Neo-lymphoid aggregates in the adult liver can initiate potent cell-mediated immunity

Abstract

Subcutaneous immunization delivers antigen (Ag) to local Ag-presenting cells that subsequently migrate into draining lymph nodes (LNs). There, they initiate the activation and expansion of lymphocytes specific for their cognate Ag. In mammals, the structural environment of secondary lymphoid tissues (SLTs) is considered essential for the initiation of adaptive immunity. Nevertheless, cold-blooded vertebrates can initiate potent systemic immune responses even though they lack conventional SLTs. The emergence of lymph nodes provided mammals with drastically improved affinity maturation of B cells. Here, we combine the use of different strains of alymphoplastic mice and T cell migration mutants with an experimental paradigm in which the site of Ag delivery is distant from the site of priming and inflammation. We demonstrate that in mammals, SLTs serve primarily B cell priming and affinity maturation, whereas the induction of T cell-driven immune responses can occur outside of SLTs. We found that mice lacking conventional SLTs generate productive systemic CD4- as well as CD8-mediated responses, even under conditions in which draining LNs are considered compulsory for the initiation of adaptive immunity. We describe an alternative pathway for the induction of cell-mediated immunity (CMI), in which Ag-presenting cells sample Ag and migrate into the liver where they induce neo-lymphoid aggregates. These structures are insufficient to support antibody affinity maturation and class switching, but provide a novel surrogate environment for the initiation of CMI.

Figure 1. Aly/aly mice are resistant to the development of EAE.

(A) EAE was induced by active immunization with MOG_35–55/CFA in aly/aly (▿) and aly/+ (♦) mice. (B) EAE was induced by adoptive transfer of pathogenic T cells derived from MOG-immunized aly/aly or aly/+ donors into aly/aly or aly/+ recipients. aly/+ into aly/+: ▴, aly/+ into aly/aly: Δ, aly/aly into aly/+: •, and aly/aly into aly/aly: ○. Shown is a representative of two individual experiments (n≥5 mice/group)±SEM. (C) Aly/+ and aly/aly mice were injected with 20×10⁶ CFSE-labeled splenocytes i.v. derived from 2D2 Tg mice and immunized s.c. with MOG_35–55/CFA. At 4 dpi, splenocytes were analyzed by flow cytometry by gating on 2D2⁺ cells. Results are representative of two individual experiments (n = 2 mice/group).

Figure 2. SLTs are crucial for B but not T cell-mediated immune responses.

(A and B) EAE progression in BM-chimeras immunized s.c. with MOG_35–55/CFA. (A) aly/+→aly/+: ▴, aly/+→aly/aly: •. (B) aly/aly→aly/+: □, aly/+→aly/+: ▴. (C) EAE was induced by active immunization with MOG_35–55/CFA of LTβR^−/− mice (▪) and wt mice (Δ). Shown are representatives of three individual experiments (n≥5/group)±SEM. (D) LN-derived cells were obtained from aly/aly→aly/+ (black bars) and aly/+→aly/+ (grey bars) BM-chimeras 21 dpi with MOG_35–55/CFA and rechallenged in vitro with 50 µg/ml MOG_35–55 peptide to reveal IFNγ- and IL-17–secreting cells using Elispot. Shown is a representative of two individual experiments (n = 3/group)±standard deviation (SD). (E) DTH responses were induced by s.c. immunization with KLH/CFA of aly/aly and aly/+ mice. At 11 dpi, the mice were challenged by intradermal injection of KLH (grey bars), or PBS (black bars) into the ear. Swelling was measured 24 h postchallenge using a precision caliper, and shown is the increase of ear swelling over baseline of a representative of three independent experiments (n≥2 mice/experiment)±SD. (F) Sera were collected from KLH-immunized aly/aly (▿) and aly/+ mice (♦) mice on 12 dpi and analyzed for the presence of total anti-KLH Abs by ELISA. Results are representative of three independent experiments (n≥2 mice/group)±SD.

Figure 3. Ab response to s.c. auto-Ag depends on the presence of dedicated lymphoid structures.

Titers of anti-MOG Abs (total Ig, IgG, IgM and IgA) determined from sera of diseased BM-chimeras immunized s.c. with MOG_35–55/CFA by ELISA. aly/+→aly/+: ▪, aly/+→aly/+^spl: □, aly/+→aly/aly: ▴, aly/+→aly/aly ^spl: Δ. (A) shows total Ig, (B) shows IgG, IgM, and IgA. Shown is a representative of 3 individual experiments (n = 3/group)±SD.

Figure 4. Ag-laden APCs migrate to the liver in the absence of SLTs.

(A) Aly BM-chimeras were injected s.c. with YG microspheres/CFA, and various organs were analyzed by FACS for the presence of fluorescently labeled CD11c⁺ cells 7 dpi. Data represent one of three individual experiments. (B) Aly BM-chimeras were injected s.c. with either a mixture of YG and PR beads (YG+PR) into both flanks or YG beads into one flank and PR beads into the other flank (YG vs. PR). At 7 dpi, livers and LNs (only in aly/+→aly/+ mice) were analyzed by FACS for single (YG or PR) or double (YG and PR) positive APCs (gated on CD11c⁺ and CD11b⁺ cells). (C) Aly BM-chimeras were painted on the shaved flanks with 100 µl of 4 mg/ml FITC dissolved in 1:1 acetone:dibutylphalate. After 24 h, livers and, in aly/+→aly/+ mice, draining and nondraining inguinal LNs were analyzed by FACS for the presence of FITC⁺ cells (CD11c⁺).

Figure 5. Extra-lymphoid aggregates in the liver host T cells and APCs.

(A) Liver cryosections from aly BM-chimeras immunized s.c. with MOG_35–55 (d7) were stained with H&E. Bar indicates 500 µm. (B) Higher magnification image of the region indicated by the square in (A) stained with H&E and mAbs against CD3 and CD11c. Bar indicates 100 µm.

Figure 6. Accumulation and Ag-specific T cell expansion in the liver.

Aly BM-chimeras were injected i.v. with 8×10⁶ Luc-2D2 Tg CD4⁺ T cells and immunized s.c. with MOG_35–55/CFA. (A) At 2 dpi, mice were injected with luciferin, and after 10 min, sacrificed. Livers and, in control mice, LNs and spleen were isolated, and images were acquired by bioluminescence imaging to reveal the accumulation of the injected luciferase-positive (Luc-2D2) cells. (B) Absolute numbers of liver-invading DCs and Ag-specific T cells assessed from the percentage of CD11c⁺, CD4⁺, and Vβ11⁺ cells analyzed by flow cytometry. Numbers above the graph indicate the fold-increase of liver-invading cells of aly/+→aly/aly ^spl (grey) over aly/+→aly/+ (black). (C) Aly/+→aly/+ and aly/+→aly/aly ^spl BM-chimeras were injected with 8×10⁶ CFSE-labeled naive (CD62L⁺) CD4⁺ T cells derived from 2D2 Tg mice and immunized s.c. with MOG_35–55/CFA. At 5 dpi, LNs (only in aly/+→aly/+) and liver-invading cells were analyzed by flow cytometry by gating on 2D2⁺ cells. (D and E) Aly/+→aly/+ and aly/+→aly/aly ^spl BM-chimeras were immunized s.c. with MOG_35–55/CFA. (D) At 7 dpi, BM-chimeras were injected with BrdU i.p. 30 min after BrdU injection, liver-invading cells were analyzed by flow cytometry for BrdU⁺ CD4⁺ cells. (E) Absolute numbers of liver-invading BrdU⁺ CD4⁺ T cells assessed by flow cytometry. aly/+→aly/aly ^spl (grey) and aly/+→aly/+ (black).

Figure 7. Surrogate liver aggregates support CMI, but not B cell maturation.

Wt→wt and wt→LTα^{−/− spl} BM-chimeric mice were immunized s.c. with MOG_35–55/CFA. (A) At 11 dpi, titers of anti-MOG Abs (IgG, IgM and IgA) were determined from sera by ELISA (n = 4 mice/group)±SD. (B) Liver sections from wt→wt, wt→LTα^−/−spl, and aly/+→aly/aly ^spl BM-chimeras were stained with Abs against CD4, CD8, CD11b, CD11c, CD19, CD62L, CD68, FDC, ICAM, Ki67, PNA, and VCAM. Positively stained infiltrated areas of 14-mm² liver sections were counted (n = 4 mice/group)±SD.

Figure 8. CD8⁺ T cell priming in the liver and lymphoid aggregates in plt/plt mice.

(A) Tumor progression of aly BM-chimeras. Mice were vaccinated s.c. with 1 ×10⁶ irradiated GM-CSF-B16.F10 cells into one flank and 12 d later, mice received 2×10⁵ live B16.F10-Luc cells into the opposite flank. Vaccinated aly/+→aly/+: ▪; nonvaccinated aly/+→aly/+: □; vaccinated aly/+→aly/aly ^spl: ▴; and nonvaccinated aly/+→aly/aly ^spl: Δ. (B) aly/+→aly/+ and aly/+→aly/aly ^spl BM-chimeras were injected i.v. with 20×10⁶ CFSE-labeled splenocytes from OTI Tg mice and s.c. injected with a mix of 1×10⁶ B16.F10-OVA and 1×10⁶ B16.F10-GM-CSF cells. At 12 dpi, LNs (only in aly/+→aly/+) and liver-invading cells were analyzed by flow cytometry for the proliferation of CD8⁺ OTI cells (Vα2⁺). (C) EAE progression of plt/plt (□) and wt (▪) mice immunized s.c. with MOG_35–55/CFA. (D) Liver cryosections from diseased plt/plt mice (C) were stained with mAbs against CD11c, CD11b, CD4, FDC, B220, and PNA. Bar indicates 100 µm.