Tim-1 is induced on germinal centre B cells through B-cell receptor signalling but is not essential for the germinal centre response

Abstract

T-cell immunoglobulin mucin-1 (Tim-1) has been proposed to be an important T-cell immunoregulatory molecule since its expression on activated T cells was discovered. To study the role of Tim-1 on T cells in vitro and in vivo we generated both Tim-1-deficient mice and several lines of Tim-1 transgenic mice with Tim-1 expression on either T cells, or B and T cells. We demonstrate that neither deficiency nor over-expression of Tim-1 on B and T cells results in modulation of their proliferation in vitro. More surprisingly, T helper type 2 cells generated either from Tim-1-deficient mice or Tim-1 transgenic mice did not show enhancement of interleukin-4 (IL-4), IL-5 and IL-10 production. Furthermore, using a Schistosoma mansoni egg challenge as a potent T helper type 2 response inducer we also show that Tim-1 is not essential for T- and B-cell responses in vivo. However, we observe induction of Tim-1 on B cells following B-cell receptor (BCR), but not Toll-like receptor 4 stimulation in vitro. We show that the induction of Tim-1 on B cells following BCR stimulation is phosphoinositide-3 kinase and nuclear factor-kappaB pathway dependent. More importantly, we conclude that Tim-1 is predominantly expressed on germinal centre B cells in vivo although the percentage of germinal centre B cells in wild-type and Tim-1-deficient mice is comparable. Identification of Tim-1 as a marker for germinal centre B cells will contribute to the interpretation and future analysis of the effects of the anti-Tim-1 antibodies in vivo.

Figure 3

Tim-1 is induced on B cells through the B-cell receptor signalling pathway. (a) B and T cells were isolated from the spleen of either wild-type or Tim-1-deficient mice and cultured in media (control) or media containing anti-immunoglobulin M (IgM) or anti-CD3 and CD28. Expression of Tim-1 on each cell type was analysed by flow cytometry 48 hr later using anti-Tim-1 antibody (solid line) or isotype control (dash line). Data shown are representative from at least three independent experiments with three mice per group. (b) Total splenocytes from naive mice were labelled with carboxyfluorescein succinimidyl ester (CFSE) and cultured in media with or without anti-IgM or lipopolysaccharide (LPS). CFSE dilution and Tim-1 expression on CD19⁺ cells were analysed by flow cytometry on day 5. Data shown are representative from three independent experiments with three mice per group. (c) Purified B cells were cultured in media with or without anti-IgM or LPS for 24 hr. Tim-1 expression in each sample was analysed by real-time polymerase chain reaction, in triplicate, using 18S as an internal control. Tim-1 expression was then normalized to the unstimulated control (with value 1). Data shown are mean ± standard error of the mean from three independent experiments with two mice per group. (d) B cells were cultured in media or media containing anti-IgM with or without various inhibitors as shown. Percentage of Tim-1-expressing B cells was analysed by flow cytometry analysis. Data shown are derived from at least three independent experiments.

Figure 1

Generation of Tim-1-deficient mice and Tim-1-over-expressing transgenic mice. (a) The structure of the Havcr-1 locus, its targeting vector, and predicted homologous recombination event. neo, neomycin resistance cassette. (b) Genotyping of genomic DNA from wild-type (+/+), Havcr-1 gene-targeted heterozygous (+/−) and homozygous (−/−) mice by polymerase chain reaction (PCR) analysis. (c) Reverse transcription (RT-) PCR analysis of Tim-1 expression in various tissues collected from either wild-type (WT) or Havcr-1^−/− mice. HPRT was used as a loading control for the analysis. Data shown are representative of three independent experiments performed. (d) Tim-1 complementary DNA was cloned into the XhoI site of the pTEXCD2 vector. The vector was then linearized by NotI digestion. (e) Genotyping of genomic DNA from Tim-1-over-expressing mouse and its littermate control by PCR analysis using primers ASEQ965 and ASEQ966. (f) RT-PCR analysis of Tim-1 expression on total splenocytes from either WT or Tim-1 transgenic (Tim1Tg) mice. (g) Flow cytometric analysis of Tim-1 expression on B and T cells from wild-type and Tim-1-over-expressing transgenic mice.

Figure 2

T-cell proliferation and differentiation is normal in Tim-1-deficient mice and Tim-1 transgenic mice. Proliferation of B and T cells from Tim-1-deficient mice (a) and Tim-1 transgenic mice (b) in media alone (control) or in media containing anti-CD3 or anti-immunoglobulin M (IgM). Thymidine incorporation assay for each sample was performed in triplicate. Data shown are from a single experiment with four or five mice per group but are representative of three repeat experiments. Cytokine production by total wild-type and Tim-1-deficient splenocytes (c), or by wild-type and Tim-1 transgenic CD4⁺ T cells (d) differentiated under T helper type 1 (Th1) or Th2 polarizing conditions and re-stimulated for 24 hr with anti-CD3ε antibody. Data shown are from a single experiment with four or five mice per group but are representative of three repeat experiments.

Figure 4

Tim-1 over-expressing mice have a normal in vivo type 2 immune response. Wild-type or Tim-1 transgenic mice received intraperitoneal injection of phosphate-buffered saline (PBS) or Schistosoma mansoni eggs. Two weeks later the mice received PBS or Schistosoma mansoni eggs intravenously. Mice were killed 3 days later. (a) Relative amount of various isotypes of Schistosoma mansoni egg-specific antibodies in each mouse group were analysed by direct enzyme-linked immunosorbent assay (ELISA). (b) Splenocytes from each mouse group were re-stimulated in vitro with various concentrations of Schistosoma mansoni egg antigen (SEA). Proliferation was measured by thymidine incorporation assay, in triplicate, after 48 hr. (c) Interelukin-4 (IL-4) and IL-5 production from the day 2 SEA-stimulated total splenocytes from each mouse group was measured by sandwich ELISA. (d) Lungs from each mouse group were fixed in formalin. Paraffin sections were prepared and stained with Giemsa for cell infiltrate and periodic acid-Schiff (PAS) for goblet cells (pictures). Mucus production was evaluated blindly by using numerical scoring expressed in arbitrary units (histogram). Data shown are from a single experiment with five or six mice per group but are representative of three repeat experiments.

Figure 5

Tim-1 is predominantly expressed on germinal centre B cells. (a) Wild-type BALB/c mice were given intraperitoneal injections of phosphate-buffered saline (PBS) or 2 × 10⁷ sheep red blood cells (SRBCs). On day 9, induction of germinal centre (GC) B cells was analysed by flow cytometry using anti-Tim-1 (solid line) or isotype control (dashed line). CD19, peanut agglutinin (PNA) and CD95 were used as markers for GC B cells. (b) Expression of Tim-1 on GC B cells and non-GC (NGC) B cells on day 9 for SRBC-challenged mice was analysed by flow cytometry using anti-Tim-1 (solid line) or isotype control (dashed line). (c) Wild-type (WT) and Tim-1-deficient (Tim1KO) mice were given intraperitoneal injections of PBS or 2 × 10⁷ SRBCs. On day 9, the percentage of GC B cells within the B-cell population was analysed (left). Expression of Tim-1 on GC B cells from day 9 SRBC challenged WT (solid line) and Tim1KO (dashed line) mice was analysed by flow cytometry using anti-Tim-1 antibody (right). All data shown are derived from a single experiment with five or six mice per group but are representative of three repeat experiments.