Chapter 2: Material and methods
3.3 Aberrant CD45 pre-mRNA alternative splicing
3.3.1 Multiple CD45 transcript variants in thu/thu T cells
As mentioned in chapter 3.2.2, thu/thu thymocytes constitutively express the B220 (CD45RABC) receptor, which is recognized by a monoclonal antibody (RA3-6B2) that binds an epitope on the extracellular domain of the CD45 glycoprotein (Coffman, R.L. & Weissman I.L. 1981; Asensi, V. et al. 1989). The binding of this antibody to the T cell surface is dependent on the expression of exon 4, 5, 6 and specific carbohydrate residues, indicating higher expression of variable exons in thu/thu mice, the expression of which is normally silenced during T cell development and upon T cell activation in the periphery.
Reverse transcript-PCR of thymus cDNA +/+ thu/thu CD45 RO CD45 RB CD45 RAB/BC CD45 RABC
CD4
CD8
B cells
thu/thuCD45RC
CD45.2
CD45RA
CD45RB
a.
b.
thu/+ +/+Figure 3.3 The thundermutation leads to disruption of alternative splicing of CD45 in multiple cell types.
(a) RT-PCR of thymocyte RNA purified from thymocytes of wild type and thu/thumice. Arrows on the left indicate the predicted sizes of the different CD45 isoforms. Data shows representative results from 3 mice of each genotype (predicted size: RO 71bp; RB 218bp; RAB 347bp; RBC 359bp; RABC 488bp).
(b) Representative histograms of CD4+, CD8+ T cells and B cells in the spleen of wild type (red line), thu/+ (green line), and thu/thu(blue line) mice stained with CD45R segment-specific antibodies and a pan- CD45.2 antibody.
To demonstrate that thu/thu thymocytes undergo aberrant CD45 pre-mRNA alternative splicing, total RNA was isolated from thymocytes of wild-type and thu/thu mutant mice and reverse transcribed to cDNA using oligo-dT primer. CD45 transcript variants were amplified by RT-PCR using a pair of primers complementary to exon 3 and exon 7. Thymocytes from wild-type mice predominantly express the CD45RO isoform and some CD45RB, in contrast with thymocytes from thu/thu mice which exhibit expression of multiple transcript variants including CD45RO, RB, RAB, and RBC, suggesting defective CD45 pre-mRNA alternative splicing during thymocytes development in thunder mice (Figure 3.3 a).
3.3.2 T cells from thunder mice cannot silence the variable exons of CD45
A panel of monoclonal antibodies specific to epitopes in the CD45RA, B, and C segments was used to examine the cell surface expression of various CD45 isoforms by T and B cells using flow cytometry. T and B cells from different lymphoid tissues including thymus, spleen, LN, and blood were examined from adult wild-type, heterozygous and homozygous mutant mice. The data shown in Figure 3.3 b and c shows that B cells cannot silence the variable exons 4, 5 or 6 of CD45 and therefore constitutively express high levels of CD45RA, RB and RC epitopes. In contrast, wild- type CD4 T cells have low expression of CD45RA and RC but maintain an intermediate level of CD45RB isoform expression. The levels of the different CD45 isoforms increases 2-4 fold after CD4+ cells leave the thymus and enter the periphery (Fig 3.3c). Also there is a 10 fold increase in the expression of CD45RA and RC segments after CD8+ cells migrate from the thymus to the periphery (Fig. 3.3c). CD8+ T cells in the spleen of wild-type mice have approximately 10 fold higher
c.
CD45.2 Expression 1 10 100 1000 10000 DN DP CD4SP CD8SP CD19 CD4 CD8 Ge o Me a n +/+ Thu/+ Thu/Thu CD45RC expression 1 10 100 1000 10000 DN DP CD4SP CD8SP CD19 CD4 CD8 G eoMean +/+ Thu/+ Thu/Thu CD45RB expression 1 10 100 1000 10000 DN DP CD4SP CD8SP CD19 CD4 CD8 G eoM ean +/+Thu/+ Thu/Thu C D 45R A expression 1 10 100 1000 D N D P C D 4S P C D 8S P C D 19 C D 4 C D 8 G eoM ean +/+ Thu/+ Thu/ThuFigure 3.3 The thundermutation leads to disruption of alternative splicing of CD45 in multiple cell types.
(c). Bar graphs representing the geometric mean fluorescence of different CD45R epitopes and total CD45 on the surface of DN, DP CD4SP and CD8SP thymocytes, as well as CD4+, CD8+ and CD19+ spleen cells. Data showing the mean and standard deviation of data were collected from n=6 mice/group. Wild type mice (open bars), thu/+(hatched bars), thu/thu(filled bars).
levels of CD45RA, RB and RC compared to CD4+ T cells (Fig. 3.3 b and c). Thus in wild-type mice the expression of CD45 isoforms changes with cell types and T cell development and maturation state of the cells.
When we examined the expression of the CD45R epitopes on thymocytes and spleen cells from thunder homozygous mutant mice, the thu/thu T cells display constitutive high level expression of all 3 segments, equivalent to the levels expressed by B cells (Fig. 3.3 b and c). In heterozygous mice the expression of all three CD45R epitopes in CD4+ cells (and CD45RA & RC in CD8+ cells) was intermediate to that observed in homozygous mutant mice (Fig. 3.3 b and c) suggesting a dose-dependent effect of the mutation on CD45 alternative splicing. The only exception was the CD45RB isoform on CD8+ T cells which showed little effect with the thunder mutation mainly because this exon is not normally silenced in CD8+ cells. However, quantitating the levels of expression of CD45 RB on multiple animals indicated that there was still a 1.5-2 fold increase in CD45RB levels on thunder homozygote mutant CD8+ cells compared to wild-type CD8+ cells (Fig. 3.3 c). These results indicate that the thunder mutation has an essential role in CD45 pre-mRNA alternative splicing in T cells, disruption of thunder gene causes failure in silencing CD45 variable exons 4, 5, and 6, and leading to the constitutive expression of high molecular weight isoforms of CD45 at the surface of T cells regardless of status of development and activation.
3.3.3 Aberrant CD45 alternative splicing in other hematopoietic cells
We were interested to know if the defects in CD45 alternative splicing were restricted to just T cells so we examined the expression of the different CD45
Gr
CD11c DC CD11b MØ
thu/thu +/+
Figure 3.3 The thundermutation leads to disruption of alternative splicing of CD45 in multiple cell types.
Representative histograms comparing CD45RA and CD45RC expression on (d) NK and TCRγδ+ cells in the thymus and (e) Gr-1+ cells, CD11c+ DCs and CD11b+ macrophages in the spleen. (red lines = +/+ cells, blue line=thu/thucells)
isoforms expression on a range of other hematopoietic cells in the thymus and spleen including NK cells, γδ T cells, dendritic cells, macrophages, and granulocytes. As shown in Figure 3.3 d and e, dysregulation of CD45 splicing was observed in all leukocyte populations as all the cell types tested constitutively expressed the high MW isoforms of CD45 containing the CD45RA and/or CD45RC epitopes. This suggests thunder mutation has a broad role in regulating CD45 pre-mRNA alternative splicing in all nucleated haematopoietic cells.