Co-modulation of T cells and B cells enhances the inhibition of inflammation in experimental hypersensitivity pneumonitis

Abstract

Background: Hypersensitivity pneumonitis (HP) is an interstitial lung disease characterized by antigen-triggered neutrophilic exacerbations. Although CD4⁺ T cells are sufficient for HP pathogenesis, this never translated into efficient T cell-specific therapies. Increasing evidence shows that B cells also play decisive roles in HP. Here, we aimed to further define the respective contributions of B and T cells in subacute experimental HP.

Methods: Mice were subjected to a protocol of subacute exposure to the archaeon Methanosphaera stadmanae to induce experimental HP. Using models of adoptive transfers of B cells and T cells in Rag1-deficient mice and of B cell-specific S1P₁ deletion, we assessed the importance of B cells in the development of HP by evaluating inflammation in bronchoalveolar lavage fluid. We also aimed to determine if injected antibodies targeting B and/or T cells could alleviate HP exacerbations using a therapeutic course of intervention.

Results: Even though B cells are not sufficient to induce HP, they strongly potentiate CD4⁺ T cell-induced HP‑associated neutrophilic inflammation in the airways. However, the reduction of 85% of lung B cells in mice with a CD19-driven S1P₁ deletion does not dampen HP inflammation, suggesting that lung B cells are not necessary in large numbers to sustain local inflammation. Finally, we found that injecting antibodies targeting B cells after experimental HP was induced does not dampen neutrophilic exacerbation. Yet, injection of antibodies directed against B cells and T cells yielded a potent 76% inhibition of neutrophilic accumulation in the lungs. This inhibition occurred despite partial, sometimes mild, depletion of B cells and T cells subsets.

Conclusions: Although B cells are required for maximal inflammation in subacute experimental HP, partial reduction of B cells fails to reduce HP-associated inflammation by itself. However, co-modulation of T cells and B cells yields enhanced inhibition of HP exacerbation caused by an antigenic rechallenge.

Keywords: Adoptive lymphocyte transfer; B cells; Biologics; CD69; Conditional knockout; Extrinsic allergic alveolitis; Hypersensitivity pneumonitis; Rituximab; S1P1.

Fig. 1

B cells synergize with T cells in the MSS-induced HP model to amplify airway inflammation. A Saline, 5 × 10⁶ B cells, 2.5 × 10⁶ T cells, or the combination of 5 × 10⁶ B cells and 2.5 × 10⁶ T cells were injected intravenously to Rag1-deficient mice. Beginning 48 h after the transfer, mice were exposed i.n. three consecutive days a week for three weeks to 100 µg MSS and euthanized 24 h after the last exposure. Upward arrow indicates time of euthanasia. Quantifications of (B) total leucocytes and (C) differential counts for leucocyte subsets in BALF. I.V.: Intravenous. Mo: Macrophages. Ly: Lymphocytes. Ne: Neutrophils. Eo: Eosinophils. BALF: Bronchoalveolar lavage fluid. MSS: Methanosphaera stadtmanae. Averages ± SEM. n = 3–6. *p < 0.05

Fig. 2

CD19-driven S1P₁ deficiency inhibits experimental HP-associated B cell accumulation in the lung. A Control and CD19^Cre± S1P₁^loxP+/+mice were exposed three consecutive days a week for three weeks to saline or 100 µg MSS and euthanized 24 h after the last exposure. Upward arrow indicates timing of euthanasia. B Gating strategy from single cell suspensions from lungs. FSC-A^low SSC-A^low cells were selected, and B cells were identified as autofluorescence low (AF⁻) B220⁺CD90⁻ lymphocytes. C Frequencies of lymphocyte subsets were multiplied by the total number of lung cells to determine the absolute numbers of B cells. D TLT area (percentage of lung area) was quantified as described in the methods after (E) hematoxylin/eosin staining of lung coronal slices. F CD69 MFI on B220⁺ cells. G Frequencies of B220⁺ cells positive for CD69. MFI: median fluorescence intensity. AF: Autofluorescence. TLT: tertiary lymphoid tissue. FMO: Fluorescence Minus One control. MSS: Methanosphaera stadtmanae. Averages ± SEM. n = 6–17. *p < 0.05

Fig. 3

Hallmarks of inflammation in the BALF are unaltered by CD19-driven S1P₁ deletion. Control and CD19^Cre± S1P₁^loxP+/+ mice were exposed to either saline or MSS as described in the Methods section and euthanized 24 h after the last MSS instillation. Quantification of (A) total leucocytes and (B) differential counts for leucocyte subsets in BALF. C BALF albumin was quantified by ELISA on BALF supernatant. Mo: Macrophages. Ly: Lymphocytes. Ne: Neutrophils. Eo: Eosinophils. BALF: Bronchoalveolar lavage fluid. MSS: Methanosphaera stadtmanae. Averages ± SEM. n = 10–17. *p < 0.05

Fig. 4

Cells from draining lymph nodes of mice with CD19‑driven S1P₁ deletion generate MSS-specific IgGs. Mice were exposed to saline or MSS as described in methods and lymph nodes were harvested 24 h after the last instillation. Cytometry was done as described in Fig. 2B to measure (A) Numbers of mLN B cells and (B) the frequency of CD69⁺ B cells. C) MSS-specific IgGs were assessed by indirect ELISA on mLN B cells supernatant diluted 1:2 and on different (D) BALF and (E) plasma dilutions. mLN: mediastinal lymph node. O.D.: optical density. MSS: Methanosphaera stadtmanae. Averages ± SEM. n = 3–6. *p < 0.05

Fig. 5

Anti-CD4 and anti-CD8 administration reduces experimental HP inflammation caused by an antigenic rechallenge. C57Bl/6 J mice were administered MSS three times a week for three weeks. Anti-CD4 and anti-CD8 or their respective control IgGs were given intraperitoneally 2 days after the MSS exposure. MSS rechallenge was performed 4 days after antibodies injections and mice were euthanized 48 h later. A Quantification of neutrophils/ml of BALF. Single cell suspension from lungs after the MSS rechallenge were analyzed using flow cytometry to determine the frequencies of (B) CD19⁺ and (C) CD90⁺CD8⁺ and CD90⁺CD4⁺ cells. Fully-labeled specimens are presented in black and FMOs (from pooled cells from all experimental groups) are in blue or red. The percentages shown in B and C are from fully-labeled specimens. α: anti, MSS: Methanosphaera stadtmanae. BALF: Bronchoalveolar lavage fluid, FMO: Fluorescence Minus One control. Averages ± SEM. n = 4–5 *p < 0.05

Fig. 6

The anti-CD19 does not dampen neutrophilic inflammation caused by an MSS rechallenge in subacute HP. C57Bl/6 J mice were administered MSS three times a week for four weeks. Anti-CD19 or its respective control IgG were given intraperitoneally 2 days after the last MSS exposure. MSS rechallenge was performed 4 days after antibody injection and mice were euthanized 48 h later. A Quantification of neutrophils/ml of BALF. Single cell suspensions from MSS-rechallenged lungs were analyzed using flow cytometry to determine the frequencies of (B) CD19⁺, (C) B220⁺ and (D) CD90⁺CD8⁺ and CD90⁺CD4⁺ lung cells. Fully-labeled specimens are presented in black and FMOs (from pooled cells from all experimental groups) are in blue or red. The percentages shown in B–D are from fully-labeled specimens. α: anti, MSS: Methanosphaera stadtmanae. BALF: Bronchoalveolar lavage fluid, FMO: Fluorescence Minus One control. Averages ± SEM. n = 4–6 *p < 0.05

Fig. 7

Co-administration of anti-CD19, anti-CD4 and anti-CD8 yields strong inhibition of inflammation caused by an antigenic rechallenge. C57Bl/6 J mice were administered MSS three times a week for four weeks. Anti-CD19, anti-CD4 and anti-CD8 or their respective control IgGs were given intraperitoneally 2 days after the last MSS exposure. MSS rechallenge was performed 4 days after antibody injection and mice were euthanized 48 h later. A Quantification of neutrophils/ml of BALF. Single cell suspensions from MSS-rechallenged lungs were analyzed using flow cytometry to determine the frequencies of (B) CD19⁺, (C) B220⁺ and (D) CD90⁺CD8⁺ and CD90⁺CD4⁺ cells. Fully-labeled specimens are presented in black and FMOs (from pooled cells from all experimental groups) are in blue or red. The percentages shown in B–D are from fully-labeled specimens. α: anti, MSS: Methanosphaera stadtmanae, BALF: Bronchoalveolar lavage fluid. FMO: Fluorescence Minus One control. Averages ± SEM. n = 4–5 *p < 0.05