IL-2Rα is dispensable for murine B cell development and humoral response

Abstract

The cytokine IL-2 plays a pivotal role in the immune system, specifically in the proliferation of T, B, and NK cells. The alpha subunit of the IL-2 receptor, IL-2Rα (CD25), is known to regulate the expansion and differentiation of T lymphocytes. CD25 is also expressed in developing B cells; however, its B cell intrinsic role remains undefined. We generated a mouse model with a B cell-specific deletion of CD25 to ascertain its role in B cell development and function. Unexpectedly, we found that the loss of CD25 had no impact on B cell development, homeostasis, or immune response to model antigens. Additionally, while CD25 expression was upregulated in activated splenic B cells, its absence did not affect class switch recombination in vitro or in vivo. We conclude that in contrast to its critical role in T cell differentiation and function, and despite its expression in developing and activated B cells, CD25 does not have any significant role in B cell development and adaptive immune functions.

Introduction

B lymphocytes play a critical role in adaptive immunity by producing antibodies upon exposure to a wide variety of pathogens. They also exhibit immunoregulatory properties through antigen presentation and cytokine secretion.¹ B cells emerge from multipotent hematopoietic stem cells located in the bone marrow (BM). Their development is guided by key differentiation steps, which are characterized by the sequential rearrangements of the immunoglobulin (Ig) heavy (IgH) and light (IgL) chain segments. First, developing pro-B cells in the BM undergo V(D)J recombination to assemble the variable (V), diversity (D), and joining (J) gene segments upstream of exons encoding the Igµ constant region.² Subsequently, the expressed Igµ chain associates with a surrogate light chain during the pre-B cell stage.³ This association forms the pre-B cell receptor (pre-BCR), serving as an essential quality control checkpoint.⁴ B cells that successfully express a functional pre-BCR then downregulate its expression and proceed to reconfigure the V and J segments of the IgL chain locus. Successful recombination of the IgL chain locus results in assembly of a functional IgH-IgL protein complex, also known as the B cell receptor (BCR), on the surface of the immature B cells.

The distinct stages of B cell differentiation are typically marked by changes in the expression of specific cell surface and intracellular markers.⁵ IL-2Rα (CD25), a component of the IL-2 receptor is routinely used to identify early pre-B cells that have completed IgH gene rearrangement.⁶ IL-2 has no effect on the proliferation or differentiation of cultured pre-B cells, and its function in developing B cells remains largely unknown.⁶ As B cells progress from the pre-B to the immature B cell stage, the expression of CD25 rapidly decreases. In the context of immune cell development, CD25 is also expressed on developing T cells at the early stages of CD4^-CD8^-CD3^- thymocytes.⁷

The receptor for IL-2 comprises 3 subunits: IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rγ (CD132).⁸ While IL-2Rα can bind IL-2 independently, it does so with low affinity, and it cannot transduce signals on its own. In contrast, the combined interaction of IL-2Rβ and IL-2Rγ forms a receptor of intermediate affinity for IL-2. The highest affinity for IL-2 is achieved when all 3 subunits unite, forming a high-affinity receptor that facilitates downstream signal transduction.^9,10 Thus, although incapable of signaling in isolation, IL-2Rα modulates the sensitivity of lymphocytes to IL-2. Constitutive loss of IL-2Rα in mice leads to massive lymphoproliferation and severe autoimmunity.¹¹ The underlying cause of these complications is the disruption in various T cell functions, including clonal expansion, differentiation, and activation-induced cell death, as well as impairment in the activity of regulatory T (T_reg) cells.^12–14

Despite extensive research on the role of CD25 in T cells, the B cell intrinsic function of CD25 has yet to be thoroughly investigated. Mouse and human B cells are known to upregulate CD25 upon stimulation.^15,16 Past studies have reported the existence of CD25-expressing B cells in various tissues such as the BM, peritoneal cavity, and the spleen, and have described these cells as having a highly activated phenotype.^17,18 Despite these findings, the in vivo significance of CD25 in B cell development and function is yet to be fully elucidated. The systemic immune perturbations caused by a global loss of CD25 preclude the accurate characterization of the B cell intrinsic role of CD25.¹¹ In our endeavor to explore the role of CD25 in B lymphocyte development and function, we generated a mouse model that allowed B cell-specific deletion of CD25. We find that the loss of CD25 in B cells had no discernible impact on B cell development within the BM and on B cell maturation within the periphery. Notably, B cells deficient in CD25 retained their ability to induce class switch recombination (CSR) ex vivo and mount robust responses to model antigens in vivo. Thus, while CD25 could be used as a convenient marker for specific differentiation stages of B cells, there appears to be no functional consequence of its B cell-specific loss in model systems.

Materials and methods

Mice

Mb1^cre/cre19 and Aicda^−/− mice²⁰ were respectively, kindly provided by Dr. Alexander Tarakhovsky (Rockefeller University, USA) and Dr. Tasuku Honjo (University of Kyoto, Japan). CD25^fl/fl mice on a C57BL/6 genetic background were generated as described in Fig. S1. Littermates were used whenever possible, otherwise age-matched controls were used. All mice were maintained in specific-pathogen free conditions and handled in compliance with the guidelines stipulated by MSKCC Research Animal Resource Center (RARC) and Institutional Animal Care and Use Committee (IACUC).

Primary B cell ex vivo CSR assays

Single cell suspension of splenocytes was prepared by passing mouse spleens through 70 µM cell strainers (Corning). Following red blood cell lysis in ACK lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA), naïve B cells were purified by negative selection with anti-CD43 magnetic beads (Miltenyi Biotec). Purified naïve B cells were cultured at a density of 1 × 10⁶ cells/ml in B cell media (RPMI-1640 supplemented with 15% FBS, 1% Pen-Strep (Gemini), 1% L-glutamine, 0.1% BME (GIBCO) in 6-well cell culture plates. The following cytokine stimulation conditions were used: (1) 30 µg/ml lipopolysaccharide (LPS) (Sigma) + 25 ng/ml IL-4 (R&D Systems); (2) 30 µg/ml LPS (Sigma); (3) 30 µg/ml LPS (Sigma) + 2 ng/ml TGF-β (R&D Systems) + 300 ng/ml anti-IgD dextran conjugate (Fina Biosolutions). IL-2 (Sigma) was used at 0.1 µg/ml. The cultures were split 1:2 at 48 h and 72 h, and harvested at 72 and 96 h for flow cytometry analysis.

Infections and immunizations

For immunization experiments, mice aged 10 to 12 weeks were first injected intraperitoneally with 100 µg of the hapten, 4-hydroxy-2-nitrophenylacetyl (NP), conjugated to chicken gamma globulin (CGG) (Biosearch Technologies), precipitated in Imject Alum adjuvant (Thermo Scientific). Mice were boosted with the same amount of NP-CGG on day 7. Spleens and lymph nodes were then collected for analysis on day 10. For LPS injections, mice were anesthetized using 3% isoflurane and injected with 100 µl (100 µg) of LPS from Salmonella minnesota R595 (Re) (TLRgrade™) (Enzo Life Sciences) into the retroorbital vein. Control mice received sterile PBS injections. Three days post-injection, the mice were sacrificed, and their spleens were collected for analysis.

Flow cytometry

Mouse spleen, lymph nodes, and Peyer's patches were processed into single cell suspensions by being passed through a 70 µM filter. BM cells were obtained by flushing the tibia bone. Spleen and BM samples underwent red blood cell lysis, followed by staining with Zombie Red fixable viability dye and rat anti-mouse CD16/CD32 Fc block antibody (clone 2.4G2). All data were collected using an LSR II flow cytometer (BD Biosciences). The following antibodies were used: B220 (BV786 and AF700; clone RA3-6B2), IgM (BUV395 and APC- eFluor780; clone II/41), IgD (BV510, clone 11-26c.2a) CD43 (FITC; clone eBioR2/60), CD19 (AF700; clone 6D5), CD21 (eFluor450; clone eBio4E3), CD23 (PerCP-eFluor710 and APC-Cy7; clone B3B4), cKit (APC; clone 2B8), CD25 (APC-eFluor780 and PE-Cy7; clones PC61.5 and PC61), CD138 (PE-Cy7; clone 281-2), CD90.2 (PE; clone 30-H12), CD4 (eFluor450; clone RM4-5), CD8a (BV510; clone 53-6.7), IgG1 (APC; clone X56), IgA (PE; 11-44-2), GL7 (PerCP-eFluor710; clone GL7), CD38 (AF700; clone 90), CD95 (PE-Cy7; clone Jo2), IgG3 (FITC; clone R40-82), NP (AF700, clone B1-8). All were purchased from eBioscience, Biolegend and BD Biosciences.

ELISA

Assays were done in Thermo Fisher Scientific MaxiSorp clear, flat-bottom, 96-well plates (439454, Thermo Fisher Scientific). Coating antibodies for binding IgM, IgG1, IgG2b, IgG2c, IgG3, and IgA (1020-01, 1070-01, 1090-01, 1079-01, 1100-01, and 1040-01, respectively; Southern Biotech) were used at 3 µg/ml in PBS pH 8. Each plate was coated with antibodies overnight at 4°C, washed with 0.05% PBST (0.05% PBS + 0.1% Tween-20), and blocked with ELISA diluent (00-4202-56; eBioscience) for 3 h at 25°C or overnight at 4°C. Plates were washed, loaded with serum samples or standards, and incubated for 2.5 h at 25°C or overnight at 4°C. The following isotype standards were used to calculate absolute concentration values: IgM (14- 4752-81; eBioscience), IgG1 (0102-01; Southern Biotech), IgG2b (14- 4732-81; eBioscience), IgG2c (0122-01; Southern Biotech), IgG3 (553486; BD Pharmingen), and IgA (553478; BD Pharmingen). Secondary antibodies for detecting IgM, IgG1, IgG2b, IgG2c, IgG3, and IgA (1020-05, 1070-05, 1090-05, 1079-05, 1100-05, and 1040- 05 respectively, Southern Biotech) were used at 1:2000 for 1.5 h at 25°C. eBioscience TMB substrate (00-4201-56, 100 µl per well) was used to develop and 1M phosphoric acid was used to stop development. Plates were read at 450 nm on a BioTek Synergy HT detector. Absolute concentrations of serum antibodies were determined by interpolation from the standard curve, while keeping within standard and sample linear ranges. All samples were done in duplicate over a 6-step dilution series. An 11-step standard curve was generated for each plate.

Autoantibody panel

Serum was collected from n = 4 pairs of 3- to 4-month-old CD25-KO and CD25-WT littermates and autoantibodies were analyzed as previously described.²¹

RNA sequencing and analysis

RNA sequencing of sorted pre-B cells (B220⁺IgD^-IgM^-CD43^-) was performed using an RNASeq-SMARTer by Integrated Genomics Operation facility at MSKCC. The obtained reads were aligned with STAR v2.7.7a²² to the mm10 genome and annotated using GATK.²³ Counts were generated using featureCounts.²⁴ DESeq2 ²⁵ was used to define differentially expressed genes and to compare the expression of WT vs. CD25-KO samples. The data were further visualized as a volcano plot, with transcripts considered differentially expressed (DE) if they showed a false discovery rate (FDR)-adjusted P-value < 0.05, and an absolute log₂ fold change of 0.5. The RNAseq data and analysis generated in this study are deposited in GEO under the accession code GSE282135.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, Inc.). FlowJo (Version 10.8.1) was used to analyze all flow cytometry data.

Results

CD25 is dispensable for B cell development and maturation

To investigate the requirement of CD25 in B cell development, we generated CD25^fl/fl mice (Fig. S1) in which exons 2 and 3 of CD25 were flanked by loxP sites. Exons 2 and 3 encode a region that binds IL-2 ²⁶ and are essential for the role of CD25 as an IL-2 receptor.¹¹ The CD25^fl/fl mice were bred to Mb1^Cre/+ mice to generate Mb1^Cre/+CD25^fl/fl progenies. The Mb1 gene encodes the Igα subunit of the BCR and is uniquely expressed in B cells from the earliest pro-B cell stage in the BM.¹⁹ The resulting Mb1^Cre/+CD25^fl/fl mice are thus expected to carry a B-cell-specific deletion of CD25. To confirm the efficacy and specificity of Mb1-Cre-mediated CD25 deletion in B cells, we carried out CD25 staining on BM pre-B cells and double negative (DN) thymocytes, as these are the respective developmental stages where CD25 expression is observed in developing B cells and T cells. As anticipated, pre-B cells from Mb1^Cre/+CD25^fl/fl mice (henceforth referred to as CD25-KO) mice displayed a total absence of CD25 staining, distinguishing them from Mb1^Cre/+CD25^fl/+ (CD25-Het) and Mb1^Cre/+CD25^+/+ (WT) mice (Fig. S2A, B). The staining data, in conjunction with the previous observation that T_reg-specific deletion of exons 2 and 3 shows a profound T_reg phenotype,¹³ suggest that the Cre-deleted CD25 allele encodes a non-functional protein. The deletion of CD25 was specific to B cells as we did not observe discernible differences in the frequency of CD25⁺ cells among CD25-KO and control double negative thymocytes (Fig. S2C, D). As expected, we also did not observe any differences in the frequencies of CD4⁺, CD8⁺, DN and DP thymocytes, and splenic CD4⁺ and CD8⁺ T cells between CD25-KO and control mice (Fig. S2C).

Next, we performed comprehensive analysis of the developing and mature B cell compartments in CD25-KO and control mice. We observed no statistically significant differences in the total cell counts and frequency of B cells in the BM and spleen of CD25-KO and control mice (Fig. 1A, B). The frequencies of immature B cells (B220^loIgM⁺), mature recirculating B cells (B220⁺IgD⁺), pro-B cells (B220⁺IgD^-IgM^-CD43⁺), and pre-B cells (B220⁺IgD^-IgM^-CD43^-) in the BM of CD25-KO mice were equivalent to those found in control mice (Fig. 1C, D).

Given that pre-B cells in the BM represent the sole developmental stage of B cells that expresses CD25, we aimed to identify potential transcriptional differences between the pre-B cell populations of CD25-KO and CD25-WT mice by bulk RNA sequencing. The top 3 differentially expressed genes (absolute log₂ fold change > 1, false discovery rate corrected P-value < 0.05) were H1f2, Junb and Adrb2 (Fig. 1E). Junb can directly bind to the Il2ra promoter and regulate CD25 expression,²⁷ while Adrb2 modulates CD25 expression by cAMP dependent transcriptional machinery^28,29 Hence, it is feasible that CD25 expression in pre-B cells is controlled by a feedback-responsive transcriptional network leading to the upregulation of these genes in the CD25-KO pre-B cells.

CD25 is expressed at the stage of B cell development when B cells pass through the pre-BCR checkpoint, which led us to explore its potential role in regulating negative B cell selection under physiological conditions. Given that altered pre-BCR signaling can lead to defects in B cell central tolerance and increased autoantibody production, we analyzed the blood plasma of 3-4-month-old CD25-KO and WT littermate controls for IgM and IgG autoantibody profiles against a panel of 128 established autoantigens. While there appeared to be a slight upward trend in the production of IgM autoantibodies in the CD25-KO mice, the differences were not statistically significant (Fig. 1F).

To examine the role of CD25 in B cell maturation, we next compared the frequencies of various mature splenic B cell subsets between CD25-KO and control mice. We found no differences in the frequencies of follicular B cells, T1 and T2 transitional cells, and marginal zone cells between the groups (Fig. 1G, H). Our findings suggest that loss of CD25 in B cells does not cause gross abnormalities in steady state B cell development and maturation.

Loss of CD25 in B cells does not impair germinal center formation at homeostasis

To evaluate the effect of CD25 deletion on B cell function in vivo, we analyzed the homeostatic germinal center (GC) response within secondary lymphoid organs. We included Aicda^-/- mice as a reference control, as these mice exhibit enlarged GCs and lymph node hyperplasia, thus serving as an indicator of atypical GC B cell frequencies. We observed no differences in the frequency of GC B cells within the Peyer’s Patches or mesenteric lymph nodes between CD25-KO and control mice (Fig. 2A–C). To investigate the potential influence of CD25 deficiency on CSR in GC B cells, we examined the prevalence of IgA and IgG1 class-switched cells within Peyer’s Patches and mesenteric lymph nodes, respectively. We did not observe any significant differences in the frequency of class-switched B cells between CD25-KO and control mice in these organs (Fig. 2D, E).

To evaluate the potential effect of CD25 deficiency on immunoglobulin production, we utilized ELISAs to analyze serum Ig concentrations at steady state. We did not observe differences in the levels of IgM, IgG1, IgG2b, IgG2c, and IgA when comparing CD25-KO and control mice. There was a minor statistical difference in the IgG3 titers when comparing CD25-Het and WT mice, but this disparity was absent when comparing CD25-KO and WT mice (Fig. 2F). In conclusion, our results indicate that the absence of CD25 in B cells does not disrupt the homeostatic GC responses and the production of serum Igs.

CD25 is expressed in activated splenic B cells but does not affect CSR

To assess the potential impact of CD25 on B cell activation, we carried out a series of ex vivo experiments using naïve splenic B cells and various cytokine cocktails known to activate B cells and induce CSR. At 96 h post-stimulation with LPS and IL-4, approximately 40% of WT B cells expressed CD25 (Fig. 3A). Addition of IL-2 did not increase the frequency of CD25-expressing B cells. We also did not observe any difference in the frequency of IgG1⁺ cells between CD25-KO and control B cells, regardless of IL-2 supplementation in the media (Fig. 3B). Likewise, while CD25 was expressed after induction of CSR to IgG3 by LPS (Fig. 3C), the frequency of IgG3 CSR remained similar between CD25-KO and control B cells and was unaffected by the presence of IL-2 (Fig. 3D).

Finally, we stimulated naïve splenic B cells with LPS + TGFβ + anti-IgD dextran to induce CSR to IgA. Addition of IL-2 to the stimulation cocktail led to a dramatic upregulation of CD25 expression in WT and CD25-Het cells (Fig. 3E, G). Yet, this substantial increase did not influence their capacity for CSR to IgA. We found equivalent levels of IgA positive cells between CD25-KO and control B cells, regardless of the presence or absence of IL-2 (Fig. 3F). Overall, loss of CD25 does not influence the ability of ex vivo stimulated B cells to undergo CSR.

Loss of CD25 does not affect immune response in vivo

To examine the role of CD25 in B cells during an immune response, we intravenously challenged mice with Salmonella-derived lipopolysaccharide (LPS), a standard model antigen for eliciting a Type-I T-independent response. We observed a modest increase in the frequency of GC B cells within the spleens of immunized mice, compared to PBS controls (Fig. 4A). There was a slight increase in the frequency of GC B cell frequency in CD25-KO mice compared to CD25-Het mice, but the differences were not statistically significant when compared to WT mice (Fig. 4A). Additionally, the frequency of splenic IgG3⁺ B cells, in response to the immunization, remained unchanged between CD25-KO and control mice (Fig. 4B).

To evaluate the T-dependent B cell response in vivo, mice were immunized intraperitoneally with the model antigen 4-hydroxy-3-nitrophenyl acetyl (NP)-conjugated chicken gamma globulin (NP-CGG). Immune response was assessed at day 10 post-immunization. As expected, there was an increased frequency of splenic GC B cells in immunized mice compared to the PBS control; however, there was no significant difference between CD25-KO and control mice (Fig. 5A). Neither were there any observed differences in the frequency of class-switched IgG1⁺ GC B cells in immunized CD25-KO mice compared to control mice (Fig. 5B). Finally, we analyzed the frequency of antigen-specific NP⁺ cells within the population of class-switched GC B cells. While a significant percentage of IgG1+ switched cells (∼60%) were specific for NP, the frequency was similar between the experimental groups (Fig. 5C). Overall, CD25 does not appear to play a cell intrinsic role in the ability of B cells to respond to T cell-dependent and T cell-independent in vivo immune challenges.

Discussion

Our phenotypic analysis of mice with a B-cell-specific CD25 deletion indicates that although CD25 is expressed in early pre-B cells, its absence does not result in any discernible developmental B cell phenotype. RNA-sequencing data from CD25-KO pre-B cells suggests that CD25 expression in these cells could be an incidental outcome of the transcriptional network directing B cell development. The transcription factor JunB, implicated in cellular proliferation—a process rapidly undertaken by pro-B cells transitioning to the pre-B stage—was upregulated in CD25-KO pre-B cells, potentially due to a negative feedback loop. An alternative explanation for the lack of a B cell-intrinsic phenotype in CD25-KO mice may lie in the absence of CD122 expression in developing B cells.³⁰ CD122 is essential for forming the high-affinity IL-2 receptor, which consists of CD25, CD122, and CD132. Consequently, developing B cells may be unable to respond to IL-2 signaling, suggesting that CD25 might not play a role outside the context of IL-2 signaling. Our data does not discount the potential role of CD25 in facilitating the expansion of pre-B cells. It is plausible that any minor impairments in CD25-KO mice could be offset during later phases of development.

Our data indicate that CD25 is expressed on ex vivo activated splenic B cells. This aligns with studies in human B cells showing that activation stimuli, such as LPS, CpG, and CD40L, can independently induce CD25 expression on B cells.^15,31 Despite the robust expression of CD25 in ex vivo activated B cells, we found that the absence of CD25 does not affect the ability of B cells to undergo CSR. Additionally, CD25-KO cells did not show any impairments in vivo as they responded robustly to standard T cell-dependent and -independent immunizations. This suggests that CD25 is not critical for optimal B cell activation and effector response. Overall, our data strongly suggest that CD25 does not exert an intrinsic role in B cells. Previous reports of elevated serum immunoglobulins in mice globally deficient in CD25, are likely a consequence of impaired T_reg cell function. Since T_reg cells are crucial for maintaining proper humoral immunity, the loss of T_reg cells in global CD25-KO mice likely underlies the observed B cell-related phenotypes.^13,32

It is interesting to note that the presence of IL-2 in the local environment may influence CD25 expression in B cells in vivo. Previous research has demonstrated that IL-2 can induce human B cells to differentiate into plasma cells, possibly via CD25 expression induction.^33,34 Future research should explore the specific conditions under which B cells across various tissue environments express and utilize the IL-2 - IL-2R signaling pathway.

Acknowledgments

We thank A. Bravo for help with maintenance of the mouse colony. We thank all members of the Chaudhuri Lab for helpful discussions and feedback. We acknowledge the use of the Integrated Genomic Operation Core and the Molecular Cytology Core Facility at MSKCC.

Supplementary material

Supplementary material is available at The Journal of Immunology online.

Funding

J.C. was supported by grants from the National Institutes of Health (grant numbers R01AI072194, R01AI124186, and P30CA008748), the Starr Cancer Research Foundation, the Ludwig Center for Cancer Immunotherapy, MSKCC Functional Genomics, and the Geoffrey Beene Cancer Center. K.T.B. was a recipient of the General Atlantic Fellowship.

Conflicts of interest

None declared.

Data availability

The GEO data underlying this article are available in the NCBI database at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE282135 and can be accessed with accession number GSE282135.

References

Author notes