Th1-driven immune reconstitution disease in Mycobacterium avium-infected mice

Abstract

Following antiretroviral therapy, a significant proportion of HIV(+) patients with mycobacterial coinfections develop a paradoxical, poorly understood inflammatory disease termed immune reconstitution inflammatory syndrome (IRIS). Here, we show that Mycobacterium avium-infected T cell-deficient mice injected with CD4 T cells also develop an immune reconstitution disease (IRD) manifesting as weight loss, impaired lung function, and rapid mortality. This form of IRD requires Ag recognition and interferonγ production by the donor CD4 T cells and correlates with marked alterations in blood and tissue CD11b(+) myeloid cells. Interestingly, disease is associated with impaired, rather than augmented, T-cell expansion and function and is not strictly dependent on lymphopenia-induced T-cell proliferation. Instead, our findings suggest that mycobacterial-associated IRIS results from a heightened sensitivity of infected lymphopenic hosts to the detrimental effects of Ag-driven CD4 T-cell responses.

Figure 1

Immune reconstitution disease in Mycobacterium avium–infected T cell–deficient mice after CD4 T-cell adoptive transfer. (A) TCRα^−/− mice were infected intravenously with 1 × 10⁶ colony-forming units of M avium. After at least 2 months, 2 × 10⁶ CD4 T cells isolated from uninfected WT mice were transferred intravenously. (B) Mice were monitored for weight loss (n > 11 mice/group) and survival (n = 8 mice/group). The shaded area between traveling error bars represents the SD. (C) Bacterial loads were measured in the lungs and spleens of control and T cell–recipient TCRα^−/− mice on day 9 of transfer. (D) Lung function was assessed by measuring percent O₂ saturation using a murine pulse oximeter on day 8 after transfer. Data are representative of at least 2 independent experiments performed, and the results shown in panel B have been repeated at least 15 times.

Figure 2

Donor CD4 T-cell expansion and activation during reconstitution of naive and M avium–infected TCRα^−/− mice. Naive and day 69 infected CD45.2⁺ TCRα^−/− mice received 1.5 × 10⁶ CFSE-labeled CD45.1⁺ CD4 T cells from naive donors, and tissues were harvested on day 7 after transfer. (A) Representative FACS plots showing the frequency of donor CD4 T cells in different tissues of naive and infected recipient mice (top panels). Numbers represent the percentage of the total recovered cells that were of donor origin. The corresponding histograms of CFSE dilution for each of the gated CD4⁺CD45.1⁺ donor cell populations are shown in the bottom panels. Numbers represent the percentage of donor cells that have fully diluted CFSE. (B) Absolute numbers of donor CD4 T cells recovered from each of the tissues shown in (A). For the PBMCs, the percentage of donor cells is shown. (C) CD69 staining of the donor CD4 T cells recovered in panels A and B. In the case of mediastinal lymph nodes, it was necessary to pool cells from 4 mice to obtain sufficient numbers for staining. Data are representative of 2 independent experiments.

Figure 3

CD4 T cell–derived IFNγ, but not IL-4 or IL-17A, plays a major role in immune reconstitution disease of M avium–infected TCRα^−/− mice. Totals of 2 × 10⁶ WT, IL-4^−/−, IL-17A^−/−, or IFNγ^−/− CD4 T cells were transferred into M avium–infected TCRα^−/− mice (n = 5 mice/group), as described in Figure 1, and (A) weight loss and (B) survival were monitored. The data shown in (A) are representative of 2-3 independent experiments, while the survival curves shown in (B) represent 9 mice pooled from 2 independent experiments. (C) TCRβ⁺CD4⁺ cells were measured in the PBMCs of the mice shown in panels A and B on day 9 after transfer. (D) IFNγ was measured by ELISA in the serum of the same animals on day 10 after transfer. (E) Levels of nitrate were measured in the same sera using a nitrate reductase assay, and TNFα levels (F) were determined by ELISA. (G) M avium–infected TCRα^−/− mice were reconstituted with WT CD4 T cells and then treated with anti-TNFα–neutralizing Ab or isotype control Ab on days 0, 3, 6, 9, and 12 after transfer, and weight loss was monitored. The data shown for each panel are representative of at least 2 independent experiments. In panels A and G, the shaded area between the traveling error bars represents the SD.

Figure 4

The naive T-cell subpopulation is largely responsible for the induction of immune reconstitution disease by bulk CD4 T cells. (A) CD4 T cells from uninfected IFNγ reporter mice were FACS sorted into eYFP⁺ and eYFP⁻ cells. Purified cells (5 × 10⁵) of each subset were then separately transferred into M avium–infected TCRα^−/− mice (n = 6 control mice, n = 5 recipients of eYFP⁻, and n = 4 recipients of eYFP⁺ CD4 T cells). On day 10 after transfer, the recipient mice were analyzed for the donor cell marker and eYFP expression. (B) CD4 T cells from foxp3 reporter mice were FACS purified into CD44^lofoxp3⁻, CD44^hifoxp3⁻, and foxp3⁺ cells. Purified cells (5 × 10⁵) of each subset were then separately transferred into M avium–infected TCRα^−/− mice, and recipients were monitored for weight loss and survival (n = 5: recipients of CD44^lofoxp3⁻ or CD44^hifoxp3⁻ cells and n = 3: recipients of foxp3⁺ T cells). In panels A and B, the shaded area between the traveling error bars represents the SD. All data are representative of 2 independent experiments performed.

Figure 5

CD4 T cells specific for M avium can drive immune reconstitution disease. (A) M avium–infected TCRα^−/− mice received CD4 T cells (2 × 10⁶ each) isolated from uninfected WT or OT-II mice. The shaded area between the traveling error bars represents the SD. (B) Uninfected or M avium–infected TCRα^−/− mice received P25 (Ag85b specific TCR Tg) CD4 T cells (2 × 10⁶) or no T cells, and survival was monitored. (C) Bacterial loads in the lungs of infected mice from panel B 7 days after transfer of P25 CD4 T cells. (D) Total nitrate and (E) TNFα were measured in the serum of mice from panel B on day 5 of P25 transfer. (F) CD11b⁺ cells were measured in the blood and (G) lung of mice from panel B on day 5 of P25 transfer. (H) Phenotype of CD11b⁺ cells in the PBMC of M avium–infected TCRα^−/− mice on day 6 of P25 transfer.

Figure 6

CD4 T cells specific for endogenous antigens can drive immune reconstitution disease. (A) M avium–infected and –uninfected male or female TCRα^−/− mice received Marilyn (antimale TCR Tg) CD4 T cells (2 × 10⁶) isolated from female donor mice. (B) Survival of mice in shown in panel A. (C) IFNγ and (D) total nitrate were measured in the serum of mice shown in panel A on day 3 after transfer. All data are representative of at least 2 independent experiments.

Figure 7

Immune reconstitution disease is associated with the prior absence of host M avium–specific T-cell responses, rather than lymphopenia-driven T-cell expansion. (A) Uninfected WT, TCRα^−/−, and OT-II recipients received CFSE-labeled Thy1.1⁺ P25 CD4 T cells (2 × 10⁶/mouse), and lymph nodes were harvested on day 7 after transfer and analyzed for CFSE dilution. Histograms are gated on CD4⁺Thy1.1⁺ donor P25 CD4 T cells. (B) M avium–infected WT, TCRα^−/−, or OT-II mice were injected with P25 CD4 T cells (2 × 10⁶), and survival was monitored. (C) CD4 T cells (2 × 10⁶) isolated from C57Bl/6 mice were transferred into M avium–infected TCRα^−/−, OT-II, or P25 recipient mice and weight loss was monitored. The shaded area between the traveling error bars represents the SD. (D) Survival of mice depicted in panel C, n = 5/group. All of the data shown were representative of 2 independent experiments performed.