Bronchoalveolar Lavage Fluid Reflects a TH1-CD21low B-Cell Interaction in CVID-Related Interstitial Lung Disease

Abstract

Background: About 20% of patients with common variable immunodeficiency (CVID) suffer from interstitial lung disease (ILD) as part of a systemic immune dysregulation. Current understanding suggests a role of B cells in the pathogenesis based on histology and increased levels of BAFF and IgM associated with active disease corroborated by several reports which demonstrate the successful use of rituximab in CVID-ILD. It is debated whether histological confirmation by biopsy or even video-assisted thoracoscopy is required and currently not investigated whether less invasive methods like a bronchoalveolar lavage (BAL) might provide an informative diagnostic tool.

Objective: To gain insight into potential immune mechanisms underlying granulomatous and lymphocytic interstitial lung disease (GLILD) and to define biomarkers for progressive ILD by characterizing the phenotype of B- and T-cell populations and cytokine profiles in BAL fluid (BALF) of CVID-ILD compared to sarcoidosis patients and healthy donors (HD).

Methods: Sixty-four CVID, six sarcoidosis, and 25 HD BALF samples were analyzed by flow cytometric profiling of B- and T-cells and for cytokines by ELISA and Multiplexing LASER Bead technology.

Results: Both sarcoidosis and CVID-ILD are characterized by a predominantly T-cell mediated lymphocytosis in the BALF. There is an increase in T follicular helper (T_FH)-like memory and decrease of regulatory T cells in CVID-ILD BALF. This T_FH-like cell subset is clearly skewed toward T_H1 cells in CVID-ILD. In contrast to sarcoidosis, CVID-ILD BALF contains a higher percentage of B cells comprising mostly CD21^low B cells, but less class-switched memory B cells. BALF analysis showed increased levels of APRIL, CXCL10, and IL-17.

Conclusion: Unlike in sarcoidosis, B cells are expanded in BALF of CVID-ILD patients. This is associated with an expansion of T_FH- and T_PH-like cells and an increase in APRIL potentially supporting B-cell survival and differentiation and proinflammatory cytokines reflecting not only the previously described T_H1 profile seen in CVID patients with secondary immune dysregulation. Thus, the analysis of BALF might be of diagnostic value not only in the diagnosis of CVID-ILD, but also in the evaluation of the activity of the disease and in determining potential treatment targets confirming the prominent role of B-cell targeted strategies.

Keywords: CD21low B cells; TFH and TPH cells; common variable immunodeficiency; cytokines; interstitial lung disease.

Figure 1

Increased percentage of B cells in bronchoalveolar lavage fluid (BALF) of common variable immunodeficiency (CVID)-interstitial lung disease (ILD) compared to sarcoidosis. The diagnostic workup of the BALF of patients with CVID or sarcoidosis for cell counts, percentages of lymphocytes, neutrophils, eosinophils, macrophages, and basophils/mast cells (A), CD3+ T cells (B), CD20+ B cells (C), as well as CD4+ and CD8+ T cells including CD4/CD8 ratio (D). The normal range is marked in grey for each population and defined genetic defects are marked by color coding. Sarc., sarcoidosis. *P <.05, **P <.01.

Figure 2

Increased percentage of T_FH1-like and T_PH cells in bronchoalveolar lavage fluid (BALF) of common variable immunodeficiency (CVID)-interstitial lung disease (ILD) compared to sarcoidosis. Memory CD4 T cells were differentiated into CXCR5^pos T_FH1-, T_FH1/17-, T_FH17-, T_FH2-like cell subsets according to their CXCR3 and CCR6 expression (A) and total memory CD4 T cells into CXCR5^negPD1^high T_PH cells. Shown are two examples with high and low amounts of T_PH cells (B). Corresponding statistics are shown below. Memory CD4 T cells were further differentiated into FoxP3⁺CD25⁺ Tregs, statistics are shown on the right (C). The mean fluorescence intensity (MFI) of FoxP3, CD25, CTLA-4 in Tregs is shown in (D) and the ratio of CXCR5^pos memory CD4 T_FH-like cells to Tregs in (E). Defined genetic defects are marked by color coding. *P <.05, **P <.01 ***P <.001, Sarc., sarcoidosis.

Figure 3

Increased percentage of CD21^low cells in bronchoalveolar lavage fluid (BALF) of common variable immunodeficiency (CVID)-interstitial lung disease (ILD) compared to sarcoidosis. B cells were further divided into CD21^low B cells, plasmablasts (PB) and CD21^pos B cells. An exemplary FACS plot is shown in (A). Naive (IgD⁺CD27^-), switched memory B cells (IgD^-CD27⁺), atypical (IgD^-CD27^-) and non-switched memory B cells (IgD⁺CD27⁺) were gated from the CD21^low B-cell compartment as well as from the CD21+ nonPB subset. IgA, IgG, IgM-only cells were gated out of the switched memory B cell gate (IgD^-CD27⁺). Corresponding statistical analysis is shown in (B). Defined genetic defects are marked by color coding. *P <.05, **P <.01 ***P <.001, Sarc, sarcoidosis.

Figure 4

Altered cytokine milieu in bronchoalveolar lavage fluid (BALF) of common variable immunodeficiency (CVID)-interstitial lung disease (ILD). (A) ELISA of BALF supernatants for APRIL production. (B) Multiplex Bead Array of BALF supernatants for CXCL10, IL-4 and IL-17. Defined genetic defects are marked by color coding. *P <.05, **P <.01 ****P <.0001 HD, healthy control; Sarc, sarcoidosis.

Figure 5

Correlations between cell subsets and cytokines of bronchoalveolar lavage fluid (BALF). (A) Correlation of IL-17 in BALF of common variable immunodeficiency (CVID) patients with neutrophil numbers (n = 28), (B) of B cells and T_PH cells (n = 16), (C) of CD21^low B cells and T_FH1 cell subset (n = 16) and (D) of IgA^pos CD21^low B cells and T_FH1 cells (n = 16). Correlation of total CD21^low B cells (n = 13) (E), naïve CD21^low B cells (n = 13) (F) and switched CD21^pos B cells (n = 13) (G) in BALF and peripheral blood. Defined genetic defects are marked by color coding. *P <.05, **P <.01 ***P <.001, ****P <.0001.