Disturbed CD4+ T cell homeostasis and in vitro HIV-1 susceptibility in transgenic mice expressing T cell line-tropic HIV-1 receptors - PubMed (original) (raw)

Disturbed CD4+ T cell homeostasis and in vitro HIV-1 susceptibility in transgenic mice expressing T cell line-tropic HIV-1 receptors

S Sawada et al. J Exp Med. 1998.

Abstract

T cell line-tropic (T-tropic) HIV type 1 strains enter cells by interacting with the cell-surface molecules CD4 and CXCR4. We have generated transgenic mice predominantly expressing human CD4 and CXCR4 on their CD4-positive T lymphocytes (CD4+ T cells). Their primary thymocytes are susceptible to T-tropic but not to macrophage-tropic HIV-1 infection in vitro, albeit with a viral antigen production less efficient than human peripheral blood mononuclear cells. Interestingly, even without HIV infection, transgenic mice display a CD4+ T cell depletion profile of peripheral blood reminiscent of that seen in AIDS patients. We demonstrate that CD4+ T cell trafficking in transgenic mice is biased toward bone marrow essentially due to CXCR4 overexpression, resulting in the severe loss of CD4+ T cells from circulating blood. Our data suggest that CXCR4 plays an important role in lymphocyte trafficking through tissues, especially between peripheral blood and bone marrow, participating in the regulation of lymphocyte homeostasis in these compartments. Based on these findings, we propose a hypothetical model in which the dual function of CXCR4 in HIV-1 infection and in lymphocyte trafficking may cooperatively induce progressive HIV-1 infection and CD4+ T cell decline in patients.

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Figures

Figure 1

(A–D) hCD4 and hCXCR4 are expressed predominantly on CD4+ subsets of PBLs and thymocytes in transgenic mice. Three-color flow cytometric analysis of hCD4 and hCXCR4 expression on PBLs (A and B) and thymocytes (C and D) of a control nontransgenic mouse (Non-Tg) and of a transgenic litter mate (Tg). Cells were stained with FITC-conjugated monoclonal antibody against hCD4 (A and C) or hCXCR4 (B and D) in the presence of monoclonal antibodies against murine CD4 (PE) and CD8 (TRICOLOR). (E) Three-color analysis of receptors for murine SDF-1 (open histograms) on gated CD4+CD8− (CD4 SP) and CD4−CD8+ (CD8 SP) subsets of lymph node T cells from control and transgenic littermates. Background levels obtained with the FITC anti–huIgG alone are shown in solid histograms.

Figure 2

T-tropic HIV infection in transgenic thymocytes. (A) PHA-stimulated thymocytes from 6-wk-old control nontransgenic mice, transgenic mice, or human PBMCs were exposed to T-tropic HIV strains IIIB and SF2, or an M-tropic SF162 for 3 h. After washing three times with PBS, cells were treated with trypsin-EDTA for 3 min, and then washed three times with complete medium. The p24 antigen was assayed in the medium immediately after cell washing (day 0) and in the culture supernatant harvested on the indicated days after infection. Background levels taken to be those on day 0 were subtracted from the amount of p24. Results shown are the mean ± SD of duplicated infections. (B) PCR amplification of HIV DNA in control or transgenic thymocytes infected with IIIB or SF162. Murine IL-1 gene primers were used for internal control. The product with LTR5/LTR6 represents the early HIV DNA product, whereas that with LTR5/5NC2 represents the late product (22). Anticipated DNA size is as follows: 139 bp (LTR5/LTR6), 202 bp (LTR5/5NC2), 308 bp (IL-1). Similar results were obtained in two independent experiments (A and B).

Figure 2

Figure 3

Abnormal CD4+ T cell distribution in transgenic mice and transient normal distribution recovery after an injection of pertussis toxin. (A) Flow cytometric analysis of lymphocytes in tissues from 5-wk-old nontransgenic (left) and transgenic (right) litter mates. (B) Percentage of CD4 SP T cells in lymphoid cells from tissues of 5-wk-old litter mates (n = 9). CD3+ CD4 SP T cells are shown for bone marrow because it contained immature CD3− CD4+ cells. Bone marrow cells gated on lymphoid cells may include immature myeloid and erythroid lineage cells. Data is the averaged value obtained with three nontransgenic and six transgenic mice. The averaged number of lymphoid cells in each tissue is as follows: peripheral blood, 9.78 × 106/ml (Non-Tg) vs. 9.38 × 106/ml (Tg); thymus, 1.97 × 108 vs. 1.45 × 108; bone marrow, 2.52 × 107 vs. 1.95 × 107; spleen, 6. 07 × 107 vs. 3.71 × 107; lymph node, 7.27 × 107 vs. 6.15 × 107. PB, peripheral blood; LN, lymph node; BM, bone marrow. (C) Before and after intraperitoneal injection of pertussis toxin (500 ng) or control saline, murine peripheral blood was analyzed using a flow cytometer on indicated days. The percentage of CD4 SP T cells in blood lymphocytes is shown. Data represents mean values obtained with two mice for each experimental set. Similar results were obtained in two independent experiments. (D) 5 d after injection of pertussis toxin (500 ng) or control saline i.p., mice were analyzed for CD3+ CD4 SP T cell counts in blood and bone marrow (two femurs). Data is mean values ± SD obtained with two mice. PT, pertussis toxin. Similar results were obtained in three independent experiments.

Figure 3

Figure 4

Hyperresponse of transgenic CD4+ T cells to SDF-1 in a chemotaxis assay. (A) Spleen mononuclear cells from nontransgenic and transgenic mice or from human PBLs were analyzed for migration toward the SDF-1 gradient. The percentage of migrated CD4 SP or CD8 SP T cells to input cells of each subset was calculated (Materials and Methods). (B) SDF-1-induced chemotaxis of murine CD4 SP T cells was eliminated with pertussis toxin. After 2 h pretreatment with medium or pertussis toxin (100 ng/ml), chemotaxis of lymphocyte subsets in response to SDF-1 (40 ng/ml) was analyzed. Results (A and B) are the mean ± SD of duplicated experiments. Similar results were obtained in two independent experiments (A and B).

Figure 5

In vivo migration of transgenic CD4+ T cells. 9 × 106 (A) or 5 × 106 (B) lymphocytes from two transgenic mice lines expressing either hCD4 alone (hCXCR4−) or both hCD4 and hCXCR4 (hCXCR4+) were injected intravenously into normal BDF1 mice. The proportions of hCD4+, mCD4+, and mCD8− in each lymphocyte preparation were similar (16–23%). Values are mean numbers (±SD) of recovered hCD4+, mCD4+, and mCD8− donor T cells in peripheral blood (per 1 ml) or bone marrow (per two femurs) of recipient mice 3 (A) or 16 (B) h after injection. Two (A) and five (B) mice were used for each cell transfer. *P < 0.05, **P < 0.01, and ***P < 0.0001 determined by Student's t test. Similar results were obtained in three independent experiments.

Figure 6

Models for the CXCR4 role in lymphocyte trafficking and in AIDS pathogenesis. (A) CXCR4high lymphocytes such as activated lymphocytes (28) preferentially migrate to bone marrow or other tissues/microenvironments whose stromal cells produce high levels of SDF-1 (29). CXCR4 expression regulation may thus involve lymphocyte trafficking between peripheral blood and bone marrow, influencing lymphocyte homeostasis in these compartments. (B) In transgenic mice, hCXCR4 is constitutively expressed on CD4+ T cells, and SDF-1 receptors are overexpressed in these cells (see text). CD4+ T cells are therefore trapped in bone marrow, resulting in their loss from peripheral blood. (C) Model for the CD4+ T cell decline in AIDS patients. When active HIV-1 replication loci are present in bone marrow (42, 43), T-tropic HIV-1 infection accelerates because of preferential recruitment of CXCR4 high lymphocytes to bone marrow. Migrated CXCR4high CD4+ T cells are destroyed after HIV-1 infection and do not return to the blood pool. Note that the trafficking balance of CD4+ T cells through bone marrow mimics that in transgenic mice in B.

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Disturbed CD4+ T cell homeostasis and in vitro HIV-1 susceptibility in transgenic mice expressing T cell line-tropic HIV-1 receptors - PubMed (original) (raw)