Article Figures & Data
Figures
- FIGURE 1.
IgM and IgT repertoire diversity in spleen and NALT in naive juvenile trout. (A) Overall IGHV family usage by IgM and IgT in Spleen and NALT of naive trouts. Bar plots showing the percentage of IgM (up) and IgT (bottom) MID barcoded consensus sequences belonging to each IGHV. (B) IgM and IgT clonotype diversity as measured by the Shannon Entropy calculated from seven IGHV families. Data were analyzed by one-way ANOVA with post hoc Tukey HSD test (***p ≤ 0.01). (C) Normalized distributions of clonotypes determined n times in the consensus sequence datasets from control fish. Red bars correspond to IgM clonotypes and blue bars to IgT. (D) Accumulated frequency for the top 20 most prevalent clonotypes for IgM and IgT repertoire observed in spleen and NALT of each control fish.
- FIGURE 2.
(A) Detection of IgM (left panel) and IgT (right panel) specific to ERM bacterin in serum from controls, i.p., or i.n. ERM-immunized fish. Serum dilutions, 1/100 for IgM and 1/20 for IgT were evaluated by indirect ELISA, and the results are represented as relative values to the most positive serum. (B) Effect of i.p. and intranasal (IN) immunization on the IGHV gene family usage by trout IgM and IgT repertoire. Mean IGHV gene family usage in spleen and NALT of controls (C) and i.p. or i.n. ERM- immunized fish (n = 6 fish per group). Graphics show mean + SD of IGHV family usage for IgM or IgT considering MID barcoded consensus sequences. Significant difference in IGHV family usage was detected by one-way ANOVA posthoc Tukey HSD test and Bonferroni. aSignificant difference p < 0.05 between C and i.p. bSignificant difference p < 0.05 between C and IN.
- FIGURE 3.
Clonotype diversity in spleen and NALT after ERM i.p. or intranasal (IN) immunization. (A) IgM and IgT repertoire diversity considering the Shannon Index calculated from seven IGHV families or (B) considering each IGHV family. Data were analyzed by one-way ANOVA with post hoc Tukey HSD test and Bonferroni (**p ≤ 0.01, *p ≤ 0.05). If the statistical significance relative to control group was obtained by Student t test, ^p ≤ 0.05. (C) IgM clonotype size distribution from control and ERM-immunized fish are represented IGHV5 and IGHV4 families. Large clonal expansions are indicated by high number of consensus sequence counts.
- FIGURE 4.
Size distribution of splenic IgM and IgT clonotypes shared by individual. dot/box plots showing the size distribution for each VH-C combination and for clonotypes found among n fish (n = 1 in dark gray, 2 in gray, 3 in yellow, 4 in blue, 5 in green, or 6 in pink) within each treatment group (control, i.p., i.n.). Dots are computed from the values corresponding to clonotypes found in one to six fish, over 10 subsamplings in the selected IGHV families that are indicated on the right. Each dot represents a clonotype in a fish. Relative clonotype size value (y-axis) in log10 scale.
- FIGURE 5.
Size distribution of NALT IgM and IgT clonotypes shared by individual. Dot/box plots showing the size distribution for each VH-C combination and for clonotypes found among n fish (n = 1 in dark gray, 2 in gray, 3 in yellow, 4 in blue, 5 in green, or 6 in pink) within each treatment group (control, i.p., i.n.). Dots are computed from the values corresponding to clonotypes found in one to six fish, over 10 subsamplings in the selected IGHV families indicated on the right. Each dot represents a clonotype in a fish. Relative clonotype size value (y-axis) in log10 scale.
- FIGURE 6.
Identification of HSR clonotypes. Expression heat maps of IgM (up) and IgT (low) clonotypes shared by at least four fish ERM i.p. immunized (A) or four fish ERM i.n. immunized (B) and found, on average, >10 times compared to controls. Heat maps were obtained with Heatmap.2, and the values were scaled by rows to have mean 0 and SD 1. Each column corresponds to one fish and each arrow to one clonotype. The identified CDR3 amino acid sequence and JH segment are indicated on the right of each heat map. Sequences belonging to IGHV5 family are shown in blue and those coming from IGHV4 (for IGHM heat maps) and IGHV6 (for IGHT heat maps) are shown in black. Sequences in bold and underlined represent clonotypes detected in Spleen and NALT. Venn diagrams showing the overlap of these clonotypes detected in Spleen and NALT are shown on the right of corresponding heat maps.
Tables
- Table I. Number of distinct clonotypes detected in controls for each VH-C combination and organ
IgM IgT MIDs Organ C1 C2 C3 C4 C5 C6 Mean Total C1 C2 C3 C4 C5 C6 Mean Total IGHV1 175 Sp 1449 1279 1076 819 786 1071 1080 6480 1217 960 1001 348 1138 1271 989 5935 NALT 744 118 244 79 201 103 248 1489 588 299 204 222 572 303 364 2188 IGHV2 100 Sp 810 782 615 293 538 585 603 3623 626 408 476 158 637 660 494 2965 NALT 436 287 123 116 88 56 184 1106 297 126 102 98 240 129 165 992 IGHV4 175 Sp 1518 1149 980 615 998 869 1021 6129 1085 707 750 187 1116 1053 816 4898 NALT 803 324 101 200 185 105 286 1718 448 66 150 185 264 151 210 1264 IGHV5 180 Sp 1339 1283 1041 825 687 652 971 5827 1163 905 922 295 1250 1230 960 5765 NALT 481 299 120 200 60 93 208a 1253 559 297 241 215 625 225 360a 2162 IGHV6 390 Sp 2724 2836 1917 1466 1262 2319 2087 12,524 2462 1722 1865 644 2312 1621 1771 10,626 NALT 1738 997 464 134 476 219 671 4028 1172 513 381 401 1017 575 676 4059 IGHV8 189 Sp 1637 1512 1217 751 757 948 1137a 6822 1005 592 780 132 1007 943 743a 4459 NALT 951 441 135 92 245 72 322 1936 358 156 97 118 280 96 184 1105 IGHV9 146 Sp 1043 863 725 465 854 761 785 4711 969 681 731 201 763 862 701 4207 NALT 374 283 121 54 129 46 168 1007 430 190 177 145 450 147 256 1539 Average values over 10 subsamplings of corresponding MIDs (see the Materials and Methods section) per IGHV family and C region (VH-C) combination and individual fish. Organ: Spleen (Sp) and NALT. Total spleen (IgM): 46,116. Total IgM clonotypes: Sp (46,116), NALT (12,537). Total IgT clonotypes: Sp (38,855), NALT (13,309).
↵a Significant difference, p < 0.1, data analyzed by Student t test.
- Table II. Number of distinct clonotypes detected in spleen and NALT from controls and after ERM vaccination
IgM IgT MIDs Organ Control i.p. i.n. Control i.p. i.n. IGHV1 175 Sp 1080.00 ± 257.06 800.167 ± 257.23 567.33 ± 106.96 989.16 ± 336.32 998.67 ± 211.74 746 ± 353.14 NALT 248.17 ± 250.89 400.33 ± 186.86 383.83 ± 327.79 364.67 ± 171.55 395.00 ± 191.32 414.33 ± 237.95 IGHV2 100 Sp 603.83 ± 187.44 518.33 ± 191.72 333.66 ± 103.69 494.16 ± 192.89 482 ± 117.46 380.67 ± 190.90 NALT 184.33 ± 147.01 245.17 ± 114.35 200.33 ± 153.05 165.33 ± 82.85 182.50 ± 102.23 185.17 ± 102.91 IGHV4 175 Sp 1021.50 ± 301.17 427.16 ± 190.29 607.83 ± 280.73 816.33 ± 355.08 725.83 ± 205.41 581.83 ± 336.46 NALT 286.33 ± 265.83 377.17 ± 178.93 334.33 ± 305.63 210.67 ± 132.66 259.33 ± 147.37 288.83 ± 195.52 IGHV5 180 Sp 971.16 ± 297.11 671.5 ± 281.35 484.83 ± 115.33 960.83 ± 359.14 943.16 ± 230.64 718 ± 372.11 NALT 208.83 ± 158.58 428.00 ± 190.11 309.83 ± 185.33 360.33 ± 182.87 357.50 ± 182.47 401.83 ± 223.07 IGHV6 390 Sp 2087.33 ± 650.50 1900.16 ± 643.83 1219.33 ± 397.88 1771 ± 644.05 1841.33 ± 511.74 1451.5 ± 724.07 NALT 671.33 ± 603.04 869.83 ± 513.50 661.83 ± 540.01 676.5 ± 335.17 719.33 ± 356.49 719.17 ± 445.93 IGHV8 189 Sp 1137.00 ± 381.08 863.5 ± 309.53 591.83 ± 166.15 743.17 ± 339.36 647.5 ± 274.45 535 ± 342.43 NALT 322.67 ± 336.48 419.16 ± 248.11 292.67 ± 181.77 184.17 ± 109.49 198.67 ± 126.56 199.50 ± 150.98 IGHV9 146 Sp 785.16 ± 191.82 625.5 ± 249.75 476.66 ± 150.32 701.17 ± 265.66 700.33 ± 231.4 584.5 ± 283.98 NALT 167.83 ± 132.11 286.83 ± 121.22 273.00 ± 312.36 256.5 ± 143.32 293.67 ± 131.00 312.33 ± 184.93 Total Sp 1098.00 ± 552.04 829.48 ± 558.67 611.64 ± 331.79 925.12 ± 518.91 905.55 ± 492.90 713.93 ± 494.09 NALT 298.50 ± 329.67 433.62 ± 298.92 350.83 ± 319.88 316 ± 236.59 343.71 ± 247.67 360.17 ± 281.09 Average values over 10 subsamplings of corresponding MIDs (see the Materials and Methods section) per IGHV family and C region (VH-C) combination and individual fish. Organ: Spleen (Sp) and NALT. Those values with significant difference p < 0.05 compared with controls are in bold. Data analyzed by Student t test.
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