Combinations of CD45 isoforms are crucial for immune function and disease

Abstract

Expression of the CD45 Ag in hemopoietic cells is essential for normal development and function of lymphocytes, and both mice and humans lacking expression exhibit SCID. Human genetic variants of CD45, the exon 4 C77G and exon 6 A138G alleles, which alter the pattern of CD45 isoform expression, are associated with autoimmune and infectious diseases. We constructed transgenic mice expressing either an altered level or combination of CD45 isoforms. We show that the total level of CD45 expressed is crucial for normal TCR signaling, lymphocyte proliferation, and cytokine production. Most importantly, transgenic lines with a normal level, but altered combinations of CD45 isoforms, CD45(RABC/+) and CD45(RO/+) mice, which mimic variant CD45 expression in C77G and A138G humans, show more rapid onset and increased severity of experimental autoimmune encephalomyelitis. CD45(RO/+) cells produce more TNF-alpha and IFN-gamma. Thus, for the first time, we have shown experimentally that it is the combination of CD45 isoforms that affects immune function and disease.

FIGURE 1

Characterization of single isoform CD45RABC and CD45RO Tg mice. Flow cytometric analysis showing the surface expression of CD45, detected with a pan-specific anti-CD45 mAb, on CD3-gated thymocytes from CD45^+/+ (filled histogram), CD45^−/−, CD45RABC^high, CD45RABC^low, CD45R0^high, and CD45R0^low mice.

FIGURE 2

Activation of lymph node T cells from CD45^+/+ and CD45 Tg mice expressing single isoforms. A, Total mesenteric and peripheral lymph node cells from CD45RABC^high and CD45RO^high mice were stimulated with varying amounts of plate-bound CD3 and 1 μg/ml CD28 Abs. Separated CD4 and CD8 lymph node cells were stimulated with 2 μg/ml CD3 and 1 μg/ml CD28 Abs. B, Lymph node cells were stimulated with T cell-depleted and irradiated allogeneic BALB/c splenocytes at ratios of 10:1, 5:1, and 1:1 (stimulator:target), or C, with PMA-ionomycin. D, CD45RABC^low and CD45RO^low lymph node cells were stimulated with 2 μg/ml plate-bound CD3 and 1 μg/ml CD28 Abs or with PMA-ionomycin. Cells were pulsed with [³H]thymidine at the indicated times and harvested 12 h later. Means and SDs of triplicate cultures from three mice are shown. Background counts were <500 cpm and have been subtracted. E, Western blot of Lck, pY505Lck, and pY394Lck in lymph node T cells stimulated with soluble CD3 at 2 μg/ml and CD28 at 1 μg/ml for the times indicated. Equal amount of cell lysate protein (10 μg) was loaded on each lane. Data are representative of six experiments.

FIGURE 3

Cytokine production by total or CD4- and CD8-separated lymph node cells of CD45 Tg mice expressing single CD45RABC or CD45RO isoforms. A, Amount of TNF-α, IFN-γ, IL-5, and IL-2 present in cell culture supernatants after 72-h stimulation with 2 μg/ml CD3 and 1 μg/ml CD28, or B, PMA-ionomycin. Histograms show the response and SD of three mice of each type. Data are representative of four experiments for total CD3-positive cells and two experiments for CD4- and CD8-separated cells. Statistically significant differences from wild-type CD45^+/+ controls are shown.

FIGURE 4

Characterization of CD45^RABC/+ and CD45^RO/+ mice. A, CD45 expression in CD45^RABC/+ and CD45^RO/+ mice. Lymph node cells were stained with pan CD45, CD45RA, and CD45RB isoform-specific Abs. Analysis was performed on CD4- and CD8-gated cells. The shaded histogram is CD45^+/+, dotted line CD45^RABC/+, solid CD45^RO/+, and the gray dotted isotype control. Examples are representative of three mice of each type. B, Activation of T cells from CD45^+/+, CD45^RABC/+, and CD45^RO/+ mice. Mesenteric and peripheral lymph node cells were activated in the presence of varying amounts of plate-bound CD3 and 1 μg/ml CD28 Abs. Separated CD4 and CD8 lymph node cells were stimulated with 2 μg/ml CD3 and 1 μg/ml CD28 Abs. Cells were pulsed with [³H]thymidine at the indicated times and harvested after 12 h. Means and SDs of triplicate cultures from three mice are shown. Background counts were <500 cpm and have been subtracted. Data are representative of five experiments. C, Annexin staining of cells cultured in the presence of 2 μg/ml CD3 and 1 μg/ml CD28 for the indicated times. Means and SDs of triplicate cultures from three mice are shown. D, Western blot of Lck, pY505Lck, and pY394Lck in lymph node T cells stimulated with 2 μg/ml CD3 and 1 μg/ml CD28 for the times indicated. Data are representative of three experiments.

FIGURE 5

Cytokine production and response of CD45^+/+, CD45^RABC/+, and CD45^RO/+ mice. A, Total lymph node cells or separated CD4 and CD8 cells were stimulated for 72 h with CD3/CD28, or B, PMA-ionomycin. The histograms show means and SD of the amounts in supernatants of triplicate cultures from three mice. Results are representative of six experiments for total lymph node cells and three for separated CD4 or CD8 cells. C, Activation of lymph node T cells with PMA-ionomycin. Cells were pulsed with [³H]thymidine at 72 h and harvested 12 h later. Means and SDs of triplicate cultures from three mice are shown. D, Western blot of STAT1, pY701STAT1, and T-bet in lymph node T cells stimulated with PMA-ionomycin for the times indicated. Data are representative of three experiments.

FIGURE 6

EAE in CD45^+/+, CD45^RABC/+, and CD45^RO/+ mice. EAE was induced in groups of five to nine mice of each strain, and disease was monitored, as described in Materials and Methods. The graph represents the mean score for each day from three experiments.