Key Points

NAbs alert the immune system against nascent neoantigen-expressing cells.

NAbs are required to induce a CTL response against neoantigen-expressing cells.

NAb-tagged cells mount immunity against immunogenic and nonimmunogenic Ags.

Abstract

Recent studies have revealed a critical role for natural Abs (NAbs) in antitumor immune responses. However, the role of NAbs in cancer immunosurveillance remains unexplored, mainly because of the lack of in vivo models that mimic the early recognition and elimination of transforming cells. In this article, we propose a role for NAbs in alerting the immune system against precancerous neoantigen-expressing cells immediately after they escape intrinsic tumor suppression mechanisms. We identify four distinct reproducible, trackable, MHC-matched neoantigen-expressing cell models that do not form tumors as the end point. This amplified readout in the critical window prior to tumor formation allows investigation of new mediators of cancer immunosurveillance. We found that neoantigen-expressing cells adoptively transferred in NAb-deficient mice persisted, whereas they were eliminated in wild-type mice, indicating that the circulating NAb repertoire alerts the immune system to the presence of transformed cells. Moreover, immunity is mounted against immunogenic and nonimmunogenic neoantigens contained in the NAb-tagged cells, regardless of whether the NAb directly recognizes the neoantigens. Beyond these neoantigen-expressing model systems, we observed a significantly greater tumor burden in chemically and virally induced tumor models in NAb-deficient mice compared with wild-type mice. Restoration of the NAb repertoire in NAb-deficient mice elicited the recognition and elimination of neoantigen-expressing cells and cancer. These data show that NAbs are required and sufficient for elimination of transformed cells early in tumorigenesis. These models can now be used to investigate how NAbs stimulate immunity via recognition receptors to eliminate precancerous cells.

Introduction

The average human cell makes ∼10,000 errors in transcriptional, translational, and posttranslational modification each day (1, 2). Intrinsic tumor suppression mechanisms repair or eliminate most of these errors. Only a small fraction of cells undergo cellular transformation, resulting in the expression of neoantigens. Once a transforming cell bypasses intrinsic tumor suppression mechanisms, extrinsic tumor suppression (i.e., cancer immunoediting) mechanisms are engaged. These mechanisms are orchestrated by immune cells and divided into three stages: elimination (cancer immunosurveillance), equilibrium, and escape (3). However, there is a period between the intrinsic escape of transforming cells and the extrinsic elimination phase when an adaptive immune response is initiated. This period, which lasts from days to weeks, is nearly impossible to capture: it occurs prior to tumor formation and release of damaged signals. To overcome this hurdle and address this knowledge gap, we created four trackable in vivo neoantigen-expressing cells that do not form tumors as the end point. This allows an investigator to amplify the readout during this critical window. Using these cells, we show that natural Abs (NAbs) are critical for early-stage recognition and induction of immune cell activation against a broad spectrum of neoantigens.

NAbs are nonspecific, broadly cross-reactive, low self-affinity Abs (2, 4–7). Functionally, they are widely known for apoptotic cell clearance and microbial pathogen recognition (8, 9), activation of complement (10–12), Ab-dependent cell cytotoxicity, phagocytosis (13, 14), dendritic cell (DC) maturation (15), Ag targeting to draining lymph nodes (LNs) (16), and facilitation of CD8+ T cell–mediated immunological responses (17). However, until recently, their fundamental role in preventing tumor formation and recognition of precancerous cells has been overlooked. This is mainly due to the use of a popular B cell–deficient mouse, the muMT mouse, which has an abnormal compensatory antitumor immune mechanism (18).

Recently, we demonstrated that NAbs recognize and bind neoantigens (i.e., new, altered self-antigens) that are not necessarily damaged or senescent (2, 18, 19). We were initially led to investigate the role of NAbs by observations in unconventional, non–cancer-related immune neoantigen models, including syngeneic male cells adoptively transferred into female mice. In this model, we observed the rejection of male cells in syngeneic wild-type (WT) female mice, but not in NAb-deficient mice, even though all other immune cells involved in neoantigen-expressing cell elimination (DCs, other myeloid cells, NK cells, and T cells) are present. The immune rejection of male cells in WT female mice is due to Y chromosome Ags, H-Y Ags, being tagged as neoantigens by NAbs in syngeneic female mice (19). This mechanism is not unique to sex-based cell elimination. We made the same observation when we transferred MHC-matched 129Sv female cells into C57BL/6 female mice (19–21). C57BL/6 mice lacking NAbs did not reject 129Sv female cells in circulation. In this setting, the immune response against 129Sv cells in C57BL/6 WT mice is due to the detection of allelic variations outside of the MHC locus by NAbs (18, 19). Both adoptively transferred cells, male cells or 129Sv female cells, express normal MHC class I levels and have no tumor-inducing damage signals, and yet an immune response is mounted in WT, but not NAb-deficient, mice. We also found the same to be true regardless of the tumor model we examined, either cell line or chemical induction (18, 19). Impending questions remain. How, then, are neoantigen-expressing cells identified and subsequently cleared? It is known that DCs need to be licensed to present Ag as an immunogen to T cells (22, 23). Therefore, what receptors or mediators, after NAb binding, are required to license the DCs in adaptive immunity? These are fundamental questions that need to be investigated and require models to parse out an invisible period when transforming cells have not yet formed clusters with hypoxic conditions and release of damage signals. The in vivo models outlined in this study will provide investigators the tools to address these fundamental questions.

Materials and MethodsMice

CD45.2 WT, C57BL/6 Ly5.1, OT-I transgenic,129Sv/SvImJ, C3H-H2b, C57BL/10J, C57BL/6-Tg(UBC-GFP)30Scha/J (B6-EGFP), B6.Cg-Thy1a/CyTg(TcraTcrb)8Rest/J (PMEL), Act-mOVA, IghelMD4 (IgHEL), Rag1−/−, B6.129P2(C)-Cd19tm1(cre)Cgn/J (CD19cre), B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J (Rosa-DTA), mouse mammary tumor virus (MMTV)-PyMT, Aicda−/−, and sIgM−/− mice were purchased from Jackson Research Laboratories and Charles River NCI. In IgHEL mice (hypo-IgM), >90% of IgM-secreting B cells are specific for hen egg lysozyme. Notably, IgHEL females were always used as breeders to avoid passive immunity to fetus and neonate through placenta and milk. All mice were bred in-house. Mice were genotyped or phenotyped prior to studies and used at 6–8 wk of age; housed in a specific pathogen-free environment at Dartmouth Hitchcock Medical College, an American Association for the Accreditation of Laboratory Animal Care–accredited institution; and used under protocols approved by the Institutional Animal Care and Utilization Committee.

Flow cytometry

Tissues were minced and digested with 2.5 mg/ml collagenase D (Roche) for 30 min at 37°C. A total of 100 μl of 100 mM EDTA was added to stop 1 ml of enzymatic digestion. Digested tissue was pipetted up and down 30 times using a glass Pasteur pipette and passed through a 70-μm nylon filter to acquire single-cell suspensions from spleen and lung draining LNs. Cells were stained with the following mAbs: PE conjugated to CD4, PerCP-Cy5.5 conjugated to IgMa, PE-Cy7 conjugated to C45.1, BUV805 conjugated to CD8, FITC conjugated to Va2, allophycocyanin conjugated to CD45.2, allophycocyanin-Cy7 conjugated to CD19, and BV510 conjugated to IgM. The viability dye DAPI (#D9542; Sigma) was added immediately before each sample acquisition on a BD Symphony A3 analyzer (BD Biosciences). Data were analyzed using FlowJo (Tree Star, Ashland, OR). Ag-specific Abs and isotype controls were obtained from BioLegend, eBioscience, and BD Biosciences.

Neoantigen rejection model: recall assay

The 129, C3H, and B10J strains have allelic variations outside the MHC locus of C57BL/6 mice. C57BL/6 CD45.1 OT-I mice were crossed with 129Sv, C3H-H2b, and C57BL/10J mice to create an F1 129/BL6 OT-I (CD45.1/2) mouse, C3H/BL6 OT-I (CD45.1/2) mouse, and BJ10/BL6 OT-I (CD45.1/2) mouse, respectively. Female 129/BL6 OT-I, C3H/BL6 OT-I mouse, and BJ10/BL6 OT-I cells were used to introduce neoantigens into female CD45.2 C57BL/6 mice. Male C57BL/6 OT-I (HY model) CD45.1/2 cells were also injected in C57BL/6 CD45.2 female mice. C57BL/6 CD45.1 OT-I cells were injected with every neoantigen model as an internal control. During this experiment, the cages were changed every 72 h to minimize the mice’s exposure to any possible pathogen-associated molecular patterns (PAMPs). The model setup included 1 million CD45.1/2 129-BL6 OT-I/C3H-BL6 OT-I/BJ10-BL6 OT-I/HY-BL6 OT-I cells, and CD45.1 BL6 OT-I cells were transferred i.v. into congenic recipients. A total of 2 mg was freshly prepared and filtered through a 0.22-μm filter to remove OVA aggregates. A total of 5 μg OVA was given intranasally, resulting in the expansion of adoptively transferred neoantigen-expressing T cells. Mice were then rechallenged with 100 μg OVA at day 16 (129 and HY neoantigen rejection model)/day 12 (C3H neoantigen rejection model)/day 28 (BJ10 neoantigen rejection model) to recall adoptively transferred cells. At 2 days after rechallenging, the lung draining LNs were examined for the presence (recall) or absence (rejection) of adoptively transferred neoantigen-expressing cells.

B and T cell reconstitution

B and T cells from naive WT mice were negatively enriched using the mouse Pan B Cell isolation kit and Pan T Cell isolation kit from Miltenyi Biotec. To reconstitute B cells, we i.v. injected different groups of IgHEL and Rag1−/− mice with enriched B cells from spleen (i.v.) and peritoneal cavity (i.p.). To reconstitute T cells, we injected enriched T cells from spleen in the Rag1−/− mice (i.v.).

In vivo endogenous cytotoxic T cell assayH-Y OVA

Female WT and IgHEL mice were immunized with either female or male OVA cells. Ten days later, CFSE-labeled (1:1) female C57BL6 CD45.2 (OVA+) and WT CD45.1 (24) target cells were adoptively transferred in the mice. On day 2, after target cell transfer, the target cell killing was analyzed in the spleen of the mice.

129-GFP

129Sv mice were crossed to B6-EGFP mice to create 129-EGFP mice. WT and IgHEL mice were primed separately with thymocytes from B6-EFP and 129-EGFP mice. Eighteen days later, CFSE-labeled target cells, i.e., thymocytes from B6-EGFP CD45.2: WT CD45.1 (1:1), were adoptively transferred in the mice. On day 2, after target cell transfer, the target cell killing was analyzed in the spleen of the mice.

Cancer modelsMelanoma model

B16F10 melanoma cells (CRL-6475) were purchased from ATCC and maintained in RPMI with 10% FCS, 1% penicillin/streptomycin/l-glutamine (Sigma-Aldrich), 1% nonessential amino acids (Sigma-Aldrich), 1% sodium pyruvate (Sigma-Aldrich), 10 mM HEPES (Sigma), and 0.1 mM 2-ME. The cell line was confirmed free of mycoplasma contamination, and the identification was authenticated through STR DNA profiling. Mice were i.v. challenged with 2 × 105 viable B16F10 cells and euthanized 16 d postinjection. The lungs of mice were inflated with 1% agarose. A blinded observer counted the B16F10 lung surface metastases.

MMTV-PyMT model

In the MMTV-PyMT model, we first crossed IgHEL(F) to MMTV-PyMT(M) to get IgHEL-PyMT female pups deficient in NAbs. The female littermates with only PyMT transgene were used as control, thus giving the same genetic background. The mice were harvested 16 wk after birth, and the breast tumors were collected. The number of tumors was counted for each mouse in both groups. Tumor volume was calculated for each mouse on the day of harvest (V = [π/6] × W2L).

Urethane model

Spontaneous lung tumors were induced with i.p. injections of 1 mg/g urethane (ethyl carbamate; Sigma-Aldrich) weekly for 6 wk (25). Mice were harvested 20 wk after the last i.p. injection of urethane. An experienced experimentally blind reader performed the tumor counts on the H&E-stained lungs. Tumor numbers of each lung were averaged and statistically analyzed.

Bioinformatics analysis of exome data

Exome data were trimmed using Cutadapt (26) to retain only reads longer than 18 bp and base quality > 20. Trimmed reads were mapped to the latest version of the mouse genome, mm39 with BWA mem, and the quality of the alignment was checked with picard tools MarkDuplicates and CollectHSMetrics. Variants were called using FreeBayes with pooled continuous and genotype quality options; we used a threshold of 15 for mapping quality, 20 for minimum coverage, and 0.2 for minimum alternate fraction with the Twist exome bed file (coordinates translated for mm39) for the target region. Variants were annotated with SnpEff (27).

Statistics

Statistical analysis was conducted using InStat and Prism software (GraphPad). All results are expressed as the mean ± SEM. Statistical tests were analyzed using ordinary one-way ANOVA (normal distribution) with Tukey’s multiple comparison test as posttest and two-tailed Student’s t test, respectively. Differences were considered statistically significant when p ≤ 0.05, as indicated with asterisks (*p ≤ 0.05, **p < 0.005, and ****p < 0.0001).

ResultsNAbs recognize neoantigen-expressing cells with distinct gene variants

First, we provide an updated diagram on the role of NAbs in cancer immunosurveillance (Fig. 1A). Previously, we demonstrated that NAbs are required to license APCs to present neoantigens as an immunogen to cognate T cells (19). Without NAbs, the immune system is not alerted that a cell is transforming, resulting in the acceptance of neoantigen-expressing cells (Fig. 1A) (18, 19). In this study, we expand our original findings and developed four models that investigators can use to examine how NAbs recognize a broad spectrum of neoantigens and promote immunity. One model is the adoptive transfer of male cells into syngeneic female mice, where the Y chromosome Ags are the neoantigens (Fig. 1B). The other three models are adoptively transferred female MHC-matched C3H.H2b, 129Sv, and C57BL/10 cells into C57BL/6 female mice, where the rejection of neoantigen-expressing cells is not sex based but due to allelic variations outside of the MHC locus (Fig. 1B).

FIGURE 1.

NAbs are required to recognize and eliminate a broad spectrum of transformed cells in the absence of PAMPs. (A) Proposed hypothesis illustrating the early events during tumorigenesis. The NAbs tag early transforming cells prior to cluster formation, thus recruiting different immune cells to the tumor site for elimination (created with Biorender.com). (B) Table enlists the number of genetic variations exhibited by C57BL6 (male), C3H.H2b (female), 129Sv (female), and C57BL10 (female) mice compared with the reference C57BL6 (female). (C) Experimental layout: splenocytes from CD45.1 BL6-OT-I (internal control) and CD45.1/2 129BL6-OT-I/C3H.H2b-OT-I/C57BL10-OT-I/HY-OT-I were adoptively transferred into CD45.2 WT and IgHEL mice followed by intranasal delivery of OVA (Ag), which expands the cells expressing neoantigen-expressing cells for detection in BL6 host. The mice were rechallenged with OVA on day 12, 16 and 28 to assess the neoantigen cell rejection on days 14–30. The table illustrates the number rejected from mice examined. (D) Experimental design as in (C). C57BL/6 WT and CD19creDTA mice were analyzed for the rejection of CD45.1/2 129BL6 cells. The table illustrates the number rejected from mice examined.

To demonstrate that NAbs are required for neoantigen recognition and elimination, we crossed CD45.2 C3H.H2b, 129Sv, and C57BL/10 mice with CD45.1 transgenic T cell mice (any transgenic T cells with C57BL/6 background can be used). This cross results in trackable CD45.1/2 neoantigen-expressing cells. In addition to the experimental cells of interest, an internal CD45.1 C57BL/6 control is used to ensure proper injection, expansion, and recall of adoptively transferred cells in CD45.2 hosts, WT, and IgHEL mice (Fig. 1C). We used IgHEL mice as our NAb-repertoire–deficient mice. These mice have >90% of IgM-secreting B cells specific for hen egg lysozyme (28). The advantage of these mice is that DC migration and lymphatic development appear normal compared with muMT mice, where DC migration is impaired (19, 29). As anticipated, 18 d after the adoptive transfer of CD45.1 control and CD45.1/2 neoantigen-expressing cells, WT mice mounted a robust cytolytic response against all four neoantigen-expressing cells. Rejection of the neoantigen-expressing cells is measured by the absence of CD45.1/2 cells in WT mice (Fig. 1C). In contrast, NAb-deficient IgHEL mice recalled all four CD45.1/2 neoantigen-expressing cells. Hence NAb-deficient mice did not reject the neoantigen-expressing cells (Fig. 1C). In addition, using a different B cell–deficient mouse model, 129Sv female cells were not rejected in CD19creDTA mice (lacking CD19+ cells) compared with WT control (Fig. 1D). Notably, the intensity of immunogenicity correlated with the number of gene variants expressed in each model. Therefore, the speed of neoantigen rejection, faster to slower, was as follows: C3H.H2b, 129Sv, H-Y Ags, and C57BL/10 (data not shown). Overall, these findings suggest that NAbs are required for the recognition and elimination of a broad spectrum of neoantigens.

Neoantigen-expressing cells are rejected in NAb-deficient mice reconstituted with either WT B cells or naive serum

To determine whether the rejection of neoantigen-expressing cells in WT mice requires NAbs to initiate adaptive immunity, we reconstituted IgHEL mice with either enriched B cells or naive serum from WT mice (Fig. 2A, 2B). As shown in (Fig. 1C, WT mice and not IgHEL mice rejected all four neoantigen-expressing cell models. However, when IgHEL mice were reconstituted with either WT B cells or naive serum, rejection of the adoptively transferred cells was completely restored (Fig. 2A). Overall, these data suggest that if the appropriate immune activator is present (i.e., replenishment of IgHEL mice with NAbs), the APCs and T cells will become inherently functional.

FIGURE 2.

IgHEL mice reject neoantigen-expressing cells in the presence of NAbs. Experimental design as in (Fig. 1C was used for the recall assays in (A), (D), and (E). (A) C57BL/6 WT, IgHEL, and IgHEL mice reconstituted with serum and B cells from naive C57BL/6 WT mice were analyzed for the rejection of CD45.1/2 129BL6-OT-I, C3H.H2b-OT-I, H-Y-OT-I, and C57BL10-OT-I cells. Top, scatterplots representing the frequency of rejection. Each dot represents one mouse. Data are pooled from three independent experiments with four to five mice in each group. Bottom, representative flow plots showing % recall of the neoantigen-expressing cells. (B) Flow plot showing the negative enrichment for B and T cells from the spleen of naive WT mouse used for reconstitution. (C) A schematic diagram highlights the key players in recognizing and eliminating neoantigen-expressing cells through a chain-link leukocyte reaction (created with Biorender.com). (D) Scatterplot represents the frequency of rejection of C3H-H2B-OTI cells in the absence of B and T cells (Rag1−/− mice), NAbs (IgHEL mice), CD4+ T cells (anti-GK1.5), and CD8+ T cells (PMEL mice). Data are representative of two independent experiments with three to five mice in each group. Each dot represents one mouse. (E) Scatterplot illustrates the frequency of rejection of CD45.1/2 129BL6 cells in Rag1−/− mice reconstituted with B cells, T cells, and both B and T cells from naive WT mice. Data are representative of three independent experiments with four to five mice in each group. Each dot represents one mouse. Mean ± SEM; ****p < 0.0001, one-way ordinary ANOVA, with post hoc Tukey’s multiple comparison test (A, D, and E). ns, nonsignificant.

We have previously outlined the immunological cascade by which neoantigen-expressing cells are eliminated and illustrate it in (Fig. 2C (19). In brief, NAbs form a cell-bound immune complex, licensing LN-trafficking monocytes and/or DC2 to prime cognate CD4+ T cells, which in turn use CD40L to license DC1 via CD40 to cross-prime CD8 CTLs against the neoantigen-expressing cells. Because the current paradigm holds that the elimination phase requires only T cells and not B cells, we decided to investigate this using our model systems. Two distinct experimental sets of mice were performed. One set used mice selectively deficient for either T and B cells (Rag−/− mice), B cells (IgHEL mice, lack B cells repertoire), CD4 T cells (mice treated with anti-CD4), or CD8 T cells (PMEL mice, lack CD8 T cell repertoire) (Fig. 2D). In the second set, Rag−/− mice were reconstituted with either T cells, B cells, or T and B cells from naive WT mice (Fig. 2E). Both sets of experimental mice demonstrated that the early rejection of neoantigen-expressing cells, C3H.H2b cells (Fig. 2D) and 129 cells (Fig. 2E), requires T and B cells. Thus, T cells without B cells could not mount immunity against neoantigen-expressing cells, even when NK cells and all myeloid cells are present.

NAbs promote an endogenous CTL response against nonimmunogenic Ags linked to neoantigen-expressing cells

Cancer cells contain nonimmunogenic and immunogenic Ags. Therefore, we examined whether NAbs are required to elicit an immune response against nonimmunogenic Ags, GFP and OVA, that are linked to neoantigen-expressing cells. To test this, we used an in vivo CTL killing assay against the linked Ag, OVA or GFP. First, OVA-expressing female or male cells were adoptively transferred in female mice: WT, IgHEL, and IgHEL reconstituted with WT naive serum mice. After 10 d, CFSE-labeled female target cells, OVA+ (CD45.2) and OVA− (CD45.1) cells, were transferred at a 1:1 ratio to measure an endogenous CTL response. Two days after target cells were transferred, the killing of the OVA+ target cells compared with the OVA− target cells was measured (Fig. 3A). Both WT and IgHEL female mice that received OVA-expressing female cells did not mount immunity against the foreign Ag, OVA, because OVA alone is nonimmunogenic. However, when WT and NAb-reconstituted IgHEL female mice received OVA-expressing male cells, immunity was mounted against the OVA Ag, but not in IgHEL mice (Fig. 3A). To ensure that the CTL response is not linked to the recognition of H-Y Ags, we repeated the endogenous CTL experiment using GFP. 129Sv GFP+ female cells were adoptively transferred into WT, IgHEL, and IgHEL reconstituted with WT naive serum mice. Twenty days after, only WT and NAb-reconstituted IgHEL mice killed GFP-expressing cells, whereas IgHEL mice did not (Fig. 3A). These data suggest that when neoantigen-expressing cells are tagged by NAbs, untagged linked neoantigens will also be presented as an immunogen.

FIGURE 3.

NAbs are required to mount an immune response against nonimmunogenic Ags linked with neoantigen-expressing cells. (A) Left, experimental design. Top, OVA-expressing female and male cells were adoptively transferred into female mice; 10 d later, CFSE-labeled CD45.2 OVA+ and CD45.1 OVA− female target cells were transferred to assess in vivo CTL response against the linked Ag. Bottom, GFP-expressing 129Sv and WT female cells were adoptively transferred into female mice; 18 d later, CFSE-labeled CD45.2 GFP+ and CD45.1 GFP− female target cells were transferred to assess in vivo CTL response against the linked Ag. Middle, flow plot represents frequency of killing of OVA+ and GFP+ target cells in WT, IgHEL, and IgHEL mice treated with serum from naive WT mice. Right, scatterplot represents the % killing of OVA+ and GFP+ target cells by individual mouse in each group. Data are representative of two to four independent experiments with three to four mice in each group. (B) Left, experimental design. Top, 129Sv cells preincubated with serum from WT or Rag−/− before adoptive transfer into WT and IgHEL mice. Ten days later, 129Sv CD45.2 and CD45.1 C57BL6 WT target cells were transferred to assess in vivo CTL response against the 129Sv neoantigen-expressing cells. Middle, target cells were plotted as 129 cells versus B6 cells. Right, scatterplot displays the % killing of 129Sv target cells. Data are representative of two to four independent experiments. Mean ± SEM; ****p < 0.0001, one-way ordinary ANOVA, with post hoc Tukey’s multiple comparison test (A and B).

To further confirm that NAbs are required to develop a CTL response against neoantigen-expressing cells, we preincubated 129Sv cells with either naive WT or Rag−/− (i.e., serum lacking Abs) serum and then adoptively transferred them into IgHEL mice. Unlike 129Sv cells preincubated with Rag−/− serum, 129Sv cells preincubated with WT serum resulted in an endogenous CTL response against 129Sv target cells (Fig. 3B). These findings demonstrate that (1) NAbs are required to license the immune cells to develop a CTL response against neoantigens and (2) NAb-tagged cells will result in the immunogenic presentation of their immunogenic and nonimmunogenic Ags regardless of whether the NAb directly binds the Ag.

NAbs are required for antitumor immunity in melanoma, breast cancer, and adenocarcinoma

Given the importance of NAbs during the early stages of tumorigenesis, we examined whether NAbs play a significant role in cancer using three distinct tumor models: B16F10 melanoma cell line, mammary gland-specific viral oncogene, and chemical induction. In the melanoma model, IgHEL mice displayed a significant tumor burden compared with WT mice (Fig. 4A) (18, 19). IgHEL mice reconstituted with either WT B cell or naive serum significantly decreased their tumor burden compared with IgHEL mice alone (Fig. 4A, Supplemental Fig. 1). Next, we investigated whether B cells were required as APCs to mount antitumor immunity against melanoma. CD19creDTA mice were reconstituted with either WT or MHC class II (MHC II)–deficient B cells and challenged with the B16F10. Regardless of reconstitution with either MHC II–sufficient or –deficient B cells, B cell–reconstituted CD19creDTA mice demonstrated a significant reduction in tumor burden compared with CD19creDTA mice alone (Fig. 4B, Supplemental Fig. 2). These findings suggest that in the context of the B16F10 melanoma model, the antitumor immune response is dependent on NAbs and not on the Ag-presenting property of B cells.

FIGURE 4.

The absence of NAbs enhances the tumor burden outcome in different tumor models. (A) At 16 d after i.v. injection of B16F10, melanoma cell lines in WT, IgHEL, and IgHEL mice reconstituted with serum and B cells from naive WT mice were examined for melanoma tumor development. Top, scatterplot displays the number of surface metastatic tumors developed per mouse. Bottom, pictures illustrate metastatic melanoma on the surface of whole lungs. Data are pooled from three independent experiments with four to five mice in each group. (B) Top, scatterplot displays the number of surface metastatic tumors developed in CD19creDTA and CD19creDTA reconstituted with the bone marrow from WT and MHC class II−/− mice. Bottom, pictures illustrate metastatic melanoma on the surface of whole lungs. (C) Breast tumors per mouse were enumerated and illustrated by scatterplot; each dot represents one mouse. (E) PyMT (littermate control) and IgHEL-PyMT mouse breast tumors were harvested exactly at 16 wk of age. Top, scatterplot illustrates the tumor volume and number of breast tumors. Bottom, pictures illustrate the representative size and number of breast tumors. Tumor volume was calculated for each mouse on the day of harvest (V = [π/6] × W2L). Each dot represents one mouse. (D) Top, Lung sections illustrating the tumor nodules in WT, IgHEL, and CD19creDTA mice 20 wk after urethane treatment. Bottom, the scatterplot displays the number of urethane-induced tumors observed per mouse. Data combine three to five independent experiments with four to five mice in each group. Mean ± SEM; **p < 0.003, unpaired t test (C); ****p < 0.0001, **p < 0.005, one-way ordinary ANOVA, with post hoc Tukey’s multiple comparison test (A, B, and D).

Next, we examined the role of NAbs in two spontaneous tumor models. First, MMTV-PyMT male mice were crossed with IgHEL female mice to prevent passive immunity, creating IgHEL-PyMT and WT-PyMT littermate controls. At 4 mo of age, NAb-deficient mice developed significantly more and bigger breast tumors compared with WT-PyMT mice (Fig. 4C, Supplemental Fig. 3). The second cancer model is a chemically induced spontaneous lung adenocarcinoma model. WT, IgHEL, and CD19creDTA mice were treated with urethane and harvested 20 wk later. Mice lacking NAbs, IgHEL and CD19creDTA mice, developed significantly more lung tumors than WT mice (Fig. 4D) (18, 19). Overall, these data support our hypothesis that NAbs recognize and initiate immunity against a broad range of neoantigen-expressing cells and cancer.

Lastly, to test our proposed hypothesis that circulating NAbs, not membrane-bound Abs, bind early to neoantigen-expressing cells, we created a mouse model deficient of circulating NAbs. Aicda−/− mice (hyper-IgM mice, lack isotype switching and somatic hypermutation) were crossed with sIgM−/− (lack secreted IgM) mice to create Aicda−/−sIgM−/− mice, which lack circulating IgG and IgM Abs but contain membrane-bound IgM. Aicda−/−sIgM−/− mice challenged with 129Sv cells did not mount a robust immune response against the neoantigen-expressing cells compared with Aicda−/−, sIgM−/−, and WT mice (Fig. 5A). In the B16F10 melanoma lung metastasis model, Aicda−/−sIgM−/− displayed significantly more tumors compared with WT counterparts (Fig. 5B, Supplemental Fig. 4). All in all, these data align with the interpretation that the circulating NAbs primarily recognize neoantigen-expressing cells to forewarn the immune system for their elimination, particularly in the absence of PAMPs and non-PAMP adjuvants (i.e., damage signals).

FIGURE 5.

NAbs in circulation are required to mount an immune response against neoantigen-expressing cells. (A) C57BL/6 WT, Aicda−/−, sIgM−/−, and Aicda−/−sIgM−/− mice were analyzed for the rejection of CD45.1/2 129BL6-OTI cells. Top, scatterplots representing the frequency of rejection. Each dot represents one mouse. Data are pooled from three independent experiments with three to five mice in each group. Bottom, representative flow plots showing % recall of the neoantigen-expressing cells. (B) Top, scatterplot displays the number of surface metastatic tumors developed in C57BL/6 WT and Aicda−/−sIgM−/− mice. Bottom, pictures illustrate metastatic melanoma on the surface of whole lungs. Mean ± SEM. ****p < 0.0001, unpaired t test (A); ****p < 0.0001, one-way ordinary ANOVA, with post hoc Tukey’s multiple comparison test (B). ns, nonsignificant.

Discussion

This study outlines four neoantigen-expressing cell models with varying degrees of gene variants. The elimination of all four neoantigen-expressing cells in mice required NAbs for recognition and elimination demonstrating the broad spectrum of recognition NAbs have. We hypothesize that NAbs detect kinks that occur when self is altered. Our findings open a new area of study to investigate how NAbs are licensing the immune system and promoting the clearance of neoantigen-expressing cells. The in vivo cellular models we provide allow for tracking of neoantigen-expressing cells with an internal control to assure proper experimental design.

NAbs are not thought of as the first line of defense against cancer because of the wide use of a popular B cell–deficient mouse, muMT mice. These mice were found to promote antitumor immunity similar to or greater than WT mice (18). However, we recently investigated and explained why the muMT mice, which lack NAbs, display a robust antitumor immune response. The muMT mice have an unexpected compensatory mechanism consisting of an exuberant differentiation of type 1 IFN-producing plasmacytoid DCs and recruitment of NK cells to the tumor microenvironment compared with WT mice (18). Diminishing the compensatory mechanisms in the muMT mice, plasmacytoid DC-type 1 IFN or NK cells, revealed that they developed uncontrolled tumors similar to the IgHEL mice, and thus demonstrating that NAbs are indeed required for the initiation of antitumor immunity.

Although our study focused on the role of NAbs, there are most likely several other unappreciated mechanisms in the early stages of elimination that require exploration. It remains to be determined how DCs become licensed in the absence of PAMPs and tumor-associated damage signals. It is possible that NAbs are required to activate the damage signals because it is easy to observe damage in tumor-burdened IgHEL mice (i.e., excess tumor burden; (Fig. 4A), yet antitumor immunity is not mounted without NAbs. This suggests that NAbs are not only required in the early stages of elimination, but in the later stages when damage signals are secreted within the tumor microenvironment.

In the future, it will be interesting to determine whether C1q-secreting interstitial macrophages play a role in the innate or adaptive response of NAb-mediated neoantigen-expressing cell clearance (30), particularly because complement-mediated immune response is downstream of Ab binding. All the investigative questions asked throughout the article can be addressed using our cellular models. Overall, future studies will begin to uncover the novel characteristics and function of the overlooked role of NAbs in antitumor immunity.

Limitations and caveats

The data presented are at this time limited to murine systems, and future work in humans will be necessary to fully establish the clinical and translational relevance of NAbs in the cancer patients. There are two experimental settings that do not fit our hypothesis. Both were a result of incorporating whole OVA into cells by either crossing act-OVA mice with 129Sv mice or inserting OVA plasmid into the B16F10 cell line. In IgHEL mice, the adoptive transfer of same-sex act-OVA 129Sv cells or OVA-expressing B16F10 resulted in their rejection. We believe there are two possible