Induction of MHC class I expression on immature thymocytes in HIV-1-infected SCID-hu Thy/Liv mice: evidence of indirect mechanisms

Abstract

The SCID-hu Thy/Liv mouse and human fetal thymic organ culture (HF-TOC) models have been used to explore the pathophysiologic mechanisms of HIV-1 infection in the thymus. We report here that HIV-1 infection of the SCID-hu Thy/Liv mouse leads to the induction of MHC class I (MHCI) expression on CD4+CD8+ (DP) thymocytes, which normally express low levels of MHCI. Induction of MHCI on DP thymocytes in HIV-1-infected Thy/Liv organs precedes their depletion and correlates with the pathogenic activity of the HIV-1 isolates. Both MHCI protein and mRNA are induced in thymocytes from HIV-1-infected Thy/Liv organs, indicating induction of MHCI gene expression. Indirect mechanisms are involved, because only a fraction (<10%) of the DP thymocytes were directly infected by HIV-1, although the majority of DP thymocytes are induced to express high levels of MHCI. We further demonstrate that IL-10 is induced in HIV-1-infected thymus organs. Similar HIV-1-mediated induction of MHCI expression was observed in HF-TOC assays. Exogenous IL-10 in HF-TOC induces MHCI expression on DP thymocytes. Therefore, HIV-1 infection of the thymus organ leads to induction of MHCI expression on immature thymocytes via indirect mechanisms involving IL-10. Overexpression of MHCI on DP thymocytes can interfere with thymocyte maturation and may contribute to HIV-1-induced thymocyte depletion.

FIGURE 1

HIV-1 infection-induced MHCI expression on immature thymocytes in the SCID-hu Thy/Liv mouse. A, Mock- or HIV-1 (JD)-infected Thy/Liv organs were harvested at 14 dpi and analyzed for surface MHCI expression (W6/32) on each thymocyte subpopulation. Total live cells were gated based on light scatter profiles. The percentage of each subpopulation is shown. MHCI expression of each subpopulation is shown as the mean channel fluorescence (MCF) in the histograms. B, As in A, NL4-3-infected Thy/Liv organs (14 dpi) showed similar induction of MHCI on immature thymocytes. The difference in basal MHCI levels was due to different flow cytometer settings in different experiments. At least five SCID-hu Thy/Liv mice mock infected or infected with each HIV-1 isolate were analyzed in more than three independent experiments, and similar results were observed. C, The kinetics of MHCI induction on DP thymocytes after infection with two HIV-1 isolates from multiple experiments are summarized. JD (diamond) and NL4-3 (square) are pathogenic viruses that replicate to peak levels at 2 wpi and deplete thymocytes after 3–4 wpi (24). Shown is the average fold induction of MHCI on DP thymocytes (MCF from HIV-1-infected/mock samples) derived from 5–10 SCID-hu Thy/Liv mice at each time point.

FIGURE 2

Induction of MHCI gene expression in HIV-1-infected Thy/ Liv organs. A, SCID-hu Thy/Liv mice infected with mock supernatant (−) or with JD (+) were harvested at 12 and 15 dpi, and total cell proteins from an equal number of thymocytes were analyzed by Western blot with the anti-MHCI mAb, HC-10. Relative MHCI levels on DP thymocytes and percentage of DP thymocytes from mock- and HIV-1-infected Thy/Liv organs are presented. B, Total RNA samples from a mock- or HIV-1-infected SCID-hu Thy/Liv mouse at 2 wpi were analyzed by Northern blot. Relative expression levels of MHCI mRNA was quantified by phosphorimager. The fold HIV-1-induced MHCI RNA expression (7.8) is shown. The β-actin mRNA bands were used to show the relative amounts of total RNA from mock- or HIV-1-infected samples. The experiment was repeated three times with similar results.

FIGURE 3

Induction of MHCI expression in HIV-1-infected HF-TOC assays. HF-TOC were infected with mock or HIV-1 (NL4-3) supernatant and analyzed at 7 dpi. Two representative HIV-1-infected HF-TOC samples were shown (HIV-1a and HIV-1b). MHCI expression on DP thymocytes is shown as the mean channel fluorescence (MCF) in parentheses. The histogram on the right shows cells expressing intracellular HIV-1 Rev detected by a separate FACS analysis (Rev-FITC/CD4-PE/CD8-TC). HIV-1 infection (percentage of Rev⁺) in each thymocyte subpopulation is presented. CEM cell expression of HIV-1 Rev proteins (49) was used as a positive control (not shown). HIV-1a and HIV-1b indicate two independent samples infected with NL4-3. Three independent experiments were performed with similar results

FIGURE 4

Indirect induction of MHCI expression by HIV-1 infection. A, Semiquantitative PCR assays (24) were used to analyze HIV-1 proviral DNA loads in total thymocytes at various times postinfection (2–6 wpi). A representative mock- or HIV-1-infected sample is shown to demonstrate the relative proviral DNA loads. The fold induction of MHCT on DP thymocytes by HIV-1 infection is indicated. Lane 3 of ACH2 control cells at 2 wpi was from PCR run in the absence of human cell DNA. Lanes 1–3 represent 100, 10, and 1% sample cells mixed with normal thymocytes, respectively. For ACH2 cells, lanes 1– 6 represent 100, 10, 1, 0.1, 0.01, and 0% ACH2 cells mixed with normal human thymocytes. A total of 1O4 cells were used in each PCR reaction. Similar results were observed in three independent experiments. B, Purified thymocyte subpopulation from a SCID-hu Thy/Liv mouse infected at 14 dpi (NL4-3). No significant thymocyte depletion was detected at this time point, and MHCI levels on DP thymocytes were induced by 7-fold in this Thy/Liv organ. Thymocyte sub-populations were purified to >90%, and PCR analysis was performed as described above. CD4⁻SP, CD4⁺CD8⁻ thymocytes; CD8⁻SP, CD4⁻CD8⁺ thymocytes; DP, CD4⁺CD8⁺ thymocytes. C, The majority of DP thymocytes with enhanced MHCI expression are not productively infected by HIV-1. Mock or HIV-1 (JD)-infected SCID-hu Ty/Liv mice were analyzed at 2 wpi. Total live cells were gated based on light scatter profiles. The percentage of the DP subpopulation is shown (left panel). MHCI expression of DP thymocytes is shown as the mean channel fluorescence (MCF) in the histograms (middle panel). Intracellular HIV-1 Rev expression was detected by FACS, and the percentage of Rev⁺ cells is shown (right panel). At least three independent experiments with duplicate samples were performed to confirm the results.

FIGURE 5

IL-10 is involved in the induction of MHCI expression on DP thymocytes. A, IL-10 is induced in HIV-1-infected thymus organs. IL-10 was measured by ELISA with thymocyte cell lysates from SCID-hu Thy/Liv organs or HF-TOC fragments. SCID-hu Thy/Liv mice infected with NL4-3 (n = 5) or JD (n = 4) at 2 wpi were analyzed. For HF-TOC assays, NL4-3 (n = 8) or JD (n = 10) infection was analyzed at 4–8 dpi. Fold IL-10 induction indicates IL-10 levels in HIV-1-infected samples divided by IL-10 levels from mock-infected samples. SEs are shown as error bars. B, Induction of MHCI expression by IL-10 in HF-TOC. HF-TOC was analyzed at 4 days postculture in the absence or the presence of IL-10. Fold MHCI induction indicates relative MHCI expression on DP thymocytes from IL-10-treated samples over that from mock-treated samples. Two independent experiments with duplicate samples were performed with similar results. SEs are shown.