Dual nature of the adaptive immune system in lampreys

Abstract

Jawless vertebrates use variable lymphocyte receptors (VLR) comprised of leucine-rich-repeat (LRR) segments as counterparts of the immunoglobulin-based receptors that jawed vertebrates use for antigen recognition. Highly diverse VLR genes are somatically assembled by the insertion of variable LRR sequences into incomplete germline VLRA and VLRB genes. Here we show that in sea lampreys (Petromyzon marinus) VLRA and VLRB anticipatory receptors are expressed by separate lymphocyte populations by monoallelic VLRA or VLRB assembly, together with expression of cytosine deaminase 1 (CDA1) or 2 (CDA2), respectively. Distinctive gene expression profiles for VLRA(+) and VLRB(+) lymphocytes resemble those of mammalian T and B cells. Although both the VLRA and the VLRB cells proliferate in response to antigenic stimulation, only the VLRB lymphocytes bind native antigens and differentiate into VLR antibody-secreting cells. Conversely, VLRA lymphocytes respond preferentially to a classical T-cell mitogen and upregulate the expression of the pro-inflammatory cytokine genes interleukin-17 (IL-17) and macrophage migration inhibitory factor (MIF). The finding of T-like and B-like lymphocytes in lampreys offers new insight into the evolution of adaptive immunity.

Figure 1. VLRA and VLRB expression define distinct lymphocyte populations

a, FACS analysis of lymphocyte-gated cells stained with anti-VLRA (R110) and anti-VLRB (4C4) antibodies. b, Percentages of VLRA and VLRB expressing lymphocytes in lamprey tissues. Larvae (left panel) n = 23, except gill n = 6; adults (right panel) n = 8 (***P < 0.001; **P < 0.01). c, Lymphocyte-gated cells were stained as in (a) and separated based on surface expression of VLRA and VLRB by fluorescence-activated cell sorting (FACS). Sorted cells were analyzed for VLRA (left) and VLRB (right) transcripts by quantitative real-time PCR (qPCR), n = 3. Error bars indicate s.e.m.

Figure 2. Monoallelic assembly of VLRA and VLRB genes

a,b, Schematic of VLRA (a) and VLRB (b) genes before (top panel) and after (middle panel) gene assembly. Forward and reverse primer locations and predicted sizes of PCR products are indicated. Lymphocytes from the indicated tissues were stained with anti-VLRA (R110) and anti-VLR-B (4C4) antibodies and lymphocyte-gated cells were FACS sorted into three populations: VLRA⁻/VLRB⁻ (DN), VLRA⁺ (A⁺), and VLRB⁺ (B⁺). VLRs were amplified from genomic DNA of the sorted lymphocyte populations (bottom panel). Germ-line (GL) and mature (M) products were verified by sequence analysis of representative DNA clones. c, CDA1 and CDA2 expression in sorted lymphocytes was measured by QPCR. Error bars indicate s.e.m., n = 3.

Figure 3. Differential gene expression profiles of VLRA⁺ and VLRB⁺ lymphocytes

Lamprey lymphocytes were FACS sorted into three populations on the basis of VLRA and VLRB surface expression. Relative transcript levels of the indicated genes were measured by QPCR and compiled into a heat map as described in Methods.

Figure 4. Antigen-activated VLRA⁺ lymphocytes do not secrete their receptors

a, Immunization with B. anthracis exosporium induces proliferation of VLRA⁺ and VLRB⁺ lymphocyte (n = 5; * P < 0.05). b, VLRA⁺ lymphocytes do not bind to B. anthracis spores before immunization or 14 days after booster immunization (n = 5; ***P < 0.001). c,d, VLRB, but not VLRA, is secreted. c, B. anthracis-specific VLRA and VLRB reactivity was evaluated by ELISA with anti-VLRA (9A6) and anti-VLRB (4C4) mAbs. d, Immunoblot of VLRA (lane 1) and VLRB (lane 2) HEK-293T cell transfectant lysates, naïve lamprey plasma (lane 3), and immunized plasma (lane 4) under reducing conditions. R110 = anti-VLRA rabbit antiserum; 9B3 = anti-VLRA mAb.

Figure 5. PHA preferentially stimulates VLRA⁺ lymphocytes

a,b, In vivo PHA stimulation. Lampreys were given 25 µg of PHA by intracoelomic injection on day 0. Proliferation (a) and absolute numbers (b) of VLRA⁺ and VLRB⁺ cells were determined on days 9 and 15 after injection; (a) n = 4; (b) n = 7; *P < 0.05; **P < 0.01; ***P < 0.001). c, Transmission electron microscopy imaging of unstimulated (left panel) and PHA stimulated (day 9) (right panel) VLRA⁺ cells. PHA stimulated cells have a larger diameter [naïve mean = 3.8±0.1µm (n = 7) versus PHA stimulated mean = 7.4±0.3µm (n = 7, P <0.01)] and increased cytoplasm. d, Cytokine expression by VLRA⁺ and VLRB⁺ lymphocytes. FACS sorted blood lymphocytes were isolated from PHA stimulated (day 7, n = 3) or unstimulated lampreys (n = 3) and analyzed for expression of IL-17 (left panel), MIF (middle panel), and IL-8 (right panel) by QPCR. Error bars indicate s.e.m.