Pancreatic cancer-associated inflammation drives dynamic regulation of p35 and Ebi3

Abstract

B cells are important modulators of immune responses both in autoimmunity and cancer. We have previously shown that B regulatory (Breg) cells promote pancreatic cancer via production of IL35, a heterodimeric cytokine comprised of the subunits p35 (Il12a) and Ebi3. However, it is not known how production of IL35 is regulated in vivo in the context of cancer-associated inflammation. To begin addressing this question, we have generated a knock-in mouse model, Il12a^GFP, where an IRES-emGFP gene was inserted within the 3' UTR of the Il12a locus. EmGFP signal in B cells from the Il12a^GFP mice correlated with expression of p35 mRNA and protein. Using this model, we observed that in addition to Bregs, expression of GFP (p35) is upregulated in several other B cell subtypes in response to cancer. We assessed the expression of the other IL35 subunit, Ebi3, using a published tdTomato reporter model. We determined that Ebi3 expression was more tightly regulated in vivo and in vitro, suggesting that stimuli affecting Ebi3 upregulation are more likely to result in production of full IL35 heterodimer. We were also able to detect GFP and Tomato signal in myeloid & T cell lineages suggesting that these reporter models could also be used for tracking IL12-, IL27- and IL35-producing cells. Furthermore, using primary B cells isolated from reporter mice, we identified BCR, CD40 and TLR pathways as potential drivers of IL35 expression. These findings highlight the importance of pancreatic cancer-associated inflammatory processes as drivers of cytokine expression and provide a tool to dissect both disease-associated regulation of IL12- and IL35-competent lineage cells as well as establish assays for pharmacological targeting of individual subunits of heterodimeric IL12 family cytokines.

Keywords: IL35 cytokine; Pancreatic cancer; Regulatory B cell; Reporter model.

Figure 1.. Generation of Il12a^GFP KI mice and in vitro analysis of B cells.

(A) Schematic of IRES-emGFP insertion into the Il12a genomic locus in the 3’ UTR of exon 7. (B) Genotyping of Il12a^GFP mice by PCR. Rxn 1 determines insertion of IRES-emGFP cassette into Il12a exon 7 3’ UTR. Rxn 2 determines presence of WT Il12a allele without insertion of IRES-emGFP cassette. (C) Splenic B cells (CD19⁺) isolated from healthy WT and Il12a^GFP stimulated with LPS + αCD40 for 48 hours. Fold change of Il12a expression in healthy WT and Il12a^GFP B cells (left) and emGFP (right) expression in IL12a-GFP B cells; n=3, ns = p>0.05. (D) Splenic CD1d^HiCD5⁺CD21^Hi B cells (top) and CD1d^LoCD5-CD21^Lo B cells (bottom) from healthy Il12a^GFP mice stimulated for 48 hours with LPS + αCD40. p35 (Il12a) and GFP protein expression were measured by flow cytometry.

Figure 2.. Pancreatic cancer alters immune cell expression of IL12a^GFP in vivo.

(A) Frequency of GFP⁺ immune cells isolated from spleens of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Il12a^GFP mice measured by flow cytometry; n=6, *=p<0.05, ns=p>0.05. (B) Mean fluorescent intensity (MFI) of GFP in GFP⁺ immune populations isolated from spleens and tumors of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Il12a^GFP measured by flow cytometry mice; n=6, *=p<0.05, ns=p>0.05. (C) Frequency of GFP⁺ immune cells isolated from orthotopic KPC pancreatic tumors of Il12a^GFP mice measured by flow cytometry; n=6. (D) Proportions of CD45⁺GFP⁺ cell populations isolated from Il12a^GFP orthotopic KPC pancreatic tumors; n=6.

Figure 3.. Differential effect of inflammatory signals from pancreatic tumors on IL12a^GFP expression in B cell subsets.

(A) Frequency of GFP⁺ B cell subsets isolated from spleens of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Il12a^GFP mice measured by flow cytometry; n=6, *=p<0.05, ns=p>0.05. (B) Mean fluorescent intensity (MFI) of GFP in GFP⁺ B cell subsets isolated from spleens and tumors of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Il12a^GFP measured by flow cytometry mice; n=6, *=p<0.05, ns=p>0.05. (C) Frequency of GFP⁺ B cell subsets isolated from orthotopic KPC pancreatic tumors of Il12a^GFP mice measured by flow cytometry; n=6. (D) Proportions of CD19⁺GFP⁺ cell populations isolated from Il12a^GFP orthotopic KPC pancreatic tumors; n=6.

Figure 4.. Expression of EbI3T^om in immune subsets in response to pancreatic cancer.

(A) Frequency of Tomato⁺ immune cells isolated from spleens of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Ebi3^Tom mice measured by flow cytometry; n=6, *=p<0.05, ns=p>0.05. (B) Mean fluorescent intensity (MFI) of Tomato in Tomato⁺ immune populations isolated from spleens and tumors of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Ebi3^Tom mice measured by flow cytometry mice; n=6, *=p<0.05, ns=p>0.05. (C) Frequency of Tomato⁺ immune cells isolated from orthotopic KPC pancreatic tumors of Ebi3^Tommice measured by flow cytometry; n=6. (D) Proportions of CD45⁺Tomato⁺ cell populations isolated from Ebi3^Tom orthotopic KPC pancreatic tumors; n=6. (E) Frequency of Tomato⁺ B cell subsets isolated from spleens of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Ebi3^Tom mice measured by flow cytometry; n=6, *=p<0.05, ns=p>0.05. (F) Mean fluorescent intensity (MFI) of Tomato in Tomato⁺ B cell subsets isolated from spleens and tumors of KPC orthotopic tumor-bearing and non-tumor bearing (naïve) Ebi3^Tom mice measured by flow cytometry mice; n=6, *=p<0.05, ns=p>0.05. (G) Frequency of Tomato⁺ B cell subsets isolated from orthotopic KPC pancreatic tumors of Ebi3^Tom mice measured by flow cytometry; n=6. (H) Proportions of CD19⁺Tomato⁺ cell populations isolated from Ebi3^Tom orthotopic KPC pancreatic tumors; n=6.

Figure 5.. In vitro stimulation of reporter B cells reveals differential regulation of Il12a and Ebi3 expression.

(A) Frequency of GFP⁺ B cells isolated from Il12a^GFP spleens after stimulation of indicated receptors for 48 hours; n=3. (B) Mean fluorescent intensity (MFI) of GFP in splenic Il12a^GFP B cells after stimulation of indicated receptors for 48 hours in vitro; n=3. (C) Frequency of Tomato⁺ B cells isolated from Ebi3^Tom spleens after stimulation of indicated receptors for 48 hours; n=3. (D) Mean fluorescent intensity (MFI) of Tomato in splenic Ebi3^Tom B cells after stimulation of indicated receptors for 48 hours in vitro; n=3.