T cell-B cell interaction in the T zone, has a major impact on the class and subclasses of Ig produced during the response.

Project description:INTRODUCTION: Interferon alpha (IFN-?) has a complex role in autoimmunity, in that it may both enhance and prevent inflammation. We have previously shown that the presence of IFN-? at sensitization protects against subsequent antigen-triggered arthritis. To understand this tolerogenic mechanism, we performed a descriptive, hypothesis-generating study of cellular and humoral responses associated with IFN-?-mediated protection against arthritis. METHODS: Arthritis was evaluated at day 28 in mice given a subcutaneous injection of methylated bovine serum albumin (mBSA), together with Freund adjuvant and 0 to 5,000 U IFN-? at days 1 and 7, followed by intraarticular injection of mBSA alone at day 21. The effect of IFN-? on mBSA-specific IgG1, IgG2a, IgG2b, IgA, and IgE was evaluated by enzyme-linked immunosorbent assay (ELISA). Cytokines in circulation and in ex vivo cultures on mBSA restimulation was evaluated with ELISA and Luminex, and the identity of cytokine-producing cells by fluorescence-activated cell sorting (FACS) analysis. RESULTS: Administration of IFN-? protected mice from arthritis in a dose-dependent manner but had no effect on antigen-specific antibody levels. However, IFN-? did inhibit the initial increase of IL-6, IL-10, IL-12, and TNF, and the recall response induced by intraarticular mBSA challenge of IL-1?, IL-10, IL-12, TNF, IFN-?, and IL-17 in serum. IFN-? decreased both macrophage and CD4+ T cell-derived IFN-? production, whereas IL-17 was decreased only in CD4+ T cells. Ex vivo, in mBSA-restimulated spleen and lymph node cell cultures, the inhibitory effect of in vivo administration of IFN-? on proinflammatory cytokine production was clearly apparent, but had a time limit. An earlier macrophage-derived, and stronger activation of the antiinflammatory cytokine transforming growth factor beta (TGF-?) was observed in IFN-?-treated animals, combined with an increase in CD4+ T cells producing TGF-? when arthritis was triggered by mBSA (day 21). Presence of IFN-? at immunizations also prevented the reduction in TGF-? production, which was induced by the intraarticular mBSA injection triggering arthritis in control animals. CONCLUSIONS: Administration of IFN-? has a profound effect on the cellular response to mBSA plus adjuvant, but does not affect antigen-specific Ig production. By including IFN-? at immunizations, spleen and lymph node cells inhibit their repertoire of antigen-induced proinflammatory cytokines while enhancing antiinflammatory TGF-? production, first in macrophages, and later also in CD4+ T cells. On intraarticular antigen challenge, this antiinflammatory state is reenforced, manifested as inhibition of proinflammatory recall responses and preservation of TGF-? levels. This may explain why IFN-? protects against antigen-induced arthritis.

Dataset Information

Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

Publications

Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

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