Cutting Edge: Persistent Viral Infection Prevents Tolerance Induction and Escapes Immune Control Following CD28/CD40 Blockade-Based Regimen

Mice and virus infections

Adult male 6- to 8-wk-old BALB/c and C57BL/6 (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6 LCMV carrier mice were bred at Emory University (Atlanta, GA) as previously described (8). Mice were infected with 2 × 10⁵ PFU of LCMV Armstrong injected i.p. to induce acute infection, or with 2 × 10⁶ PFU of LCMV clone 13 (i.v.) to induce chronic infection. Virus stocks were grown and quantitated as previously described (7). Infectious LCMV in serum and tissues was measured by plaque assay on Vero cell monolayers as described (7).

Skin grafting

Full thickness skin grafts (∼1 cm²) were transplanted on the dorsal thorax of recipient mice and secured with a band-aid for 7 days. Graft survival was then followed by daily visual inspection. Rejection was defined as the complete loss of viable epidermal graft tissue.

Bone marrow preparation and treatment protocols

The mixed chimerism tolerance regimen, involving treatment with donor bone marrow, busulfan (Busulfex; Orphan Medical, Minnetonka, MN), CTLA4-Ig, and anti-CD40 ligand (CD40L) (MR1), has been described elsewhere (4). The dose of MR1 (2 mg total over four treatments) has been shown to generate therapeutically effective levels for >50 days (9).

Cell preparations and flow cytometry

Intracellular IFN-γ expression was induced in response to ex vivo restimulation with LCMV peptides as described (8). Cells were stained with anti-IFN-γ and anti-CD8 (BD PharMingen, San Diego, CA) using the Cytofix/Cytoperm kit according to the manufacturer’s instructions (BD PharMingen). Peripheral blood was analyzed by staining with the indicated fluorochrome-conjugated Abs (BD PharMingen), followed by RBC lysis and washing with a whole blood lysis kit (R&D Systems, Minneapolis, MN). Flow cytometry was performed on a FACSCalibur and data were analyzed using CellQuest software (BD Biosciences, Braintree, MA).

Ongoing chronic infection, but not prior acute infection, prevents tolerance induction, mixed chimerism, and deletion of donor-reactive T cells

We have previously described a method for establishing indefinite allospecific tolerance and 50–70% mixed hemopoietic chimerism following treatment with the stem cell-selective toxin busulfan, administration of donor bone marrow, and blockade of the CD28 and CD40 costimulatory pathways (4). However, we have recently reported that tolerance induction could be inhibited by a concurrent acute LCMV infection (6). In the present experiments, we sought to determine the effects of preexisting long-term chronic viral infections on tolerance induction, the development of hemopoietic chimerism, and the deletion of donor-reactive T cells. Mice infected with LCMV clone 13 typically develop high viral titers in the serum that are gradually controlled by the host T cell response over the course of 2–3 mo. Virus is generally controlled and cleared from the serum by day 90 postinfection and persists at very low levels in peripheral organs indefinitely (7). We assessed the efficacy of the tolerance regimen during the course of a chronic infection by administering the tolerance induction protocol to mice 15, 60, and 120 days postinfection. As expected, viral titers in mice infected with LCMV clone 13 gradually decrease and disappear from the serum by day 120 postinfection (Fig. 1⇓, a, d, and g). However, low levels of virus persisted in the kidneys of infected mice as late as 250 days postinfection (data not shown). Conversely, mice infected with LCMV Armstrong, an acutely infecting strain, rapidly clear the virus from the serum (Fig. 1⇓j) and peripheral tissues (data not shown).

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FIGURE 1.

Chronic infection with LCMV clone 13 prevents tolerance induction and chimerism over a long time course. B6 mice were infected with 2 × 10⁶ PFU of LCMV clone 13. At 15 (a–c), 60 (d–f), or 120 (g–i) days postinfection, mice were treated with the tolerance induction regimen and given a BALB/c skin graft. j–l, LCMV immune mice also received the tolerance regimen 30 days after an acute infection with LCMV Armstrong. a, d, g, Mice were bled on days 15, 60, or 120 postinfection, respectively, and tested for the presence of LCMV in the serum. The dotted line represents the limit of detection. Mice transplanted at these time points were assessed for skin graft survival (b, e, and h) and donor cell chimerism (c, f, and i) over a long time course (at day 15, n = 15 for infected, n = 10 for uninfected; at days 60 and 120, n = 9 for infected, n = 8 for uninfected). Similar assays were performed for LCMV-immune mice. j, Serum titers were measured 30 days after acute infection with LCMV Armstrong (n = 5). Mice received the tolerance regimen and a BALB/c skin allograft >30 days postinfection and were assessed for skin graft survival (k) and chimerism (l) (n = 10 for infected, n = 8 for uninfected). All error bars represent the SEM (p < 0.001 for all infected vs uninfected groups). Skin graft survival and chimerism data are pooled from two separate experiments.

B6 mice receiving a BALB/c skin graft along with donor bone marrow, costimulation blockade, and busulfan 15 days postinfection rejected their grafts promptly (Fig. 1⇑b, median survival time (MST) = 18 days). In contrast, 10 of 10 uninfected control mice displayed indefinite graft survival (MST >200 days). Infected mice also failed to develop detectable levels of stable mixed chimerism, while uninfected controls generally developed ≥50% chimerism in the peripheral blood by day 60 (Fig. 1⇑c). Mice also rejected skin grafts promptly when receiving the tolerance regimen 60 (MST = 23 days) or 120 (MST = 27 days) days postinfection (Fig. 1⇑, e and h). Failure to accept skin grafts was further reflected in a failure to generate mixed chimerism following transplantation of infected mice, while uninfected controls established high levels of stable mixed chimerism (Fig. 1⇑, f and i). In comparison, 8 of 10 mice with a prior acute LCMV Armstrong infection (>30 days postinfection) accepted donor skin grafts (>200 days) indefinitely and developed stable mixed chimerism (Fig. 1⇑, k and l).

In aggregate, while 25 of 26 uninfected B6 mice were rendered tolerant and chimeric, none of the 33 infected animals accepted skin allografts or developed chimerism. It is notable that even mice with no detectable virus in the serum rapidly rejected skin allografts following administration of donor bone marrow, busulfan, and costimulation blockade.

To determine whether LCMV-induced skin graft rejection was associated with impaired peripheral deletion of donor-reactive T cells, we compared the use of Vβ11 and Vβ5.1/2 by CD4⁺ T cells from B6 recipients in the uninfected group (accepted both bone marrow and skin grafts) and from the infected groups (rejected bone marrow and skin grafts). BALB/c mice delete Vβ11- and Vβ5-bearing T cells in the thymus due to their high affinity for endogenous retroviral superantigens (mouse mammary tumor virus) presented by I-E MHC class II molecules, whereas B6 mice do not express I-E and thus use Vβ11 on ∼5–7% of CD4⁺ T cells and Vβ5.1/2 on ∼3–5% of CD4⁺ T cells. Treatment with costimulation blockade and the subsequent development of chimerism in B6 recipients results in the deletion of the donor-reactive Vβ5 and Vβ11 subsets (2, 3). Mice receiving the tolerance regimen 15, 60, or 120 days postinfection were bled 120 days posttransplant and tested for the presence of Vβ5- and Vβ11-bearing CD4⁺ T cells. Chronic infection with LCMV prevented the deletion of donor-reactive T cells at all time points (Fig. 2⇓). In contrast, tolerant control animals deleted these subsets. We concluded that inhibition of tolerance induction by chronic LCMV infection was associated with a failure to delete donor-reactive T cells from the periphery.

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FIGURE 2.

Chronic LCMV infection prevents deletion of donor-reactive T cells. B6 mice were infected with 2 × 10⁶ PFU of LCMV clone 13, and then received the tolerance regimen and a BALB/c skin graft 15 (a), 60 (b), or 120 (c) days postinfection. Mice were bled 120 days posttransplant and peripheral leukocytes stained for the presence of Vβ5⁺CD4⁺ and Vβ11⁺CD4⁺ cells. The y-axis is the percentage of CD4⁺ cells that are also Vβ5⁺ or Vβ11⁺ for infected (n = 5 for day 15, n = 4 for day 60, n = 4 for day 120), uninfected tolerant (n = 4 for all time points), B6 (n = 3), and BALB/c (n = 3) mice. Error bars represent the SEM (p < 0.001 between all infected and uninfected tolerant groups).

Costimulation blockade impairs ongoing antiviral immunity

The observation that chronic LCMV infection could induce alloresponses sufficient for graft rejection suggested that administration of costimulation blockade-based immunosuppressive therapy was unlikely to disrupt antiviral responses and prevent effective viral clearance. To test this, we measured viral titers in the serum of animals that had received either no treatment or the full tolerance regimen (skin allograft, donor bone marrow, busulfan, CTLA4-Ig, and anti-CD40L) either 15, 60, or 120 days postinfection. Eight of eight mice receiving the regimen at day 15 and four of eight mice receiving the regimen at day 60 failed to clear virus from the serum by day 150 postinfection (Fig. 3⇓a) and throughout the course of the experiment (>200 days, data not shown). Significantly, mice treated 120 days postinfection that had already resolved viremia but maintained low levels of virus in the kidney did not display reemergence of the virus in the peripheral blood at day 150 postinfection (Fig. 3⇓a) or at later time points (data not shown). As a treatment control, mice given bone marrow and busulfan without costimulation blockade on day 15 postinfection successfully controlled viremia with normal kinetics (data not shown). We further found that treatment with costimulation blockade alone (CTLA4-Ig and anti-CD40L), beginning at day 0 or 20 postinfection, also prevented normal viral clearance from the serum by day 90 postinfection (Fig. 3⇓b).

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FIGURE 3.

Costimulation blockade-based therapies prevent control of ongoing chronic infection. a, B6 mice were infected with LCMV clone 13 and received either no further treatment (No Tx; n = 7) or the tolerance regimen on days 15 (d15 Tx; n = 8, p < 0.01 vs No Tx), 60 (d60 Tx; n = 8, p < 0.05 vs No Tx), or 120 (d120 Tx; n = 6) postinfection. Mice were bled at day 150 postinfection and serum was tested for the presence of LCMV. The y-axis displays plaque forming units per milliliter. The dotted line indicates the limit of detection for this assay. b, B6 mice were treated with LCMV and received either no further treatment (No Tx; n = 10) or administration of CTLA4-Ig and anti-CD40L starting on day 0 (CB day 0; n = 10, p < 0.05 vs No Tx) or day 20 (CB day 20; n = 10, p < 0.05 vs No Tx) postinfection. Mice were bled on day 90 postinfection and serum was tested for the presence of LCMV. The y-axis displays plaque forming units per milliliter in the serum. The dotted line indicates the limit of detection for this assay. Results are pooled from two separate experiments.

To determine whether the treatment regimen could specifically blunt ongoing antiviral CD8 T cell responses to chronic LCMV, we treated mice with the full tolerance regimen or costimulation blockade alone 20 days postinfection, then assessed by IFN-γ staining the frequency of LCMV-specific T cells in the spleen 15 days later. We observed ∼3- to 4-fold decreased numbers of CD8 T cells specific for the gp33–41, gp276–286, and gp118–128 epitopes in the spleens of mice receiving the tolerance regimen or costimulation blockade treatment alone, as compared with untreated controls (Fig. 4⇓). Responses to the nuclear protein 396–404 epitope were virtually nonexistent in all groups, as previously documented (10).

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FIGURE 4.

Costimulation blockade blunts ongoing antiviral CD8 T cell responses. B6 mice were infected with LCMV clone 13. At 20 days postinfection, they either received no further treatment (No Tx; n = 3), the tolerance regimen (Tol. reg.; n = 5), or costimulation blockade alone (CB; n = 4). Fifteen days later, splenocytes were harvested, restimulated with the indicated peptides in the presence of brefeldin A, and stained for intracellular expression of IFN-γ. Splenocytes were restimulated with the gp33–41, nuclear protein 396–404, gp276–286, or gp118–128 peptides, as indicated on the x-axis. The total number of CD8⁺IFN-γ⁺ cells per spleen of each treatment group is indicated on the y-axis, as calculated based on the percentage of CD8⁺IFN-γ⁺ and the total number of splenocytes harvested. Error bars represent the SEM (p < 0.001 for uninfected group vs either infected group for gp33–41, gp276–286, and gp118–128).

We concluded from these experiments that treatment with costimulation blockade-based tolerance regimens during the course of a chronic viral infection can significantly impair antiviral CD8 T cell immune responses and control of viremia even well after initial priming, expansion, and differentiation. Importantly, however, this treatment does not lead to reemergence of the virus in the serum when initiated after viremia has been controlled, and the virus is confined to peripheral tissues.