Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice

Abstract

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.

Figure 1

Generation of TC-PTP–deficient mutant mice by gene targeting. (A) Targeting construct of TC-PTP. Diagram of wt TC-PTP genomic locus encompassing four exons (nc 227–771, aa 54–235, in TC-PTP cDNA; reference 14). Probe A hybridizes to a 5.2-kb fragment generated by NdeI digestion. The targeting construct using a 5.5-kb genomic sequence with the neo resistance gene eliminates ∼9 kb of genomic sequence including 1.5 exons. The resulting targeted locus after a correct homologous recombination event is shown. In the targeted allele, probe A hybridizes to a 7.6-kb fragment generated by NdeI digestion. H3, HindIII. The thick line in the targeting construct diagram represents pBluescript^TM IIKS(+) backbone sequences. (B) Southern blot analysis of representative ES cell clones after electroporation. For each clone, DNA was extracted from one well of a 96-well plate and digested NdeI (+/+, wt; +/−, heterozygously targeted clone [arrow]). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb fragments, respectively. (C) Southern blot analysis of mouse tail DNA from progeny of heterozygous mouse matings. After DNA extraction, 50 μg of tail DNA were digested with NdeI (+/+, wt; +/−, heterozygotes; −/−, homozygotes). Hybridization was with probe A. Targeted allele and normal allele correspond to the 7.6-kb and 5.2-kb fragments, respectively. (D) Northern blot analysis of total RNA extracted from spleen of wt (+/+) and homozygous (−/−) mice. Wt TC-PTP message was detected as a 1.5-kb transcript using TC-PTP cDNA as a probe. The same blot was stripped and reprobed with a glyceraldehyde 3-phosphate dehydrogenase probe to assess the level of loading. (E) Western blot analysis of total cellular protein extracted from spleen of wt (+/ +) and homozygous (−/−) mice using the anti TC-PTP monoclonal antibody 3E2. TC-PTP migrates at 45 kD. Other bands represent immunogloblin chain subunits that are recognized by the secondary goat anti– mouse horseradish peroxidase conjugated antibody. (F) Table of progeny survival in littermates from the crossing of heterozygous mice for the TC-PTP disruption.

Figure 2

Total cellularity and percentages of B cell populations in the LNs, spleen, and BM of TC-PTP–deficient mice. Total cellularity on different days after birth from the (A) LNs, (B) spleen, and (C) BM. Data are presented as the mean of 3–6 mice/group. Pre-B (B220⁺sIgM⁻) and mature B cell (B220⁺sIgM⁺) populations were determined by two-color flow cytometry. Each column and row of FACS^® profiles represents the different groups (+/+, +/−, −/−) and different days after birth (D7, D13, and D21), respectively. Numbers in the upper quadrants of each FACS^® profile represent the percentage of the population from the (D) LNs, (E) spleen, and (F) BM.

Figure 2

Total cellularity and percentages of B cell populations in the LNs, spleen, and BM of TC-PTP–deficient mice. Total cellularity on different days after birth from the (A) LNs, (B) spleen, and (C) BM. Data are presented as the mean of 3–6 mice/group. Pre-B (B220⁺sIgM⁻) and mature B cell (B220⁺sIgM⁺) populations were determined by two-color flow cytometry. Each column and row of FACS^® profiles represents the different groups (+/+, +/−, −/−) and different days after birth (D7, D13, and D21), respectively. Numbers in the upper quadrants of each FACS^® profile represent the percentage of the population from the (D) LNs, (E) spleen, and (F) BM.

Figure 2

Total cellularity and percentages of B cell populations in the LNs, spleen, and BM of TC-PTP–deficient mice. Total cellularity on different days after birth from the (A) LNs, (B) spleen, and (C) BM. Data are presented as the mean of 3–6 mice/group. Pre-B (B220⁺sIgM⁻) and mature B cell (B220⁺sIgM⁺) populations were determined by two-color flow cytometry. Each column and row of FACS^® profiles represents the different groups (+/+, +/−, −/−) and different days after birth (D7, D13, and D21), respectively. Numbers in the upper quadrants of each FACS^® profile represent the percentage of the population from the (D) LNs, (E) spleen, and (F) BM.

Figure 2

Total cellularity and percentages of B cell populations in the LNs, spleen, and BM of TC-PTP–deficient mice. Total cellularity on different days after birth from the (A) LNs, (B) spleen, and (C) BM. Data are presented as the mean of 3–6 mice/group. Pre-B (B220⁺sIgM⁻) and mature B cell (B220⁺sIgM⁺) populations were determined by two-color flow cytometry. Each column and row of FACS^® profiles represents the different groups (+/+, +/−, −/−) and different days after birth (D7, D13, and D21), respectively. Numbers in the upper quadrants of each FACS^® profile represent the percentage of the population from the (D) LNs, (E) spleen, and (F) BM.

Figure 2

Total cellularity and percentages of B cell populations in the LNs, spleen, and BM of TC-PTP–deficient mice. Total cellularity on different days after birth from the (A) LNs, (B) spleen, and (C) BM. Data are presented as the mean of 3–6 mice/group. Pre-B (B220⁺sIgM⁻) and mature B cell (B220⁺sIgM⁺) populations were determined by two-color flow cytometry. Each column and row of FACS^® profiles represents the different groups (+/+, +/−, −/−) and different days after birth (D7, D13, and D21), respectively. Numbers in the upper quadrants of each FACS^® profile represent the percentage of the population from the (D) LNs, (E) spleen, and (F) BM.

Figure 2

Total cellularity and percentages of B cell populations in the LNs, spleen, and BM of TC-PTP–deficient mice. Total cellularity on different days after birth from the (A) LNs, (B) spleen, and (C) BM. Data are presented as the mean of 3–6 mice/group. Pre-B (B220⁺sIgM⁻) and mature B cell (B220⁺sIgM⁺) populations were determined by two-color flow cytometry. Each column and row of FACS^® profiles represents the different groups (+/+, +/−, −/−) and different days after birth (D7, D13, and D21), respectively. Numbers in the upper quadrants of each FACS^® profile represent the percentage of the population from the (D) LNs, (E) spleen, and (F) BM.

Figure 3

Immune function of TC-PTP–deficient mice. (A) Proliferative responses of splenic cells to B- and T cell–specific mitogens, LPS, and Con A, respectively. Mice were killed 21 d after birth. Data are presented as the mean ± SE of 3–6 mice/group. (B) Response of splenic PFCs against SRBCs. Mice of 21 d of age were injected intravenously with SRBCs and killed 4 d after immunization. Data are presented as the mean PFC ± SE/10⁶ spleen cells.

Figure 4

Immune function and hematocrit of irradiated wt recipients rescued with wt or TC-PTP–deficient BM. (A) Immune function of irradiated wt recipients reconstituted with TC-PTP wt or −/− BM. Immune function was assessed by the ability of splenic cells to proliferate in response to Con A and LPS. Data represents the mean ± SE of 3–6 mice/ group. (B) Hematocrit level of irradiated wt recipients rescued with TC-PTP −/− BM. Data are presented as the mean ± SE of 3 mice/group.

Figure 5

Bone marrow histology. Histology of the BM microenvironment of nonirradiated TC-PTP–deficient mice. Analysis of the BM was performed on 21-d-old mice. (A) Stromal cells (arrows) are attached to, or clustered between bone trabeculae in +/− control mice. (B) By contrast, a marked paucity of stromal cells is observed in the BM of −/− mice in those areas normally rich in stromal cells. The hematopoietic cell population is comparable in both groups of mice. Periodic–acid Schiff stain; original magnification: 150.

Figure 5

Bone marrow histology. Histology of the BM microenvironment of nonirradiated TC-PTP–deficient mice. Analysis of the BM was performed on 21-d-old mice. (A) Stromal cells (arrows) are attached to, or clustered between bone trabeculae in +/− control mice. (B) By contrast, a marked paucity of stromal cells is observed in the BM of −/− mice in those areas normally rich in stromal cells. The hematopoietic cell population is comparable in both groups of mice. Periodic–acid Schiff stain; original magnification: 150.