Immune Surveillance by Natural IgM Is Required for Early Neoantigen Recognition and Initiation of Adaptive Immunity

Abstract

Early recognition of neoantigen-expressing cells is complex, involving multiple immune cell types. In this study, in vivo, we examined how antigen-presenting cell subtypes coordinate and induce an immunological response against neoantigen-expressing cells, particularly in the absence of a pathogen-associated molecular pattern, which is normally required to license antigen-presenting cells to present foreign or self-antigens as immunogens. Using two reductionist models of neoantigen-expressing cells and two cancer models, we demonstrated that natural IgM is essential for the recognition and initiation of adaptive immunity against neoantigen-expressing cells. Natural IgM antibodies form a cellular immune complex with the neoantigen-expressing cells. This immune complex licenses surveying monocytes to present neoantigens as immunogens to CD4⁺ T cells. CD4⁺ T helper cells, in turn, use CD40L to license cross-presenting CD40⁺ Batf3⁺ dendritic cells to elicit a cytotoxic T cell response against neoantigen-expressing cells. Any break along this immunological chain reaction results in the escape of neoantigen-expressing cells. This study demonstrates the surprising, essential role of natural IgM as the initiator of a sequential signaling cascade involving multiple immune cell subtypes. This sequence is required to coordinate an adaptive immune response against neoantigen-expressing cells.

Keywords: Ly6C+ monocytes; XCR1+ Batf3+ dendritic cells; cancer; natural IgM; neoantigens.

Figure 1.

Key players involved in the coordinated, immunological cascade against neoantigen-expressing cells: B cells, T cells, lymph node (LN) monocytes, and dendritic cells (DCs). (A) Schematic diagram of our stated and examined hypothesis. The rejection and elimination of neoantigen-expressing cells requires a chain-link leukocyte reaction. (B) Top: the experimental scheme used to determine, if minor antigen–mismatched cells (neoantigen-expressing cells) are accepted or rejected in female mice. Bottom: flow plots show the recall of adoptively transferred CD45.1 male cells into wild-type (WT) male or female mice (left) compared with CD45.1 female cells into WT female mice (right). (C) Flow data illustrate three controls: two negative controls (acceptance, male cells into male WT and knockout [KO]/transgenic mice, referred to as experimental [Exp] mice) and one positive control (rejection, male cells into WT female mice). Experimental mice are PMEL (CD8⁺ T cell transgenic, hypo-CD8⁺ T cell repertoire), Batf3^−/− (lack Batf3⁺ DCs), CD4^−/− (lack CD4⁺ T cells), CCR2^−/− (lack LN monocytes), and μMT (lack peripheral B cells) female mice. Data are representative of two to three individual experiments with n = 3–5 mice per group. Table displays the combined experiments with the number of mice that rejected male cells (numerator) over the number of total mice examined (denominator). Acceptance is shown in blue, rejection in red. Ag = antigen; CTL = cytotoxic T lymphocytes; IN = intranasal; IV = intravenous; MHCII = major histocompatibility complex class II; Va2 = Vα2.

Figure 2.

Natural IgM antibodies are essential for the initial recognition and elimination of neoantigen-expressing cells. (A) Flow data illustrate three controls: two negative controls (acceptance, male cells into male WT and KO/transgenic [Tg] mice) and one positive control (rejection, male cells into WT female mice). Exp mice are IghelMD4 (hypo-IgM repertoire) AID^−/− (hyper-IgM repertoire) female mice. Data represent two independent experiments consisting of n = 3–5 animals per group. Table displays combined experiments with the number of mice that rejected male cells (numerator) over the number of total mice examined (denominator). Acceptance is shown in blue, rejection in red. (B) Lung-draining LN (LLN) flow plots. Top row: live cells plotted as CD11c versus MHCII to illustrate migratory DCs, gated blue. Middle row: migratory DCs are plotted as XCR1 versus CD11b to illustrate the two overarching DC subtypes: XCR1⁺, CD11b^low Batf3⁺ DCs (red arrow) and CD11b⁺, Irf4⁺ DCs. Batf3^−/− mice lack Batf3⁺ DCs, whereas IghelMD4 (Ighel) mice contain Batf3⁺ DCs. Bottom row: LN cells pregated on low side scatter (SSC), CD64⁺, CD11b⁺ cells and plotted as Ly6C versus MHCII to illustrate LN Ly6C⁺ monocytes. CCR2^−/− mice display significantly diminished Ly6C⁺ monocytes (green arrow), whereas IghelMD4 mice display a normal quantity of LN monocytes.

Figure 3.

Female serum immunoglobulins bind male cells, resulting in its immunogenic uptake and antigen presentation. (A) Top: experimental design: ovalbumin (OVA)-expressing female and male cells were adoptively transferred into female mice; 6 days later, CD45.2 OVA⁺ and CD45.1 OVA⁻ female target cells were transferred to assess in vivo CTL response against the linked antigen, OVA. Bottom: target cells were plotted as CD45.2 OVA⁺ versus CD45.1 OVA⁻ cells. Right bar graph displays the percent killing of OVA⁺ target cells. (B) Top: experimental design (schematic diagram in E6), OVA-expressing male cells were either untreated (−) or preincubated WT male, WT female, or Rag^−/− female serum before adoptive transfer into male mice; 6 days later, CD45.2 OVA⁺ and CD45.1 OVA⁻ female target cells were transferred to assess in vivo CTL response against the linked antigen, OVA. Bottom: target cells were plotted as CD45.2 OVA⁺ versus CD45.1 OVA⁻ cells. Right bar graph displays the percent killing of OVA⁺ target cells. (C) Western blot and densitometry analysis of IgM binding on male cells preincubated with female, male, or no serum. Positive control is WT serum. Data are representative of three to five independent experiments. **P < 0.005, ***P < 0.001. CFSE = carboxyfluorescein succinimidyl ester; WB = Western blot.

Figure 4.

Surveying monocytes require MHCII expression to prime CD4⁺ T helper cells, which then licenses Batf3⁺ DCs via CD40 to cross-present antigen as an immunogen. Flow data illustrate three controls: one negative control (acceptance, male cells in 100% chimeric WT male mice) and two positive controls (rejection, male cells in 100% chimeric WT female mice and [A] 80:20 bone marrow [BM] cell replenishment of circulating WT monocytes [CCR2^−/−:WT], [B] WT CD4⁺ T cells [CD4^−/−:WT] or [C] WT Batf3⁺ DCs [Batf3^−/−:WT]). Exp chimeric female mice consist of 80:20 BM replenishment of 100% circulating (A) MHCII-deficient monocytes (CCR2^−/−:IAb^−/−) (APC MHCII expression, E6), (B) CD40L-deficient CD4⁺ T cells (CD4⁻:CD40L^−/−), or (C) CD40-deficient Batf3⁺ DCs (Batf3^−/−:CD40^−/−). Data represent two independent experiments consisting of n = 4–5 animals per group. (D) Table displays the combined experiments with the number of mice that rejected male cells (numerator) over the number of total mice examined (denominator). Acceptance is shown in blue, rejection in red.

Figure 5.

129SvEv female cells were not rejected in C57BL/6 female mice lacking a diverse natural IgM antibody repertoire or Batf3⁺ DCs. Schematic diagram illustrates experimental design. At Day 20, CD45.1 female 129/BL6 cells were recalled in CD45.2 IghelMD4 (Ighel) and Batf3^−/− female mice compared with WT female mice, where complete rejection of 129/BL6 cells occurred. Flow plot represents n = 8 mice per group.

Figure 6.

A complete natural IgM antibody repertoire is required for antitumor immunity. (A) The scatter plot displays the number of urethane-induced tumors observed per mouse in WT, IghelMD4 Tg, CCR2^−/−, and Batf3^−/− mice after 6 months post–urethane treatment. (B) At 16 days after intravenous injection of B16F10, melanoma cell line in WT, IghelMD4 Tg, CCR2^−/−, and Batf3^−/− mice was examined for melanoma tumor development. Left: pictures illustrate metastatic melanoma on the surface of whole lungs. Right: scatter plot displays the number of surface metastatic tumors developed per mouse. Circles indicate pure KO mouse, and squares indicate WT recipient mice reconstituted with 100% BM from the indicated KO mouse. Data combine three to five independent experiments. ****P < 0.0001.