Role of Programmed Cell Death Protein-1 and Lymphocyte Specific Protein Tyrosine Kinase in the Aryl Hydrocarbon Receptor- Mediated Impairment of the IgM Response in Human CD5+ Innate-Like B Cells

Abstract

Innate-like B cells (ILBs) are a heterogeneous population B cells which participate in innate and adaptive immune responses. This diverse subset of B cells is characterized by the expression of CD5 and has been shown to secrete high levels of immunoglobulin M (IgM) in the absence of infection or vaccination. Further, CD5⁺ ILBs have been shown to express high basal levels of lymphocyte specific protein tyrosine kinase (LCK) and programmed cell death protein-1 (PD-1), which are particularly sensitive to stimulation by interferon gamma (IFNγ). Previous studies have demonstrated that activation of the aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, results in suppressed IgM responses and is dependent on LCK. A recent study showed that CD5⁺ ILBs are particularly sensitive to AHR activation as evidenced by a significant suppression of the IgM response compared to CD5^- B cells, which were refractory. Therefore, the objective of this study was to further investigate the role of LCK and PD-1 signaling in AHR-mediated suppression of CD5⁺ ILBs. In addition, studies were conducted to establish whether IFNγ alters the levels of LCK and PD-1 in CD5⁺ ILBs. We found that AHR activation led to a significant upregulation of total LCK and PD-1 proteins in CD5⁺ ILBs, which correlated with suppression of IgM. Interestingly, treatment with recombinant IFNγ reduced LCK protein levels and reversed AHR-mediated IgM suppression in CD5⁺ ILBs in a similar manner as LCK inhibitors. Collectively, these results support a critical role for LCK and PD-1 in AHR-mediated suppression of the IgM response in human CD5⁺ ILBs.

Keywords: AhR (aryl hydrocarbon receptor); IFNgamma; IgM; Lck; PD1 (programmed cell death protein 1); TCDD; innate-like B cells.

Figure 1

TCDD-mediated increase in the percentage of LCK⁺ human CD5⁺ ILBs and the suppression of the IgM response. Human CD5^+/- B cells were activated with CD40L, IL-21, and IL-2 and treated with Veh (0.02% DMSO), or TCDD (10 nM) on day 0 and cultured for 7 days. Cells and culture supernatants were collected and assessed for LCK and LCK Y505 phosphorylation by flow cytometry and IgM secretion via ELISA and ELIspot. (A) Correlation of percent CD5⁺ B cells and percent LCK⁺ B cells; (B) Representative flow cytometry plots of CD5⁺ and LCK⁺ cells; (C) Percentage of LCK⁺ B cells within the CD5^+/- populations on day 0; (D) Representative flow cytometry plots of LCK⁺ cells within CD5^+/- populations; (E) Percentage of LCK⁺ B cells within CD5^+/- populations on day 1, 3, 4 and 7 with Veh or TCDD treatment; (F) Percentage of pLCK (Y505)⁺ B cells within CD5^+/- populations on day 1, 3, 5 and 7 with Veh or TCDD treatment; (G) Representative ELIspot wells showing IgM secreting cells measured within CD5^+/- populations with Veh or TCDD (10 nM) treatment on day 7; (H) Number of IgM secreting cells; and (I) IgM concentration from culture supernatants from CD5^+/- B cells treated with Veh or TCDD. Determinations were made using B cells from 6 human donors (N = 6). For (E, F), data were normalized to the CD5^- Veh on day 1. For (H, I), data were normalized to CD5^- Veh. Significant differences are indicated by *p < 0.05 and **p < 0.01 (Student’s T test or two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 2

The TCDD-mediated upregulation of LCK and suppression of the IgM response in CD5⁺ ILBs is blocked by AHR antagonist treatment. Human CD5^+/- B cells were pre-treated with 1 or 10 μg/ml of AHR antagonist (CH-223191 abbreviated as CH), for 30 minutes and then followed by activation and treatment with Veh (0.02% DMSO) or TCDD (10 nM) on day 0 and cultured for 7 days as previously described. (A) Representative flow cytometry plots of CD5⁺ ILBs with or without AHR agonist and/or antagonist treatment; (B) Percentage of LCK⁺ cells; (C) Mean florescence intensity (MFI) of LCK in CD5^+/- populations; (D) Number of IgM secreting cells; and (E) IgM concentration within CD5^+/- populations with Veh or TCDD treatment. Determinations were made using B cells from 6 human donors (N = 6) from 2 independent experiments. Data were normalized to the CD5^- Veh control group. Significant differences are indicated by **p < 0.01 (two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 3

LCK inhibitor treatment reversed the AHR-mediated suppression of the IgM response in CD5⁺ ILBs. Human CD5^+/- B cells were activated and treated with Veh or TCDD on day 0 and cultured for 7 days as described previously. LCK inhibitor (RK24466) was added on day 5 to the cell culture. (A) Representative ELIspot wells showing IgM secreting cells measured within CD5^+/- B populations on day 7; (B) Averaged number of IgM secreting cells and; (C) Averaged IgM concentration within culture supernatants collected from Veh or TCDD-treated CD5+/- cultures with and without LCK inhibitor; (D) Representative flow cytometry plots of LCK⁺ CD5⁺ ILBs with or without LCK inhibitor treatment; and (E) Averaged percentage of LCK⁺ cells within CD5^+/- populations with or without LCK inhibitor treatment. Determinations were made using B cells from 6 human donors (N = 6) from two independent experiments. Data presented in the figures were normalized to the CD5^- Veh without LCK inhibitor treatment. Significant differences are indicated by *p < 0.05 and **p < 0.01 (two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 4

TCDD treatment of human CD5⁺ B cells significantly increases the frequency of PD-L2 cell surface protein positive cells. Human CD5⁺ and CD5^- B cells were isolated, activated, and treated with TCDD (10nM) for 7 days as previously described. After the culture period, cells were collected and surface stained with anti-PD-L1 and PD-L2 antibodies and cell surface protein expression was quantified by flow cytometry. Representative flow plots for Veh and TCDD treated CD5^+/- B cells are shown in panels (A, C). PD-L1⁺ and PD-L2⁺ cells were identified in the lymphocyte, singlet gate by gating on live CD19⁺ cells. The frequency of PD-L1 and PD-L2 positive cells was normalized to CD5^- Veh control. Averaged data from 2 independent experiments assessing a total of 6 human donors are shown in panel (B) (PD-L1) and (D) (PD-L2). Significance was determined by a repeated measures two-way ANOVA with a Dunnett’s posttest. **p<0.01.

Figure 5

Kinetics of PD-L1 and PD-L2 cell surface protein expression by activated human CD5⁺ ILB. Human CD5^+/- B cells were activated and cultured for 7 days as described previously and cells were collected daily and assessed for surface expression of either PD-L1 or PD-L2 by flow cytometry. (A) Representative PD-L1 flow plots at days 0, 1, 3, and 7; (B) Averaged frequency of PD-L1⁺ cells; (C) Averaged PD-L1 PerCP-Cy5.5 geometric mean fluoresence intensity; (D) Representative PD-L2 flow plots at days 0, 1, 3, and 7; (E) Averaged frequency of PD-L2⁺ cells; and (F) Averaged PD-L2 APC geometric mean fluoresence intensity. Determinations were made using B cells from 6 human donors (N = 6) across 3 independent experiments. Signficant differences between CD5^+/- B cells were determined at each time point using a paired, two way t-test. Significant differences are indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6

Treatment with soluble PD-1 ligands (PDL1 and PDL2) suppressed the IgM response and LCK inhibitor treatment blocked PD-1-mediated IgM suppression in CD5⁺ ILBs. Human CD5^+/- B cells were activated as previously described and treated with soluble PD-L1, PD-L2 or the combination of both on day 0 and cultured for 7 days. LCK inhibitor (RK24466) was provided on day 0 or day 5 to the cell culture. (A) Averaged ELIspot data showing the number of IgM secreting cells; (B) Averaged IgM concentration from culture supernatants; (C) Averaged frequency of LCK⁺ cells within CD5^+/- populations on day 7; (D) Averaged number of IgM secreting cells; and (E) Averaged IgM concentration from CD5^+/- culture supernatants with PDL2 and LCK inhibitor treatments. Determinations were made using B cells from 6 human donors (N = 6) across two independent experiments. For (A–C), data presented in the figure were normalized to CD5^- B cells without PD-1 ligand treatment. For (D, E), results were normalized to CD5⁻ B cells without PDL2 or LCK inhibitor treatment. Significant differences are indicated by *p < 0.05 and **p < 0.01 (two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 7

Soluble PDL2 treatment did not further suppress the IgM response compared to AHR activation alone in CD5⁺ ILBs. Human CD5^+/- B cells activated as described were treated with Veh or TCDD in the presence or absence of soluble PD-L2. Soluble PDL2 was added to the culture on day 0 or day 3. (A) Averaged number of IgM secreting cells; and (B) Averaged IgM concentrations from CD5^+/- culture supernatants on day 7. Determinations were made using B cells from 6 human donors (N = 6). Data presented in the figure were normalized to Veh treated CD5^- B cells without PDL2 treatment. Significant differences are indicated by **p < 0.01 (two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 8

PD-1 antibody blockade prevented TCDD-mediated suppression of the IgM response in human CD5⁺ ILBs. Human CD5+/- B cells were isolated, activated, and treated with Veh or TCDD as described previously. To determine the contribution of PD-1 to TCDD-mediated IgM suppression, 10 ng/mL anti-PD-1 blocking antibody (S228P) was added to CD5^+/- B cell cultures. Following 7 days of culture, cells and culture supernatants were collected and assessed for IgM secretion. (A) Representative ELIspot wells showing the number of IgM secreting cells within CD5^+/- populations with or without anti-PD-1 treatment; (B) Averaged number of IgM secreting cells; and (C) Averaged IgM concentration from CD5^+/- culture supernatants. Determinations were made using B cells from 7 human donors (N = 7) across three independent experiments. Data presented in the figure were normalized to CD5^- Veh without anti-PD-1 treatment. Significant differences are indicated by **p < 0.01 (two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 9

Antibody blockade of PD-L1 or PD-L2 promotes IgM secretion by TCDD-treated CD5⁺ B cells. To determine the contribution of PD-1 ligands to the TCDD-mediated suppression of IgM secretion, we leveraged PD-1 ligand antibody blockade. Briefly, human CD5^+/- B cells were isolated, activated, and treated (Veh or TCDD) as described. At the initiation of culture, 1 μg/mL of either αPD-L1 or αPD-L2 was added and cells were cultured for 7 days. Following the culture period, cells and culture supernatants were collected for IgM enumeration. Representative ELIspot wells from a single donor are shown in (A). Averaged raw numbers of IgM secreting cells are shown in (B) and normalized data in (C). Averaged IgM concentrations from culture supernatants are shown in (D) and normalized data are shown in (E). Determinations were made using B cells from 8 human donors (N = 8) across three independent experiments. Data presented in the figure were normalized to CD5^- Veh without anti-PD ligand treatments. Significant differences between Veh and TCDD are indicated by *p < 0.05 and **p < 0.01. Significant differences between TCDD (no ligand blockade) and indicated TCDD-treated groups are indicated by ^# p < 0.05 and ^## p < 0.01. Significance was determined using a two-way ANOVA followed with a Tukey’s posttest. Significant differences between Veh and Veh treated with PD ligand are indicated by 'a' p < 0.05.

Figure 10

IFNγ treatment reversed the AHR-mediated suppression of the IgM response through a decrease of LCK in CD5⁺ ILBs. Human CD5^+/- B cells were activated/treated with soluble PDL1 or PDL2 on day 0 and cultured for 7 days as described previously. In addition, IFNγ treatment (1 U/mL) was provided on day 0. (A) Averaged frequency of LCK⁺ total B cells; (B) Averaged number of IgM secreting cells in CD19⁺ B cells; (C) Representative flow cytometry plots of IFNγR1⁺ and IFNγR2⁺ cells; (D) Averaged frequency of IFNγR1⁺ and IFNγR2⁺ cells; (E) Averaged MFI of IFNγR1 and IFNγR2 within CD5^+/- populations; (F) Averaged number of IgM secreting cells; and (G) Averaged IgM concentration within CD5^+/- culture supernatants treated with IFNγ; (H) Averaged percentage of LCK⁺ cells and; (I) averaged LCK MFI within CD5^+/- populations treated with IFNγ. Determinations were made using B cells from 6 human donors (N = 6) from two independent experiments. For (A, B), data presented in the figure were normalized to the Veh group without IFNγ treatment. For F-I, data presented were normalized to the CD5^- Veh without IFNγ treatment. Significant differences are indicated by *p < 0.05, **p < 0.01 and ***p < 0.001 (two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 11

IFNγ treatment blocks PD-1-mediated IgM suppression in CD5⁺ ILBs. Human CD5^+/- B cells were activated, treated with soluble PDL1 or PDL2 on day 0 and cultured for 7 days as previously described. In addition, IFNγ treatment (1 U/mL) was provided on day 0. (A) Average number of IgM secreting cells; (B) Average IgM concentration from culture supernatants treated with soluble PDL1 within CD5^+/- cultures in the presence of IFNγ treatment; (C) Averaged number of IgM secreting cells; (D) Average IgM concentration from culture supernatants treated with soluble PDL1 within CD5^+/- cultures in the presence of IFNγ treatment; (E) Average frequency of PD-1⁺ cells; (F) Average frequency of PDL1⁺ cells; and (G) Average frequency of PDL2⁺ in CD5^+/- populations with or without IFNγ treatment. Determinations were made using B cells from 6 human donors (N = 6) across two independent experiments. For (A–D), results were normalized to CD5^- B cells without soluble PD-1 ligand treatment. For (E–G), results were normalized to the Veh control group in CD5⁻ B cells. Significant differences are indicated by *p < 0.05 and **p < 0.01 (two-way ANOVA following with Fisher’s LSD post hoc test).

Figure 12

Schematic of potential mechanism. (A) Effect of TCDD-mediated AHR activation; and (B) Effect of IFNγ on CD5⁺ ILBs.