Novel Teleost CD4-Bearing Cell Populations Provide Insights into the Evolutionary Origins and Primordial Roles of CD4+ Lymphocytes and CD4+ Macrophages

Abstract

Tetrapods contain a single CD4 coreceptor with four Ig domains that likely arose from a primordial two-domain ancestor. Notably, teleost fish contain two CD4 genes. Like tetrapod CD4, CD4-1 of rainbow trout includes four Ig domains, whereas CD4-2 contains only two. Because CD4-2 is reminiscent of the prototypic two-domain CD4 coreceptor, we hypothesized that by characterizing the cell types bearing CD4-1 and CD4-2, we would shed light into the evolution and primordial roles of CD4-bearing cells. Using newly established mAbs against CD4-1 and CD4-2, we identified two bona-fide CD4(+) T cell populations: a predominant lymphocyte population coexpressing surface CD4-1 and CD4-2 (CD4 double-positive [DP]), and a minor subset expressing only CD4-2 (CD4-2 single-positive [SP]). Although both subsets produced equivalent levels of Th1, Th17, and regulatory T cell cytokines upon bacterial infection, CD4-2 SP lymphocytes were less proliferative and displayed a more restricted TCRβ repertoire. These data suggest that CD4-2 SP cells represent a functionally distinct population and may embody a vestigial CD4(+) T cell subset, the roles of which reflect those of primeval CD4(+) T cells. Importantly, we also describe the first CD4(+) monocyte/macrophage population in a nonmammalian species. Of all myeloid subsets, we found the CD4(+) population to be the most phagocytic, whereas CD4(+) lymphocytes lacked this capacity. This study fills in an important gap in the knowledge of teleost CD4-bearing leukocytes, thus revealing critical insights into the evolutionary origins and primordial roles of CD4(+) lymphocytes and CD4(+) monocytes/macrophages.

FIGURE 1

Characterization of leukocyte populations expressing surface CD4-1 and CD4-2. (A) Flow cytometry of HK leukocytes double-stained with anti-CD4-1 and anti-CD4-2b mAbs. Representative dot plot shows CD4-2 vs. CD4-1 expression on whole HK leukocytes. Three different cell populations are circled: CD4-1⁺/CD4-2⁺ (CD4 DP), CD4-1⁻/CD4-2⁺ (CD4-2 SP), and CD4-1⁺/CD4-2⁻ (CD4-1 SP) cells. (B) Representative dot plot profiles (FSC vs. SSC) of CD4 DP, CD4-2 SP, and CD4-1 SP cells. The distributions of CD4 DP (top), CD4-2 SP (middle) and CD4-1 SP (bottom) cells are shown in blue, red and green dots respectively, while negatively stained cells are in gray dots. (C) The morphology of sorted CD4 DP (top), CD4-2 SP (middle), and CD4-1 SP cells (bottom), visualized by WG staining. Scale bar = 10 μm. (D and E) Staining of HK leukocytes with anti-CD4-1 (D) or anti-CD4-2b (E) mAbs in combination with anti-IgM, anti-IgT, or anti-CD8α mAbs. Representative dot plots show stained cells within the lymphocyte gate. Data in A–E are representative of three independent experiments (n = 12 fish). (F–H) Transcription profile of sorted lymphocyte populations. CD4 DP, CD4-2 SP, CD8α⁺, and CD4-1⁻/CD4-2⁻/CD8α⁻ lymphocytes (Neg), were sorted from HK leukocytes. Gene expression analysis of these sorted lymphocyte populations was performed by real-time PCR for the genes encoding for: (F) T-cell co-receptors (cd4-1, cd4-2a, cd4-2b, lag3, cd8a, and cd8b); (G) B-cell receptors (membrane bound form of ighm and ight); and (H) TCR/CD3 complex and Lck (tcra, tcrb, cd3g/d, and lck1). The transcript levels of indicated genes in F–H are shown relative to the expression levels in the Neg lymphocyte population (set to 1) and are expressed as mean ± SEM (n = 4 fish). Data in F–H are representative of two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Bonferroni correction).

FIGURE 2

Distribution of CD4⁺ lymphocyte subsets in lymphoid tissues. (A-C) Flow cytometry of leukocytes from blood (PBL), spleen (SPL), HK (HKL), and thymus (THY) stained with anti-CD4-1, anti-CD4-2b, and anti-CD8α mAbs. Representative dot plots show CD4-2 vs. CD4-1 (A), CD4-1 vs. CD8α (B), and CD4-2 vs. CD8α (C) expressions within the lymphocyte population. The values adjacent to outlined areas indicate percentage of each subset within the lymphocyte gate. (D) The percentage of CD4 DP and CD4-2 SP lymphocytes within the lymphocyte gate of indicated tissues. (E) Frequency of CD4 DP and CD4-2 SP lymphocytes among total CD4⁺ lymphocytes of indicated tissues. (F) The percentage of indicated lymphocyte subsets in thymus. The values shown in D–F are expressed as mean ± s.d. (n = 8 fish). (G–I) Immunofluorescence microscopy of CD4-1 (green) and CD4-2 (red) expressions on CD4 DP (G) and CD4-2 SP (H) lymphocytes from spleen. Spleen lymphocytes stained with isotype-matched control mAbs are shown in I. Nuclei are counterstained with DAPI (blue). Scale bar = 5 μm. Data are representative of two independent experiments.

FIGURE 3

CDR3 length analysis of TRVβ transcripts from CD4⁺ T cells. (A) CDR3 length profiles from sorted CD4-2 SP and CD4 DP lymphocytes and from whole HK leukocytes for selected TRBV-TRBC combinations. Data are representative of three healthy fish. X-axis: length of run-off products (in bp); Y-axis: fluorescence arbitrary units. (B) PCA projection of CD4-2 SP, CD4 DP, and whole HK leukocyte samples according to the first two components using diversity scores computed from all TRBV-TRBC combinations. (C) Heatmap of diversity scores for selected TRBV-TRBJ combinations from three healthy animals. Scores are represented on pale-yellow to red scale color, corresponding to increasing diversity of TRBV-TRBJ profiles.

FIGURE 4

Proliferative capacities of CD4 DP and CD4-2 SP cells upon mitogen, MLR, and antigen-specific stimulations. (A) Gating strategy used to identify proliferating and non-proliferating CD4 DP and CD4-2 SP cells. We first selected singlets using forward scatter area (FSC-A) versus forward scatter height (FSC-H) parameters (left dot plot). From the singlet population, we selected all CD4-2⁺ leukocytes from living cells (leukocytes were stained with the anti-CD4-1 anti- CD4-2b mAbs in combination with 7-AAD). Thereafter CD4-2⁺ singlets were gated (middle dot plot), and the living and single CD4-2⁺ cells were further separated into CD4 DP and CD4-2 SP cells (right dot plot). The percentage of dividing CD4 DP and CD4-2 SP cells was determined by analyzing their degree of CellTrace Violet staining (upper and lower histogram, respectively). (B) Representative histograms of splenic CD4 DP (left) and CD4-2 SP (right) cell proliferation 7 days after incubation with PHA (1 μg/ml), LPS (100 μg/ml), or medium alone (Control). (C) The percentage of dividing CD4 DP and CD4-2 SP cells from experiments shown in B. (D) Representative histograms of splenic CD4 DP (left) and CD4-2 SP (right) cell proliferation 7 days after MLR cultures upon stimulation with allogenic PBLs (Allo-PBL), autologous PBLs (Self-PBL), or no PBLs (Control). (E) The percentage of dividing CD4 DP and CD4-2 SP cells from experiments shown in D. (F) Representative histograms of splenic CD4 DP (left) and CD4-2 SP (right) cell proliferation upon antigen-specific stimulation for 7 days with KLH (100 μg/ml). Controls included irrelevant protein (OVA, 100 μg/ml) or culture medium alone (Medium). (G) The percentage of dividing CD4 DP and CD4-2 SP cells from experiments shown in F. The percentage of dividing CD4 DP and CD4-2 SP cells was determined by the dye-dilution method with CellTrace Violet stain and measured by flow cytometry as shown in A, B, D and F. Data are representative of three independent experiments and are expressed as mean ± s.e.m. (n = 11–13 fish/group). *P < 0.05, **P < 0.01, and ***P < 0.001 (repeated-measures one-way ANOVA with Bonferroni correction was used for comparison among different stimulants for each CD4⁺ cell population; and one-way ANOVA with Bonferroni correction was used for comparison between CD4⁺ cell population incubated with PHA or alloantigen).

FIGURE 5

Expression analysis of cytokine transcripts in CD4⁺ lymphocytes after Y. ruckeri infection. CD4 DP, CD4-2 SP, CD8α⁺, and CD4-1⁻/CD4-2⁻/CD8α⁻ (Neg) lymphocytes were sorted from spleen leukocytes of PBS-injected control fish and Y. ruckeri-injected fish at day 4 post-injection. Transcript levels of indicated cytokine genes were analyzed by real-time PCR in the sorted lymphocyte populations as well as the unsorted whole spleen leukocytes (WSL). Data are shown relative to the expression levels of WSL from control fish (set to 1). Data are representative of two independent experiments and are expressed as mean ± s.e.m. (n = 5~6 fish/group). *P < 0.05 and **P < 0.01 (Mann-Whitney test). ND, not detected.

FIGURE 6

Gene expression and cytochemical staining analyses of CD4-1⁺ and CD4-1⁻ myeloid cells. (A) Transcription profile of sorted CD4-1⁺ and CD4-1⁻ myeloid cells. CD4-1⁺ and CD4-1⁻ myeloid cells (Mye) as well as CD4⁺ (comprising of CD4 DP and CD4-2 SP) and CD4⁻ lymphocytes (Lym) were sorted from HK leukocytes. Gene expression analysis of these sorted leukocyte populations were performed by real-time PCR for the indicated genes. The transcript levels of indicated genes are shown relative to the expression levels in the CD4-1⁻ myeloid cell population (set to 1) and are expressed as mean ± s.e.m. (n = 4 fish). Data are representative of two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Bonferroni correction). ND, not detected. (B and C) Cytochemical staining of CD4-1⁺ (B) and CD4-1⁻ (C) myeloid cells sorted from HK leukocytes. Cytospin preparations of sorted CD4-1⁺ and CD4-1⁻ myeloid cells were stained with Wright-Giemsa-like (WG), β-glucuronidase (BG), naphthol AS-D chloroacetate esterase (NCAE), myeloperoxidase (MPO), and Sudan Black B (SBB) stains (From left to right). Scale bar = 10 μm. (D) The percentage of sorted CD4-1⁺ and CD4-1⁻ myeloid cells (Mye) positive for BG, NCAE, MPO, and SBB stains. At least 200 cells were counted per preparation to obtain the percentage of cells positive for each cytochemical staining. The values shown are expressed as mean ± s.e.m. (n = 4 fish). (E) The percentage of CD4-1⁺ myeloid cells within the myeloid cell population (gray bars) and the whole leukocyte population (white bars) from indicated tissues. Data are expressed as mean ± s.e.m. (n = 4 fish). Data in D and E are representative of two independent experiments.

FIGURE 7

Phagocytosis by CD4-1⁺ and CD4-1⁻ myeloid cells with fluorescent latex beads. (A) Phagocytosis of 1.0 μm green fluorescent latex beads by CD4-1⁺ myeloid (Mye) cells (top), CD4-1⁻ myeloid cells (middle), and CD4⁺ lymphocytes (Lym) (bottom) from HK. HK leukocytes were incubated in vitro with the beads for 3h and then stained with anti-CD4-1 and anti-CD4-2b mAbs. Bead phagocytosis was thereafter measured by flow cytometry. The figure shows histograms of cell number (y-axis) vs. fluorescence intensity (x-axis) representative of uptake activity by the indicated cell populations. Increased peak fluorescence denotes an increased number of ingested fluorescent beads. The values within histograms represent the percentage of phagocytic cells in each cell population. Phag⁻, non-phagocytic; Phag⁺, phagocytic. (B) The percentage of phagocytic cells in CD4-1⁺ myeloid (Mye), CD4-1⁻ myeloid, and CD4⁺ lymphocyte (Lym) populations from HK leukocytes incubated with the beads (n = 6 fish). (C and D) Immunofluorescence microscopy of HK leukocytes incubated in vitro with 1.0 μm red fluorescent latex beads and then stained with anti-CD4-1 mAb (C) or isotype-matched control mAb (D). Nuclei are counterstained with DAPI (blue). From top to bottom: bright field; CD4-1 (green) or isotype-matched control antibody staining (Control mAb); beads (red); nuclei (blue); and merged fluorescence images (Merge). Scale bar = 5 μm. (E) The percentage of CD4-1⁺ and CD4-1⁻ myeloid cells ingesting various number (1–6+) of beads (n = 6 fish). Data are representative of at least two independent experiments. Data shown in B and E are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Bonferroni correction [B] or unpaired Student’s t-test [E]).

Figure 8

Evolution of CD4 molecules and CD4-bearing leukocytes in vertebrates. (A) Evolution of CD4 molecules and CD4-bearing leukocytes. Tetrapods contain a single CD4 co-receptor with four Ig domains that is thought to have arisen from a primordial two Ig-domain ancestor. In support of this hypothesis, a CD4-like gene encoding only two Ig domains has been identified in lamprey, and thus, it was proposed to represent the prototypic two Ig-domain CD4 co-receptor. This molecule however, lacks a CXC motif (critical for T-cell function and development). While lamprey lymphocytes express transcripts of CD4-like, the protein characterization of this molecule as well as the role of the cells bearing it remain to be elucidated. Recent genome sequence and transcriptome analyses of cartilaginous fish have failed to identify a molecule with classical CD4 features, thus making teleosts the oldest living species with bona fide CD4 co-receptors. In contrast to the situation of tetrapods, which possess a single CD4 gene, rainbow trout and other teleost fish contain two CD4 genes, cd4-1 and cd4-2. Like tetrapod CD4, trout CD4-1 contains four Ig domains while trout CD4-2 contains only two. In this study, we have found two CD4⁺ lymphocyte populations and one CD4⁺ myeloid subset (described in B). Amphibians appear to contain only a four Ig-domain CD4 co-receptor, although the lack of antibodies against this molecule has precluded the phenotypic and functional characterization of amphibian CD4⁺ T cells. Thus far, nothing has been reported on the characterization of CD4 and CD4-bearing cells in reptiles. Birds and mammals contain a single four-Ig-domain CD4 co-receptor. Both birds and mammals contain T-cell subsets expressing surface CD4. However, while mammalian (e.g., human and rat) monocytes/macrophages express CD4, such cells have not been identified in birds. (B) Main findings of this study. Using newly generated mAbs against trout CD4-1 and CD4-2, we identified a predominant trout lymphocyte population co-expressing both CD4 molecules (CD4 DP), and a minor subset expressing only CD4-2 (CD4-2 SP). While both subsets exhibited conserved CD4⁺ T-cell functions (i.e., production of Th1, Th17, and Treg cytokines), CD4-2 SP lymphocytes were less proliferative and displayed a more restricted TCRβ repertoire than CD4 DP cells. Here we also identified the first non-mammalian CD4⁺ monocyte/macrophage population, which represented the leukocyte subset with the highest phagocytic capacity.