IgD ligation allows peritoneal cavity B cell proliferation

Abstract

Atypical cytokine production and immune cell subset ratios, particularly those that include high proportions of macrophages, characterize tumor microenvironments (TMEs). TMEs can be modeled by culturing peritoneal cavity (PerC) cells which have a high macrophage to lymphocyte ratio. With TCR or BCR ligation, PerC lymphocyte proliferation is tempered by macrophages. However, PHA (T cells) and anti-CD40 (B cells) are activators that induce proliferation. Herein, we report that ligating IgD, in contrast to IgM, triggers PerC B cell proliferation. IL-4 addition enhanced the IgD response for BALB/c PerC B cells but suppressed that of C57BL/6 mice. Intriguingly, concurrent ligation of IgD and CD3_ε rescued a PerC T cell proliferative response. These results serve to expand the list of targets for promoting cellular and humoral immunity in conditions that model macrophage-rich TMEs.

Introduction

Freedom from external and internal challenges, infectious disease and cancer, relies on the collaborative integration of innate and adaptive immunity. Regulation, mediated through receptors and signaling molecules, ensures the collaborative harmony of myeloid and lymphoid cells up to, and through, the reproductive “window” of mammalian life¹. Organized microenvironments within lymphoid organs optimize responses to diverse antigens. Proportionally, lymphocytes, affording a diverse collection of receptor specificities, outnumber myeloid cells in these organs. However, with aging, immune dysregulation can emerge, most notably in cancer. Carcinomas often harbor tumor-specific lymphocytes whose function is impaired by aberrant immune regulation mediated by a disproportionate influx of myeloid cells, particularly macrophages². The composition of such tumor microenvironments (TMEs) has become the focus of intensive research in the emerging discipline that is immuno-oncology.

Evolving understanding of the aberrant immunity in cancer has fostered strategies that target components of the immune system in union with those that simply focus upon transformed cells. Logically, early success came by modulating T cell biology via neutralization of suppressive receptor-ligand signalling and inhibitory cytokine activity³. Fundamentally, T cells rely on myeloid cells for their function, ergo, modulation of macrophage and dendritic cell biology has become the latest focus in cancer immunotherapy⁴. B cells, much less studied in this capacity, should be investigated considering their efficacy as both APCs and ASCs⁵. Expansion of the oncologist’s toolkit increases the probability of favorable patient outcomes.

In this report, experiments demonstrating B cell activation in the context of immunosuppression and its potential impact on T cell biology are described. The atypical myeloid:lymphoid ratio found in the tumor microenvironment can be modeled by peritoneal cavity (PerC) cell culture. PerC cells contain a high ratio of myeloid to lymphoid cells, a low percentage of T cells, and an immunochemical milieu that tempers lymphocyte proliferation. Traditional B and T cell activators, including mitogens (LPS, ConA) and BCR or TCR ligands (F’[ab’]₂ anti-IgM, anti-CD3_ε) lead to a suppressive response in this culture system6–10. However, there are activators that permit B (anti-CD40) and T (PHA) cell proliferation in this model9,10.

Mature B cells typically co-express two isotypes of membrane-bound immunoglobulin, IgM and IgD. Excluding PerC B cells, ligation of either BCR activates most B cells11,12. Herein, IgD ligation is shown to induce PerC B cell expansion and, with concurrent CD3_ε ligation, rescues T cell proliferation. These findings are discussed in the context of exploring the dynamics of B - T lymphocyte interaction for advancing cancer immunotherapy.

Materials and methods

Mice

Two to four-month old male and female BALB/c and C57BL/6 mice were obtained from the Jackson Laboratory, Bar Harbor, ME. Mice, bred and maintained at Rider University, were handled in accordance with NIH, Animal Welfare Act, and Rider University IACUC guidelines.

Preparation of cell suspensions and cell culture

Spleen cells were prepared by gentle disruption of the organ between the frosted ends of sterile glass slides. RBC depletion was performed with Ack lysis buffer followed by washing with Hanks Balanced Salt Solution (HBSS) buffer. Peritoneal cavity (PerC) cells were obtained by gentle lavage of the peritoneum with warm, sterile HBSS. Viable cell counts were obtained by Trypan blue exclusion, and cells were resuspended to reach final concentrations ranging from 0.5 – 4.0 × 10⁶/mL in RPMI 1640 culture media (Life Technologies) supplemented with 10% FBS (Hyclone), 0.1 mM nonessential amino acids,100 U/ml penicillin, 100 lg/ml streptomycin, 50 lg/ml gentamicin, 2 mM L-glutamine, 2 10⁻⁵ M 2-ME, and 10 mM HEPES, and were plated in 96-well “V”-, “U”, or half area flat-bottom microtiter plates (Corning Costar, Fisher Scientific, Pittsburgh, PA). Cells were incubated in a humidified atmosphere of 5% CO₂ at 37° C for 48–72 hours. For anti-IgM stimulation, F(ab’)² fragment of goat anti-mouse IgM, μ-chain-specific (Jackson ImmunoResearch, West Grove, PA) polyclonal antibody was added at 20 μg/ml. LPS from E. coli, serotype R515 (Enzo Life Sciences, Farmingdale, NY or Sigma) was added at 5 μg/ml. Polyclonal goat anti-mouse IgD (Ebioscience) was plated at 10 μL/ml. The monoclonal, allotype-specific mouse IgG_2b αIgD^a/Igh-5a reagent (AMS 9.1, BioLegend) was plated at 10 μL/mL, with or without the addition of polyclonal αIgG_2b (Southern Biotech) at 0.1 μg/mL. To inhibit COX, indomethacin (INDO; Sigma) was added at 0.3 μM. IL-4 (Peprotech) was added at 100U/mL. Neutralizing antibodies to IL-10 (Ebioscience) were added at culture initiation. After 44 or 68 hours of incubation, plates were labeled with 1μCi/well [³H] thymidine (Moravek Inc., Brea, CA) and left to incubate for four hours. Cells were harvested on filter paper mats using a semi-automated cell harvester (Skatron Instruments, Richmond, VA). Radioactivity was measured by liquid scintillation spectrometry. For each experiment, 5 wells were established per condition. All experiments repeated 3–6 times.

Immunofluorescence staining and flow cytometry

Cultured PerC and spleen cells were harvested using PBS + 2mM EDTA. Cell suspensions were blocked with Fc block of rat anti-mouse CD16/32 (Fc Block, eBioscience) and 2% normal rat serum (Jackson ImmunoResearch, West Grove, PA). Cells were stained with FITC-, PE-, or PerCP-Cy5.5- labeled rat anti-mouse CD45R/B220, CD11b, CD8, CD4, IgM, IgD, and/or F4/80 mAbs (eBioscience). For carboxyfluorescein succinimidyl ester (CFSE) cell proliferation, cells were labeled with CellTrace CFSE Cell Proliferation Kit as described in the protocol by Thermo Fisher (Thermo Fisher, Eugene, OR) prior to cell culture.

B lymphocytes, T lymphocytes, or myeloid cells were identified through expression of markers described above and determined through multiparameter flow cytometric analyses on a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) by FSC/SSC gating on lymphocytes and myeloid populations using CellQuest software.

Statistical Analyses, Stimulation, Percent Gated, and Mean Fluorescent Intensity Indices

Lymphocyte proliferative responses are presented as average counts per minute (CPM) +/− standard error or the mean (SEM). Data sets were compared using the Student’s t-test with p-values below 0.05 considered statistically significant. * = P<0.05, ** = P<0.005, *** = P<0.0005 compared to each relative control. The stimulation index (SI) represents the average CPM for each condition divided by the average CPM for the control (such as complete media [CM], anti-IgD alone, etc.). Mean fluorescent intensity (MFI) index represents average MFI for the condition divided by the average MFI for each relative control. Likewise, percent gated SI represents average percent gated cell population for each condition divided by the percent gated for its relative control.

Results

IgD ligation triggers PerC B cell proliferation

Initial in vitro studies of peritoneal cavity (PerC) leucocyte biology showed that, due to their higher representation in this body site, macrophages (Mϕs) restrain T cell proliferation via arginine catabolism (Fig. 1A)^6–8. PerC Mϕs were also found to temper B cell responses to LPS and BCR (F(ab’)₂ -anti-IgM) ligation via production of immunosuppressive IL-10 and prostaglandin respectively (Fig. 2)⁹. However, PerC B cells exhibited a strong response to CD40 ligation (Fig. 2)⁹. Unlike the majority of B cells (“B2”) that comprise organized lymphoid tissue (spleen, lymph nodes), PerC B cells are enriched for IgM^hiIgD^lo/− “B1” B cells (Fig. 1C)^13,14. Since B2 B cells express considerably more IgD than IgM (IgM^loIgD^hi) [Figs. 1B,C], IgD ligation was tested as a means to trigger PerC B cell proliferation. Where SP B cells responded to both IgM and IgD ligation, PerC cells only responded to anti-IgD treatment (Fig. 3). The addition of IL-4 increased SP B cell responses to IgM and IgD ligation and helped recover a PerC B cell anti-IgM response (Fig. 3). In contrast, IL-4 suppressed the anti-IgD response of PerC B cells through arginase (ARG) as shown by the recovery of this response in the presence of the ARG inhibitor N omega-hydroxyl-L-arginine (1-NA) (Fig. 4). These results reveal the complexity of activating B cells in a Mϕ-rich environment where the specifics of B cell subset, BCR type, and cytokine milieu dictate the response.

Fig. 1.

FACs analyses of C57BL/6J spleen (SP) and peritoneal cavity (PerC) cells shows higher macrophage representation in PerC and differences in IgM/IgD-defined B cell subsets. A, macrophage (F4/80⁺) representation; B, IgD expression, MFI = mean fluorescence intensity; C, correlated IgM/IgD subset composition of SP and PerC cells. Panel C profiles and data are for CD19⁺ cells.

Fig. 2.

C57BL/6J spleen and peritoneal cavity (PerC) cell proliferative response to LPS and BCR or CD40 ligation. Numbers depict the stimulation index (SI = experimental CPM /relevant control). * = p < 0.05, ** = p < 0.005, *** = p < 0.0005.

Fig. 3.

IgD ligation triggers PerC B cell proliferation. C57BL/6J PerC and SP cells were cultured with polyclonal αIgM or αIgD, +/− IL4.

Fig. 4.

Arginase suppresses PerC B cell proliferation. C57Bl/6J PerC cells were cultured with αIgM or αIgD, +/− IL4. Arginase inhibition via 1-NA addition recovered IL4 enhancement of the αIgM and αIgD response.

Cell density impacts the response to BCR ligation

Prior studies found that, with increasing PerC cell density, T cell proliferation decreases^6–8. The different outcome for IgM versus IgD ligation invited assessment of how culture density might impact these responses for PerC cells. The marginal response to IgM ligation seen at low density was lost as PerC cell density increased (Fig. 5, left panel, ● symbol).; likewise, the enhanced response seen by adding IL4 was lost (Fig. 5, left panel, ◻ symbol). In contrast, the response to IgD ligation was retained as cell density increased as well as its suppression when adding IL4 (Fig. 5, right panel). Thus, cell density is an additional, critical factor to consider when attempting to activate B cells in an unconventional (non-lymphoid organ), macrophage-rich microenvironment.

Fig. 5.

Culture density impacts the response to BCR ligation. C57Bl/6J PerC cells were cultured with polyclonal αIgM or αIgD (solid line, filled circle) +/− IL4 (dashed line, unfilled box) at the cell concentrations listed. Horizontal dotted line indicates stimulation index value of 1.0 (response = control).

Mouse strain differences with IgD ligation

Although initial experiments focused on assessing C57BL/6 B cell responses, prior reports of differences in lymphocyte biology between this strain and BALB/c mice invited their comparison¹⁵. BALB/c B cell responses paralleled those of C57Bl/6 mice (Fig. 3) with the exception that their PerC B cell response to IgD was enhanced, rather than suppressed, by the addition of IL4 (Fig. 6). This result is consistent with cytokine biology distinctions reported for these strains, notably associating Th1, IFNγ-driven (“cellular”) responses with C57BL/6 and Th2, IL4-driven (“humoral”) responses with BALB/c mice¹⁵.

Fig. 6.

IgD ligation triggers PerC B cell proliferation. BALB/c PerC and SP cells were cultured with polyclonal αIgM or αIgD, +/− IL4.

Monoclonal anti-IgD response requires extensive crosslinking or “co-ligation”

Most studies of B cell activation via BCR ligation rely on polyclonal antisera (goat-anti-mouse IgM or IgD) to optimize crosslinking¹². A monoclonal anti-IgD^a antibody (AMS-9.1) alone was insufficient to promote BALB/c PerC B cell proliferation. However, by increasing crosslinking using a second, polyclonal anti-mouse IgG_2b antibody B cell expansion was markedly enhanced (Fig. 7). These results validate the need for strong cross-linking to activate B cells through IgD.

Fig. 7.

PerC B cell proliferation following IgD^a ligation. CFSE-labeled BALB/c PerC cells were first treated with monoclonal IgG_2b anti-IgD^a and then cultured with polyclonal anti-IgG_2b. Positive values represent B2 B cells gated for CFSE dye dilution. A representative experiment of 6 is shown.

T cell expansion in PerC cell preparations cultured with both αCD3 and αIgD

There is considerable interest in developing strategies to release T cells from the suppression found within TMEs. Intriguingly, prior research has suggested that IgD ligation protects T cells from apoptosis and enhances humoral immunity^16,17. To test this premise in the PerC culture TME model, CFSE-labeled PerC cells were assessed for proliferation when CD3_ε and IgD were simultaneously ligated. As anticipated, CD3 ligation alone led to limited T cell proliferation, particularly in C57BL/6 mice (Fig. 8, Panel A, SI < 1.0). However, both CD4+ and CD8+ T cells expanded when concurrent IgD ligation was included. This was true for PerC cells from both strains of mice with greater expansion of CD8+ T cells in the C57Bl/6 and CD4+ T cells in the BALB/c mice. This effect was not observed with IgM ligation (data not shown). These data suggest that strategies that concurrently engage both the BCR and the TCR might serve in rescuing T cell activation in microenvironments that restrain T cell expansion.

Fig. 8.

IgD ligation permits T cell expansion following TCR ligation. CFSE-labeled C57BL/6 (Panel A) and BALB/c (Panel B) PerC cells were incubated with αCD3 +/− polyclonal αIgD. After 48 hrs, cells were stained for CD8 and CD4 and analyzed by FACs. % Positive values represents CD4⁺ or CD8⁺ cells gated for CFSE dye dilution.

Discussion

In this report we show that B cells in a macrophage-rich, immunosuppressive environment can be induced to proliferate by targeting their IgD, but not IgM, BCR. The difference in response between these BCRs grew as culture density increased. Multivalent ligation of IgD was essential to optimize B cell proliferation. IL4 addition, known to enhance the B cell response to IgD ligation, did so for BALB/c PerC B cells, but not those of C57BL/6 mice^15,18. Intriguingly, IgD ligation rescued the suppressed PerC T cell proliferative response with concurrent TCR ligation. These results reveal an additional target for inducing humoral immunity within putative TMEs and a strategy to recover, or enhance, cellular immunity therein.

What explains the IgM/D BCR response dichotomy? With respect to BCR targeting, the PerC B cell pool has a greater proportion of IgM^hiIgD^-/lo B1 cells relative to the IgM^loIgD^hi B2 cells that are the predominant subset in organized lymphoid tissue such as the spleen or lymph nodes^11–14. Based solely on BCR expression, B1 cells would respond optimally to IgM ligation and B2 cells to IgD. However, B1 cell activation is restrained by constitutive expression of CD5/SHP-1, CD22, and Siglec G, as well as autocrine IL10 production¹⁹. IgM ligation on B1 cells leads to insufficient calcium mobilization, limited proliferation, increased IL10 production, and apoptosis^20–24. This would also be the case for IgD ligation on B1 cells which express little, or no, IgD¹¹. In contrast, IgM ligation induces marked calcium efflux in, and proliferation of, B2 cells. With INDO present to inhibit cyclo-oxygenase (COX) generation of PGE₂, PerC B2 cells respond to IgM ligation (Fig. 2)⁹. However, INDO was not required for PerC B2 cells to respond to IgD ligation. This strategy targets naïve B cells and avoids triggering the B1, B10, or B_reg anti- inflammatory cytokine (IL10, TGF_β) and prostaglandin (PGE₂) production found with CD40 ligation^9,25–27. Thus, IgD ligation is much less likely to limit effector T cell function or foster T_reg and TAM development, but rather enhance the development of T_FH cells which have become targets for immune-oncology development^28,29.

Regardless of which BCR is ligated, IL4 enhances B cell proliferation by upregulating Ig_α and Ig_β coreceptor expression and affording protection from apoptosis^30–32. However, IL4 also induces macrophage ARG expression leading to the arginine deletion that suppresses lymphocyte proliferation^9,33. This was seen with the IgD response of C57BL/6 but not that of BALB/c PerC cells, results consistent with the C57BL/6 (TH1/M1), BALB/c (TH2/M2) T cell/macrophage immunobiology described for these strains¹⁵. Although IL4 enhances B cell proliferation, IL4R expression by TAMs and epithelial cancers likely confound clinical application unless an antibody-cytokine fusion protein targeting strategy can be developed^34–36.

How might concurrent IgD ligation “rescue” the PerC T cell proliferative response? A role for IgD in T cell biology, with T cells bearing IgD receptors (Fcδ) interacting with IgD+ B cells to enhance humoral immunity, was reported^37,38. This cognate interaction was proposed to protect T cells from dexamethasone-induced apoptosis, which would bode well for protection during AICD^16,39. However, molecular characterization of Fcδ -IgD did not follow. Further understanding of cognate T cell – APC receptor-ligand interactions will provide insight as to how IgD-activated B cells could help T cells avoid the suppression characteristic of TMEs⁴⁰.

PerC IgD++ B2 cells are phenotypically analogous to follicular B cells that rely upon T follicular helper cells (T_FH) for their terminal differentiation²⁹. The ICOS-ICOSL interaction is a key receptor-ligand combination involved in this cognate interaction⁴¹. ICOS is rapidly induced following TCR ligation while ICOSL is constitutively expressed by APCs, the highest level found on B2 cells^41–44. BCR ligation increases ICOSL expression and shedding which provides “bystander” B cell help for T cells^45–47. Reciprocal signaling not only facilitates naïve T cell proliferation, differentiation, and survival but also reactivation of memory T cells^48,49. These observations provide insight as to how increased B cell ICOS-L expression correlates with improved chemotherapeutic responses in colon and breast cancer^50–52. To assess the role of ICOS in the IgD ligation- T cell protection response, PerC cells from ICOSL^−/− mice will be studied⁵³.

Advances in defining TMEs have expanded targeting strategies for cancer immunotherapy^54,55. The early focus on T cells was logical, with initial success treating tumors with inflammatory or “hot” signatures: TH₁ infiltration, PDL1/PD1 and CTLA-4 expression, etc [56]. Still, many tumors either lack these signatures or are refractory to current protocols⁵⁷. Many carcinomas form tertiary lymphoid tissue, including B cells, which can be a positive diagnostic criterion⁵⁸. Such structures permit a more natural form of multicellular, polyclonal immunity. Strategies to orchestrate the activation of specific cell types in such microenvironments will continue to advance. Our findings suggest that further definition of key B - T cell interactions could serve in this capacity.

Abbreviations:

AICD

Activation-induced cell death

APC

Antigen presenting cell

ARG

arginase

ASC

antibody secreting cell

BCR

B cell receptor

INDO

indomethacin

INOS

inducible nitric oxide synthase

PerC

peritoneal cavity

SI

stimulation index

TAM

tumor associated macrophage

TCR

T cell receptor

TME

tumor microenvironment

T_regs

regulatory T cells

1-NA

1 N omega-hydroxyl-L-arginine