Ectopic T cell receptor expression causes B cell immunodeficiency in transgenic mice

Abstract

Mice expressing transgenic T cell receptors (TCR) are used to explore important questions in immunity. However, transgene expression may have unexpected effects. We previously reported a B cell immunodeficiency, comprising decreased B cell numbers and diminished antibody responses, in mice that express a transgenic TCR specific for nicotinic acetylcholine receptor; the mice were generated using cassette vectors designed specifically for transgenic TCR expression [see Kouskoff et al. J. Immunol. Methods 1995. 180: 273-280]. We now show data suggesting that this defect is due to the expression and accumulation of TCR § and g chains inside B cells and induction of an endoplasmic reticulum stress response, causing apoptosis at the pre B-I and later B cell stage. Thus, inappropriate transgene expression can profoundly affect B cells, leading to a previously undescribed mechanism of immunodeficiency.

Ectopic T Cell Receptor-α Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once

“…Transgenes under the control of TCRα gene regulatory elements have previously been reported to be ectopically expressed in B cells [2], [3]. Based on the results of our dual-reporter transgene described above, we hypothesized that the Dad1 gene normally present 3′ of the TCRα LCR in the genome might serve to suppress such ectopic B cell expression.…”

“…The second is a TCRα/Dad1 bacterial artificial chromosome (BAC) construction in which separate Vα and Dad1 promoter driven reporter genes flank the LCR DNA in its natural context of DNA sequence spanning from the Cα exons through the entire Dad1 genomic locus. Among the significant findings we report here is that neither of the 5′-flanking genes in these “two-gene” reporter systems displayed the ectopic B cell expression that was observed from “single-gene” Vα promoter-driven [2], [3] and heterologous promoter driven [4] reporter constructs studied previously. These data are consistent with a model in which flanking the TCRα LCR with two distinct, active genes results in a T cell restriction to its activity on the upstream-linked gene.…”

“…Similar results were obtained from simultaneous analyses of endogenous TCRα mRNA. Thus, unlike the Vα promoter-driven (single transcription unit) transgenes studied previously [2], [3], the Vα promoter-driven hCD2 reporter gene in the dual-reporter TCRα/Dad1 BAC does not display ectopic expression in B cells. Figure 9B shows RT-PCR data confirming that, as expected, the Dad1 promoter driven rCD2 reporter gene is active in B cells of the BAC transgenic mice.…”

“…Therefore, despite these important advances, a clear explanation for the restriction of TCRα gene expression to T cells has remained elusive. Nevertheless, this existing information may explain why TCRα transgenes removed from their natural genomic context [3] as well as other transcription units under the control of TCRα gene regulatory elements, such as the TCRα LCR, become expressed in both T and B cells. The latter is true whether or not transcription from the transgene construct is driven by a natural Vα promoter [2] or a heterologous promoter [4].…”

The TCRα Locus Control Region Specifies Thymic, But Not Peripheral, Patterns of TCRα Gene Expression

“…Subsequent transgenic studies have detected B cell expression in transgene configurations involving TCR␣ LCR elements. However, these studies used either indirectly linked, cointegrated TCR␣ LCR (45) or incomplete TCR␣ LCR cointegrated with a TCR␤ transgene (46). We directly addressed this question at single-cell resolution in a directly linked, complete LCR-driven reporter transgene system free of integration site-dependent position effects.…”