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Molecular Genetics & Genomic Medicine logoLink to Molecular Genetics & Genomic Medicine
. 2019 Jun 7;7(7):e00792. doi: 10.1002/mgg3.792

A comprehensive immune repertoire study for patients with pulmonary tuberculosis

Yingyun Fu 1,2,, Bo Li 3, Yazhen Li 1, Minlian Wang 2, Yongjian Yue 2, Lan Xu 1, Shulin Li 1, Qijun Huang 1, Song Liu 4, Yong Dai 4
PMCID: PMC6625341  PMID: 31173489

Abstract

Background

Tuberculosis (TB) is a major global health problem and has replaced HIV as the leading cause of death from a single infectious agent.

Methods

Here, we applied high throughput sequencing to study the immune repertoire of nine pulmonary tuberculosis patients and nine healthy control samples.

Results

Tuberculosis patients and healthy controls displayed significantly different high express clones and distinguishable sharing of CDR3 sequences. The TRBV and TRBJ gene usage showed higher expression clones in patients than in controls and we also found specific high express TRBV and TRBJ gene clones in different groups. In addition, six highly expressed TRBV/TRBJ combinations were detected in the CD4 group, 21 in the CD8 group and 32 in the tissue group.

Conclusion

In conclusion, we studied the patients with tuberculosis as well as healthy control individuals in order to understand the characteristics of immune repertoire. Sharing of CDR3 sequences and differential expression of genes was found among the patients with tuberculosis which could be used for the development of potential vaccine and targets treatment.

Keywords: CDR3 sequences, high express clones, high throughput sequencing, TRBJ gene, tuberculosis

1. INTRODUCTION

Tuberculosis (TB) is a major global health problem and has replaced HIV as the leading cause of death from a single infectious agent in 2016. In 2015, an estimated 10.4 million people developed TB and 1.4 million died from the disease. Tuberculosis is a disease caused by mycobacterium tuberculosis infection. The immunology reaction caused by pathogens during the infection and reproduction of mycobacterium tuberculosis has been studied which leads to the development of vaccine, diagnosis, drug resistance (Horwitz, Lee, Dillon, & Harth, 1995; Lindenstrøm et al., 2009; Meintjes et al., 2009; Vanham et al., 1997). Since, the TCR repertoire is a mirror of the human immune response, its characteristics have been widely investigated in infectious and other diseases to study the state of the immune system and the progression of these diseases (Chaudhry, Cairo, Venturi, & Pauza, 2013).

The diversity within the TCR repertoire is ensured through somatic recombination of germline‐encoded variable (V), diversity (D), and junctional (J) gene segments. Nucleotide deletions at the coding ends and nucleotide additions at the V(D)J junctions also contribute substantially to the TCR repertoire diversity (Nikolich‐Žugich, Slifka, & Messaoudi, 2004). The TCR diversity is a function of the third hypervariable complementary‐determining (CDR3) region, which lies at the intersection between the V, D, J and V, J gene segments within the TCR and TCR chains, respectively. The CDR3 region encodes that part of the TCR which predominantly interacts with antigenic peptide/MHC complexes. Thus, even when T cell clones express the same V/J genes rearrangement, they can be identified by the unique combination of their CDR3 sequences (TCR clonotypes) (Toivonen, Arstila, & Hänninen, 2015). Accordingly, the complexity and distribution of TCRs within specific T cell populations will reflect the degree of complexity of the T cell response.

In the present study, we studied the immune repertoire of CD4+, CD8+ T cells of patients and healthy controls and tissue sample of patients to elucidate the effect of tuberculosis on patients’ immune system. The characteristics of diversity and stability, CDR3 length distribution and CDR3 sequences sharing were analyzed. Besides, the usage of TRBJ, TRBV as well as the combination of TRBV/TRBJ were studied. The different repertoire features between tuberculosis patients and controls were then found as future targets for further study.

2. METHODS

2.1. Clinical samples

Tuberculosis tissue samples and blood samples of nine patients and blood samples of nine healthy controls were collected at the Second Clinical Medical College of Jinan University (Shenzhen People's Hospital, Guangdong, China). All patients gave written informed consent and the present study was approved by the Medical Ethics Committee of Shenzhen People's Hospital.

2.2. DNA extraction and mixing

T cell was isolated using superparamagnetic polystyrene beads (Miltenyi) coated with monoclonal antibodies specific for T cells. DNA was prepared from 0.5 to 2 × 106 T cells from each sample (patients and controls), which was sufficient for analyzing the diversity of TCR in the T cell subsets. DNA was extracted from PBMCs using GenFIND DNA (Agencourt, Beckman Coulter, Brea, CA) extraction kits following the manufacturer's instructions.

Ten milligrams of tuberculosis tissue was obtained from each patient sample and DNA was extracted using standard methods. Briefly, dewaxing was done using xylene and followed by over‐night proteinase K digestion for tissues. QIAamp DNA Mini kit (Qiagen GmbH, Hilden, Germany) was further used for DNA extraction following the manufacturer's instructions. DNA quality was evaluated by loading on a 0.8% agarose gel electrophoresis and DNA concentration was quantified by Qubit fluorometer. DNA from nine patients' peripheral blood samples were mixed together by 1:1:1:1:1:1:1:1:1 according to Qubit value, renamed one blood sample. Meanwhile, DNA from nine patients' tuberculosis tissues and control blood samples were mixed separately in the same way.

2.3. Multiplex‐PCR amplification of TCR‐β CDR3 regions

The human TCR‐β sequences were downloaded from IMGT (http://www.imgt.org/). A relative conserved region in frame region 3, upstream of CDR3, was selected for the puta‐ tive forward primer region. A cluster of primers corresponding to the majority of the V gene family sequence was selected. Similarly, reverse primers corresponding to the J gene family were designed. In total, 30 forward primers and 13 reverse primers were used for multiplex PCR to amplify the rearranged TCR‐β CDR3 regions. The reaction mixtures (50 μl total) comprised 2 μl of pooled TCR‐β variable gene (TRBV; 10 μM), 2 μl of pooled TCR‐β joining gene (TRBJ; 10 μM), 25 μl of 2X Qiagen Multiplex PCR Master Mix, 5 μl of 5X Q‐solution, 500 ng of template DNA (10 μl) and 6 μl of H2O. The PCR conditions comprised 95 °C for 15 min; followed by 25 cycles of 94 °C for 15 s and 60 °C for 3 min; followed by a final extension for 10 min at 72 °C. The PCR products were purified using AMPure XP beads to remove primer sequences (Beckman Coulter, Inc., Brea, CA, USA). A second round of PCR was performed to add a sequencing index to each sample. In this round, each reaction mixture (50 μl total) consisted of 13.5 μl of H2O, 0.5 μl of 2X Q5 DNA polymerase, 10 μl of 5X Q5 buffer, 1 μl of dNTPs (10 mM), 1 μl of P1 (10 μM), 23 μl of DNA, and 1 μl of index (10 μM). The PCR conditions comprised 98 °C for 1 min; followed by 25 cycles of 98 °C for 20 s, 65 °C for 30 s and 72 °C for 30 s; and a final extension for 5 min at 72 °C. The library was separated on an agarose gel, and the target region was isolated and cleaned using QIAquick Gel Extraction kits (Qiagen).

2.4. NGS and data analysis

The library was quantitated using the Agilent 2100 Bioanalyzer instrument (Agilent DNA 1,000 reagents) and real‐time quantitative PCR (TaqMan probes) and sequenced by Illumina MiSeq. Briefly, the adaptor reads and low‐quality reads were filtered from the raw data, the clean data was used in further alignments. Subsequently, the clean data was aligned to the human IGH database and analyzed using the online IMGT/HighV‐QUEST tool. The data included V, J assignment, CDR3 length distribution, clustering and other analyses.

3. RESULT

3.1. Quality control of all sequencing data

Using high‐throughput NGS, we sequenced repertoires CD4+ cells, CD8+ cells and tissue samples of nine pulmonary tuberculosis patients and nine normal controls. All data passed the QC process with an average Q20 >99.99%, Q30 >97.04%. A total sequencing data of total reads number (34094942), immune sequences number (33504380), unknown sequences number (590562), productive sequences number (24345651), nonproductive sequences number (9158729), In‐frame sequences number (27088243), out‐of‐frame sequences number (6326420), total CDR3 sequences number (23670829), Unique CDR3 nucleotide sequences number (1995707) and Unique CDR3 amino acids sequences number (1723256), details including data from each sample are listed in Table 1.

Table 1.

Sequence quality of CD4+, CD8+, tissue sample of tuberculosis patients and normal controls

  M1‐CD4 M1‐CD8 M1‐Tissue M2‐CD4 M2‐CD8 M2‐Tissue M3‐CD4 M3‐CD8 M3‐Tissue M4‐CD4 M4‐CD8 M4‐Tissue M5‐CD4 M5‐CD8 M5‐Tissue M6‐CD4 M6‐CD8 M6‐Tissue M7‐CD4 M7‐CD8 M7‐Tissue M8‐CD4 M8‐CD8
Total reads number 582217 459996 677310 903177 575955 335774 889156 643710 573785 1065180 859602 253607 889229 427415 495999 971852 627698 336292 776087 801797 313179 1016582 892231
Immune sequences number 550596 449546 650380 901272 573975 288998 887379 640989 539209 1062359 857908 195758 886866 424561 458664 970694 625998 286683 774495 799883 256256 1014869 890412
Unknown sequences numebr 31621 10450 26930 1905 1980 46776 1777 2721 34576 2821 1694 57849 2363 2854 37335 1158 1700 49609 1592 1914 56923 1713 1819
Productive sequences number 353828 250365 474701 699906 433221 162872 685579 492654 356619 841185 670586 98480 691340 327224 314326 764656 413577 82356 611495 700658 102129 810266 738949
Nonproductive sequences number 196768 199181 175679 201366 140754 126126 201800 148335 182590 221174 187322 97278 195526 97337 144338 206038 212421 204327 163000 99225 154127 204603 151463
In‐frame sequences number 377268 289584 502949 746867 461590 187810 731817 527307 377228 900754 717545 110971 738864 344971 323396 816839 435942 88467 654902 743398 106464 861847 783627
Out‐of_frame sequences number 163201 158365 145359 153605 111898 94557 154889 113184 159463 160716 139871 81018 147303 78997 129220 153394 189766 178497 119143 55925 143195 152559 106321
Total CDR3 sequences number 327059 233666 441542 677038 420582 153780 674805 484420 347379 817310 622440 84721 680369 321140 304849 752939 407462 46143 602093 691660 75485 798558 728731
Unique CDR3 nt sequences number 14090 12645 20153 60934 26558 16661 65880 32174 18011 65934 51955 11956 51934 18470 6677 123828 18610 4807 69335 22394 4851 74991 48370
Unique CDR3 aa sequences number 11656 10237 17021 51459 21942 15302 56729 27010 15661 54642 43502 11351 43869 15337 5265 112096 15068 4416 60466 18165 4371 63893 40937
Highly expressing clone number all 41 38 53 2 18 28 5 16 48 0 11 21 4 20 26 2 24 40 1 11 30 0 8
Highly expressing clone ratio all 0.68255575 0.52631534 0.63128989 0.02411238 0.31779772 0.71275849 0.05190685 0.34143305 0.68128471 0 0.15724086 0.6742012 0.05387518 0.50208009 0.90383436 0.05658493 0.4683357 0.7194374 0.00541112 0.6621259 0.78404981 0 0.30130597
Shannon entropy all 0.39719054 0.45082821 0.41825992 0.68833416 0.54280623 0.43039658 0.67979693 0.51286718 0.40022774 0.66933299 0.63017905 0.41873046 0.59746329 0.42595744 0.26421886 0.76166681 0.45595582 0.45273164 0.72728526 0.30304497 0.37782448 0.69998144 0.53009339
  M8‐Tissue M9‐CD4 M9‐CD8 M9‐Tissue N1‐CD4 N1‐CD8 N2‐CD4 N2‐CD8 N3‐CD4 N3‐CD8 N4‐CD4 N4‐CD8 N5‐CD4 N5‐CD8 N6‐CD4 N6‐CD8 N7‐CD4 N7‐CD8 N8‐CD4 N8‐CD8 N9‐CD4 N9‐CD8 Sum
Total reads number 345124 920454 612229 644546 734122 1015119 487365 1090624 948621 1738242 609,319 790,880 691,014 675,691 528,099 811,278 1,360,226 1,129,827 1,023,104 569,829 907,554 1,093,845 34,094,942
Immune sequences number 263278 918689 609826 615559 731341 1005773 486477 1089108 945180 1733207 598,136 778,657 689,656 671,243 526,754 806,631 1,355,308 1,122,850 1,015,052 558,523 905,671 1,089,711 33,504,380
Unknown sequences numebr 81846 1765 2403 28987 2781 9346 888 1516 3441 5035 11,183 12,223 1,358 4,448 1,345 4,647 4,918 6,977 8,052 11,306 1883 4,134 590,562
Productive sequences number 29271 740140 365631 448227 572690 632787 392543 748995 771280 478426 461,805 655,143 543,636 540,734 421,049 459,848 1,144,179 892,295 785,584 447,734 751,448 985,234 24,345,651
Nonproductive sequences number 234007 178549 244195 167332 158651 372986 93934 340113 173900 1254781 136,331 123,514 146,020 130,509 105,705 346,783 211,129 230,555 229,468 110,789 154,223 104,477 9,158,729
In‐frame sequences number 31239 791611 389714 478815 611479 674818 419284 803236 826442 1639631 491,438 700,926 581,739 579,260 447,370 493,172 1,214,667 940,753 837,153 476,306 801,996 1,026,787 27,088,243
Out‐of_frame sequences number 223550 126583 219578 132408 119590 330324 67051 285651 118315 93141 105,943 76,022 107,717 91,455 79,183 312,915 140,233 181,609 177,241 81,689 103,394 62,382 6,326,420
Total CDR3 sequences number 4069 729342 359173 437648 562036 620952 386428 737296 758769 471122 452,601 637,937 526,892 524,563 414,324 451,689 1,122,330 875,610 772,434 440,452 733,117 957,874 23,670,829
Unique CDR3 nt sequences number 2560 59322 22290 23141 58045 59010 47160 63725 123378 36185 27,333 27,072 117,546 33,206 87,434 39,496 52,585 27,685 57,173 28,979 127,052 34,112 1,995,707
Unique CDR3 aa sequences number 2529 49316 18368 20092 49690 51530 41236 55749 110754 30566 22,684 21,878 108,153 27,439 80,135 33,719 42,691 21,744 48,099 23,821 115,038 27,630 1,723,256
Highly expressing clone number all 14 0 7 47 0 5 3 10 4 14 15 10 4 12 2 10 7 16 3 12 3 10 655
Highly expressing clone ratio all 0.16515114 0 0.24276881 0.72208259 0 0.09839569 0.03277972 0.33245806 0.04359034 0.21227835 0.24436535 0.49173821 0.07930088 0.31473055 0.01548547 0.24206478 0.41441287 0.64089949 0.10992525 0.21462044 0.06475638 0.61016585 14.5499069
Shannon entropy all 0.8638929 0.66876197 0.52750687 0.40509952 0.67976672 0.59714855 0.7164873 0.55144703 0.74832534 0.57724095 0.49459581 0.37677137 0.78381492 0.52494136 0.79603452 0.58333764 0.44022788 0.34205038 0.5889533 0.56018664 0.75464691 0.33202264 24.7484339

3.2. Diversity and stability of repertoire in different groups

The distribution characteristics of the sequences and clone expansion were analyzed firstly. In the current study, the expression level of certain CDR3 clones higher than 0.5% of total clones was defined as high expansion clones (HECs). In tuberculosis patients, the HEC number was higher in the tissue group than that in the CD8 group or the CD4 group, while the comparison between the CD4 and CD8 groups showed no statistical difference. In the control groups, the HEC number in the CD8 group was significantly higher than that of the CD4 group (Figure 1a). In the comparison of HEC ratio, tuberculosis patients’ tissue group showed higher ratio than CD8 or CD4 groups, and that of CD8 group was higher than in CD4 group. In consistent with HEC number, the HEC ratio of CD8 group was higher than in CD4 group in control group (Figure 1b). The Shannon entropy measures multiplex of the immune system. It ranges from 0 to 1, “1” represents the most diversity and “0” represents the least diversity of immune system. In tuberculosis patients, Shannon entropy in CD4 was higher than CD8 or tissues, while tissue group showed the lowest Shannon entropy, although entropy of CD8 was not statistically higher than tissue group. In controls, Shannon entropy in CD4 was also higher than that of CD8 group (Figure 1c).

Figure 1.

Figure 1

CDR3 clones in patients and controls. (a) HEC number comparison of patients and controls. MCD4: CD4+ cells of tuberculosis patients, MCD8: CD8+ cells of patients, MTis: tissue samples of patients. NCD4: CD4+ cells of controls, NCD8: CD8+ cells of controls. (b) HEC ratio analysis of patients and controls. (c) Shannon entropy of patients and controls

The Gini coefficient was then calculated to further understand the stability of tuberculosis patients’ immune system. In patients, Gini coefficient in CD8 was higher than in CD4 group, while other comparisons showed no significant change. In controls, Gini coefficient in CD8 group was higher than that of CD4 group (Figure 2).

Figure 2.

Figure 2

Gini coefficient of patients and controls. (a) Comparison of CD4+ cell group, CD8+ cell group and tissue group of patients. (b) Comparison of CD4+ cell group in patients and CD4+ cell group in controls. (c) Comparison of CD8+ cell in patient group and CD8+ group in control. (d) Comparison of control CD8+ cell group and CD4+ cell group

3.3. CDR3 length distribution mode analysis

In addition, we made further analysis of CDR3 length distribution in all samples and the differences between groups. We first fit the Gaussian distribution of each sample and compared the R2 value between each sample and each group. The R2 value ranged from 0 to 1, suggesting the worst fitted Gaussian distribution to the best fitted distribution. According to the R2 value, the length distribution of all samples was fitted to Gaussian distribution, although no statistical significance was found for comparing between groups, as shown in Figures 3 and 4.

Figure 3.

Figure 3

Gaussian distribution of R 2 value in all samples. The X‐axis depicts each CDR3 length (1–30), and the Y‐axis depicts the total percentage of each length

Figure 4.

Figure 4

Gaussian distribution of R 2 value between groups. (a) Comparison among CD4+, CD8+ and tissue sample groups in patients. (b) Comparison among CD4+ and CD8+ cell groups in controls

The nucleotides and amino acids length of all samples were analyzed. As shown in Figures 3 and 5, the length distribution of CDR3 sequences ranged from 1–30 nucleotides and followed a Gaussian distribution. Besides, both nucleotides and amino acids length distribution of the high expression clones showed a significant difference between the control and tuberculosis patients. In both tuberculosis patients and controls, the amino acids sequence ranged from 1 to 30 amino acids and the highest percentage for both was 13 amino acid sequences. The CDR3 length of CD4, CD8 and tissue groups was analyzed, and it was observed that all presented with a similar pattern with the whole patient group. However, we found that the amino acid length of 1, 2, 5, 25, 27, 28, 29 were absent in more than seven samples in tissue group (n = 9), which is rare in CD4 and CD8 groups. In healthy controls, the amino acids sequence also ranged from 1 to 30 amino acids and the highest percentage was 13 amino acid sequences. For the distribution of CDR3 length, there were no statistically significant differences as has been found between groups. All samples showed a Gaussian distribution, the highest percentage centralized at 13 amino acids. Besides, CDR3 length of tissue group showed more skewed as length 1, 2, 5, 25, 27, 28, 29 were absent in most of tissue sample (Figure 6).

Figure 5.

Figure 5

CDR3 length distribution in all samples. X‐axis represents length distribution, Y‐axis depicts the percentage of sequences of the corresponding length

Figure 6.

Figure 6

CDR3 expression status in all samples. X‐axis is sample ID and M1‐M9 represents tuberculosis patient number 1‐9. N1‐N9 represent control sample number 1‐9. CD4, CD8 and Tissue mean different sample types. Y‐axis is the percentage of CDR3 clones in each sample's different sequences. Red dots mean highly expressing clones (≥0.5%) and black dots represent normal expressing clones (<0.5%). (a) CDR3 clones (b) amino acid of CDR3 clones

3.4. CDR3 sequence sharing modes analysis

Different individuals sharing an identical TCR sequence corresponding to the same antigenic epitope, termed public T cell response, were observed in a variety of immune responses, including tumorigenesis, autoimmunity, and viral infections. So, we counted the public T cell clones in each group based on the nucleotide and amino acid sequences of CDR3 (Li, Ye, Ji, & Han, 2012). In order to understand the immunological reaction to the common tuberculosis pathogens, the sharing pattern of CDR3 sequence were analyzed between patients. According to the sequence data, there were 586,248 nucleotide sequences and 504,126 amino acids sequences in CD4 group of patients, and 697,706 nucleotide sequences and 618,480 amino acids sequences in CD4 group of controls. There were 253,466 nucleotide sequences and 210,566 amino acids sequences in CD8 group of patients, 349,470 nucleotide sequences and 294,076 amino acids sequences in CD8 group of control. In addition, in tissue samples of patients, we obtained 108,817 nucleotide sequences and 96,008 amino acids sequences.

To elucidate the characteristics of sharing sequences, we compared the amino acid sequences and nucleotide sequences of highly expressed clones which were expressed in more than 0.5% in either patient group or control group (Figure 7). In patient group, eight amino acid sequences and eight nucleotide sequences were shared in all samples from CD4, CD8 and tissue groups. However, 35 amino acid sequences and 33 nucleotide sequences were shared in CD4 and CD8 samples of patient, while there were 61 amino acid sequences and 61 nucleotide sequences that were shared in CD4 and CD8 samples of control. No amino acid or nucleotide sequences were shared in CD4 and CD8 in both patients and controls. All shared sequences are displayed in Table 2.

Figure 7.

Figure 7

Top 60 expressing clones in all samples. Red rectangle: expression percentage ≥0.5%, green rectangle: expression percentage >0, gray rectangle = 0. (a) Nucleotide sequences of top 60 expressed clones in CD4+ and CD8+ group of patients. (b) Nucleotide sequences of top 60 expressed clones in CD4+, CD8+ and tissue group of patients. (c) Amino acids sequences of top 60 expressed clones in CD4+ and CD8+ group of patients. (d) Nucleotide sequences of top 60 expressed clones in CD4+ and CD8+ group of controls.

Table 2.

Shared sequence in all patient samples, in CD4+ and CD8+ cells of patients, in CD4+ and CD8+ cells of controls

Group Shared sequences (NT) Shared sequences (AA)
Shared sequences in all patients GCCTGGAGCTTCGGGAGAACTGAAGCTTTC AWSFGRTEAF
  GCCAGCATGGGTAACACCGGGGAGCTGTTT ASMGNTGELF
  GCCAGCAGTTACTCTGGGACAGGGGGCGAGCAGTAC ASSYSGTGGEQY
  GCCAGCAGTGAGAGCGGGGACTCCTCCTACGAGCAGTAC ASSESGDSSYEQY
  GCCTGGGTAAGGGACTACCCGTCGGACGAGCAGTAC AWVRDYPSDEQY
  GCCTGGAGTCCCCGTACGAAGTTAGCTTTC AWSPRTKLAF
  GCCTGGGTTAGCGGGAGCACGGACACCGGGGAGCTGTTT AWVSGSTDTGELF
  GCCAGCAGTGTCGGGACTCTCATCAATGAGCAGTTC ASSVGTLINEQF
CD4 and CD8 of patient GCCTGGAGCTTCGGGAGAACTGAAGCTTTC AWSFGRTEAF
  GCCAGCATGGGTAACACCGGGGAGCTGTTT ASMGNTGELF
  GCCAGCAGTTACTCTGGGACAGGGGGCGAGCAGTAC ASSYSGTGGEQY
  GCCAGCAGAGATATTGACAGGGAAGACAATGAGCAGTTC ASRDIDREDNEQF
  GCCAGCAGTGAGAGCGGGGACTCCTCCTACGAGCAGTAC ASSESGDSSYEQY
  GCCTGGAGTGATGTGGGGGAGACCCAGTAC AWSDVGETQY
  GCCAGCAGCCAAGAGGGTAGCGGGAGTCAGGAGACCCAGTAC ASSQEGSGSQETQY
  GCCAGCAGTTACTCGGACAGGAGCTCCTACGAGCAGTAC ASSYSDRSSYEQY
  GCCAGCAGATTTGACAGGGACCATTCACCCCTCCAC ASRFDRDHSPLH
  GCCAGCAGTTACAGGCCGAACACCGGGGAGCTGTTT ASSYRPNTGELF
  GCCAGCAGTTGGGGGGAGACCCAGTAC ASSWGETQY
  GCCTGGGTTAGCGGGAGCACGGACACCGGGGAGCTGTTT AWVSGSTDTGELF
  GCCAGCAGTTACAGGTCAGGATCCTACGAGCAGTAC ASSYRSGSYEQY
  GCCAGCGCAACCGGGACAGGGGTTCAAGAGACCCAGTAC ASATGTGVQETQY
  GCCTGGGTAAGGGACTACCCGTCGGACGAGCAGTAC AWVRDYPSDEQY
  GCCTGGAGAACTGGGAACTATGGCTACACC AWRTGNYGYT
  GCCAGCAGGCCAAGGGGCGGGGGAGGTTTCGGGGAGCTGTTT ASRPRGGGGFGELF
  GCCTGGAGTGGCAGGGTCTTGTGGGACACCGGGGAGCTGTTT ASSSEQAVREKLF
  GCCAGCAGCTCGGAACAGGCAGTACGGGAAAAACTGTTT AWSGRVLWDTGELF
  GCCAGCAGTTTATCGTGGGGAGAGACCCAGTAC ASSLSWGETQY
  GCCAGCAGTGTGGGGAGGAACACTGAAGCTTTC ASSVGRNTEAF
  GCCAGCAGCTTGGAGCAGACGGCACGCAGCAATGAGCAGTTC ASSLGWGTGSTNEKLF
  GCCTGGAGTTTGGGGGTGTCTAACTATGGCTACACC ASSLEQTARSNEQF
  GCCTGGAGCGGACAGGGCTACGAGCAGTAC AWSLGVSNYGYT
  GCCAGCAGGCGGGGGTGGGACACCGGGGAGCTGTTT AWSGQGYEQY
  GCCTGGAACCGGGACACTGAAGCTTTC ASRRGWDTGELF
  GCCTGGAGTCCCCGTACGAAGTTAGCTTTC AWNRDTEAF
  GCCAGCAGCTTAGAGGGGGGGAGTTTTAATCATGAAAAACTGTTT AWSPRTKLAF
  GCCTGGACTGAGTTCGGAGGCACCGGGGAGCTGTTT ASSLEGGSFNHEKLF
  GCCAGCAGTTTACGTCCCGTTGAGGTCAATGAGCAGTTC AWTEFGGTGELF
  GCCTGGAGTCTCCGGACAGGGTTCTGGGGGCAGGGCGCGGGTACGGGAGAGACCCAGTAC ASSLEQGARSDEQF
  GCCAGCAGTTACGGGCGGGAGAAGTCCGGGGAGCTGTTT ASSLRPVEVNEQF
  GCCAGCAGTTTCTCTCTAAACACCGGGGAGCTGTTT AWSLRTGFWGQGAGTGETQY
    ASSYGREKSGELF
    ASSFSLNTGELF
CD4 and CD8 of controls GCCTGGAGTGTAGGGACAGGGGACTCCTACGAGCAGTAC AWSVGTGDSYEQY
  AGTGCTAGAGATGCGAGGGTTCAAGACACCGGGGAGCTGTTT SARDARVQDTGELF
  GCCTGGAGTCGAGTGATGAACACTGAAGCTTTC AWSRVMNTEAF
  GCCTGGAGGATAGGGAGTCTAGTAGGCGAGCAGTAC AWRIGSLVGEQY
  AGTGCTCATGCCGGACAGGGAGAAACAGATACGCAGTAT SAHAGQGETDTQY
  AGTGCTAGACCTTACGACAGGGGGACCACCGGGGAGCTGTTT SARPYDRGTTGELF
  AGTGCTAGTCGGGATTGGGACGATACTAATGAAAAACTGTTT SASRDWDDTNEKLF
  GCCAGCAGCTTGGAACAGGGGGCTCGCACAGATACGCAGTAT ASSLEQGARTDTQY
  GCCTGGAGGGGAAAGGGTTTCACTGAAGCTTTC AWSVLAGETGELF
  GCCTGGAGCGTCCTAGCGGGAGAAACCGGGGAGCTGTTT AWRGKGFTEAF
  GCCAGCAGTTACAGGGGCCTATTGACTGAAGCTTTC ASSYRGLLTEAF
  GCCAGCACTCCGGACAGGGGACGAGGCTACACC ASTPDRGRGYT
  GCCAGCAGTTTAAAGGGACCTTACACTGAAGCTTTC ASSLKGPYTEAF
  GCCAGCAGTTACTCAGGCGAGCGGCCCTACAATGAGCAGTTC ASSYSGERPYNEQF
  GCCTGGAGTGTGACAGCCTACGAGCAGTAC AWSVTAYEQY
  GCCTGGAGTGTACAGGTCGGGTTTGGAGAGACCCAGTAC AWSVQVGFGETQY
  GCCAGCAGTTACAAGGGAAACAACTATGGCTACACC ASSYKGNNYGYT
  GCCTGGGGGGCGGAGACCTACGAGCAGTAC AWGAETYEQY
  GCTAGTGCCACAGACTCCTACAATGAGCAGTTC ASATDSYNEQF
  GCCAGCAGTTACGGGGCTACTGAAGCTTTC ASSYGATEAF
  GCCAGCAGTTTAGCACCCACCTCCTACAATGAGCAGTTC ASSLAPTSYNEQF
  GCCAGCAGTTGGAGGGTAAGGAAGCCTGGAAACACCATATAT ASSWRVRKPGNTIY
  GCCAGCAGCTTGGTGATCGGGGATCGCCCCTACGAGCAGTAC ASSLVIGDRPYEQY
  GCCAGCAGCCAAGGAAGACAGGGCTACGAGCAGTAC ASSQGRQGYEQY
  GCCAGCAGTTACAGTTTTAGCAATCAGCCCCAGCAT ASSYSFSNQPQH
  GCCAGCAGTTCCCCCACCTACGAGCAGTAC ASSSPTYEQY
  AGTGCCGAGGACAGTTCGTTGGGGAGCAATCAGCCCCAGCAT SAEDSSLGSNQPQH
  GCCAGCAGTCCGACAGGGGGCGGGGAGTATGGCTACACC ASSPTGGGEYGYT
  GCCTGGGAACCCCCGACTAGCGGGGGGTACGAGCAGTAC AWEPPTSGGYEQY
  GCCAGTAGCGGGAGTAACACCGGGGAGCTGTTT ASSGSNTGELF
  GCCTGGAGGAGTTTACGGGGCGTAAGGTCCTACGAGCAGTAC AWRSLRGVRSYEQY
  GCCAGCAGGACTAGCTCCACGAACACCGGGGAGCTGTTT ASRTSSTNTGELF
  GCCAGCAGTTTAGATGAGGGGGGGCCGAACACTGAAGCTTTC ASSLDEGGPNTEAF
  GCCTGGAGCTCGGGAGGCCTCAATCAGCCCCAGCAT AWSSGGLNQPQH
  GCCAGCGGGAGGACGGGACAGGGCTACGAGCAGTAC ASGRTGQGYEQY
  GCCTGGACCGAGGGACCGAACACCGGGGAGCTGTTT AWTEGPNTGELF
  AGTGCTAGAGTTTCTTCGGGTGGAGGGATGAACACTGAAGCTTTC SARVSSGGGMNTEAF
  GCCAGCAGTTCTGGGACTAGCAGTTACAATGAGCAGTTC ASSSGTSSYNEQF
  GCCTGGAGACAGGCGAACACTGAAGCTTTC ASSLGTDRSTEAF
  GCCAGCAGCTTGGGGACAGATCGCTCGACTGAAGCTTTC AWRQANTEAF
  GCCAGCAGCCTCGTGGGCGACAGACACTACTCTGGAAACACCATATAT ASSLVGDRHYSGNTIY
  GCCAGCAGCTCGCCCTACAGGGGCGGCCACTCTGGGGCCAACGTCCTGACT ASSSPYRGGHSGANVLT
  GCCAGCAGTGCAGGGTCCACTGAAGCTTTC ASSAGSTEAF
  GCCTGGAGTGTTGGAGGGAGGTTTGAAGATACGCAGTAT AWSVGGRFEDTQY
  GCCAGCAGCCAAGATCTCGGGGTTTCGTCAGGGGGAGGGGTGGGGGAGCAGTAC ASSQDLGVSSGGGVGEQY
  GCCTGGAGTTTAAGCGGGAGGGCCGGCGAGCAGTAC AWSLSGRAGEQY
  GCCTGGAGTGTACCAGGGGAGGACACCGGGGAGCTGTTT AWSVPGEDTGELF
  GCCAGCAGTTTAGTCCCAGGGGGAAATGGCTACACC ASSLVPGGNGYT
  GCCTGGAGTGGTCCCCCAACTAATGAAAAACTGTTT AWSGPPTNEKLF
  GCCAGCAGTTGGGGGGGGACTCCCTACGAGCAGTAC ASSWGGTPYEQY
  GCCAGCAGTCGCGGGGCAGGGGGTTACAATGAGCAGTTC ASSRGAGGYNEQF
  GCCTGGAGTGAGGGGGTCGGAAACACCATATAT AWSEGVGNTIY
  GCCTGGAGTTCGACAGGGTCTAGCACAGATACGCAGTAT AWSSTGSSTDTQY
  GCCAGCAGTTATGGACAGTTCTCCATTCAGTAC ASSYGQFSIQY
  GCCAGCAGCTCCTACTTCCGACAGGGCCCCCAAGAGACCCAGTAC ASSSYFRQGPQETQY
  GCCAGCAGCAGTCATGGGGGGCGAGAGGACGAGCAGTAC ASSSHGGREDEQY
  GCCTGGGCCGACAGCTCTGGAAACACCATATAT AWADSSGNTIY
  GCCTGGAGGGACGGGGGGCGGGACACCGGGGAGCTGTTT AWRDGGRDTGELF
  GCCAGCAGCTCGGGGACAGGGAGAGGGGATGGCTACACC ASSSGTGRGDGYT
  GCCAGCAGCTTGGGCCCATTAGGGGCGGCTAACTATGGCTACACC ASSLGPLGAANYGYT
  GCCAGCAGCTTGGGCCTACTAGCAGATACGCAGTAT ASSLGLLADTQY

3.5. Significance of TRBV and TRBJ usage in patients and controls

In addition, to elucidate the potential specific immune reaction to tuberculosis, usage of TRBV and TRBJ was analyzed in all groups.

In patients, TRBV13 and TRBV6‐4 were significantly highly expressed in CD8 group than in CD4 group, on the contrary TRBV10‐3, TRBV20‐1, TRBV3‐1, TRBV5‐1, TRBV5‐4, TRBV6‐6 showed significantly lower expression in CD8 group than in CD4 group. The expression of TRBV10‐3, TRBV18, TRBV24‐1, RBV24/OR9‐2, TRBV28, TRBV3‐1, TRBV5‐5, TRBV5‐8, TRBV6‐1, TRBV6‐6, TRBV6‐7, TRBV6‐9, TRBV7‐6, TRBV7‐7 was also higher in CD4 than in tissue group. TRBV10‐3, TRBV13, TRBV27, TRBV28, TRBV6‐2, TRBV6‐3, TRBV6‐4, TRBV6‐7, TRBV6‐9 were highly expressed in CD8 samples than in tissues while TRBV20‐1, TRBV5‐1 showed lower expression in CD8 samples than in tissue samples. In controls, TRBV11‐1, TRBV14, TRBV3‐1, TRBV6‐6, TRBV7‐2 and TRBV7‐7 exhibited significant higher expression in CD4 group than in CD8 group, while TRBV27, TRBV7‐6, TRBV9 all showed lower expression in CD4 group than in CD8 group. Moreover, TRBV3‐1 and TRBV4‐1, TRBV5‐8 and TRBV7‐3 in controls’ CD4 group showed statistically lower expression than that of in patients. TRBV11‐1 and TRBV7‐2 exhibited higher expression in patients’ CD8 group than that of control's (Figure 8).

Figure 8.

Figure 8

Comparison of TRBV gene usage in groups. X‐axis represents the genes of TRBV. Y‐axis represents the expressing percentage of corresponding gene: (a) CD4+ cell and CD8+ cell groups in patients. (b) CD4+ cell and tissue groups in patients. (c) CD8+ cell and tissue groups in patients. (d) CD4+ cell and CD8+ cell groups in controls. (e) CD4+ cell group of patients and CD4+ cell group of controls. (f) CD8+ cell group of patients and CD8+ cell group of controls

In patients, TRBJ1‐3, TRBJ2‐6 were significantly highly expressed in CD8 group than in CD4 group, on the contrary TRBJ1‐5 showed lower expression in CD8 group than in CD4 group. The expression of TRBJ1‐3, TRBJ1‐5, TRBJ2‐4, TRBJ2‐5 was also higher in CD4 than in tissue group, and TRBJ2‐7 exhibited lower expression in CD4 than in tissue group. TRBJ1‐2 showed lower expression and TRBJ2‐5 presented higher expression in CD8 group than in tissue group respectively. In controls, TRBJ1‐2, TRBJ1‐6, TRBJ2‐4, TRBJ2‐5 exhibited significant higher expression in CD4 group than in CD8 group. Besides, TRBJ1‐6 and TRBJ2‐7 in controls’ CD4 group showed statistical higher expression than that of patients. While TRBJ1‐3 in controls’ CD4 group showed lower expression than patients’ expression. TRBJ2‐5 exhibited higher expression in patients’ CD8 group than that of control's (Figure 9). Top 20 genes in each group are shown in Figure 10.

Figure 9.

Figure 9

Comparison of TRBJ gene usage in groups. X‐axis represents the genes of TRBJ. Y‐axis depicts expressing percentage of corresponding gene. (a) CD4+ cell and CD8+ cell groups in patients. (b) CD4+ cell and tissue groups in patients. (c) CD8+ cell and tissue groups in patients. (d) CD4+ cell and CD8+ cell groups in controls. (e) CD4+ cell group of patients and CD4+ cell group of controls. (f) CD8+ cell group of patients and CD8+ cell group of controls

Figure 10.

Figure 10

Top 20 used TRBV genes in each group. X‐axis is sample ID, N1‐N9 represent control sample number 1–9. CD4, CD8 and Tissue mean different sample types. Y‐axis represents the frequencies of corresponding reads. (a) Top 20 used TRBV genes in CD4+ cell group of patients. (b) Top 20 used TRBV genes in CD8+ cell group of patients. (c) Top 20 used TRBV genes in tissue group of patients. (d) Top 20 used TRBV genes in CD4+ cell group of controls. (e) Top 20 used TRBV genes in CD8+ cell group of controls. (f) Top 20 used TRBV genes in all sample groups

Additionally, we combined the expression data of all samples on TRBV or TRBJ to understand the correlation between the expression in the samples. The heatmaps are shown in Figure 11.

Figure 11.

Figure 11

Heatmap of TRBV and TRBJ usage in all samples. Heatmap of gene usage in comparison of all samples for (a) TRBV (b) TRBJ

3.6. Combination of usage of TRBV and TRBJ in tuberculosis patients and control samples

TRBV/TRBJ combination was an important source of CDR3 sequence diversification. Within all TRBV/TRBJ combinations, we first counted the highly expressed which represent more than 0.5% of all combinations in each group. For CD4 group in controls, there were six TRBV/TRBJ combinations which were used more than 0.5%, and the number is 22 in CD8 group in controls. In the tuberculosis patient group, there were also six TRBV/TRBJ combinations which were used more than 0.5% in CD4 group, and 21 high expression TRBV/TRBJ in CD8 group, and 32 high expression TRBV/TRBJ in tissue group (Table 3).

Table 3.

Combination of usage of TRBV and TRBJ in tuberculosis patients and control samples

V_type J_type count individual Percent
TRBV30 TRBJ2‐7 243,097 N7‐CD4 21.6600287
TRBV20‐1 TRBJ2‐2 233,941 N7‐CD4 20.8442259
TRBV30 TRBJ2‐7 77,626 N4‐CD4 17.1510889
TRBV30 TRBJ2‐5 51,206 N5‐CD4 9.71850019
TRBV6‐1 TRBJ1‐2 66,181 N8‐CD4 8.56785175
TRBV6‐6 TRBJ1‐2 49,082 N9‐CD4 6.69497502
TRBV30 TRBJ2‐7 285,440 N4‐CD8 44.7442302
TRBV20‐1 TRBJ2‐2 361,501 N9‐CD8 37.7399324
TRBV30 TRBJ1‐1 164,581 N2‐CD8 22.3222424
TRBV20‐1 TRBJ2‐3 190,384 N7‐CD8 21.7430134
TRBV20‐1 TRBJ1‐4 178,703 N9‐CD8 18.6562116
TRBV30 TRBJ1‐1 62,806 N6‐CD8 13.9046999
TRBV30 TRBJ2‐2 108,417 N7‐CD8 12.3818823
TRBV30 TRBJ2‐7 80,921 N2‐CD8 10.9753749
TRBV5‐1 TRBJ2‐3 87,949 N7‐CD8 10.044312
TRBV30 TRBJ2‐7 48,860 N5‐CD8 9.31441981
TRBV6‐5 TRBJ1‐1 48,079 N5‐CD8 9.16553398
TRBV12‐3 TRBJ1‐1 73,219 N7‐CD8 8.36205617
TRBV6‐5 TRBJ2‐1 41,462 N5‐CD8 7.90410303
TRBV12‐3 TRBJ2‐1 32,958 N8‐CD8 7.4827677
TRBV30 TRBJ2‐7 63,002 N7‐CD8 7.19521248
TRBV30 TRBJ1‐1 42,289 N1‐CD8 6.81034927
TRBV30 TRBJ2‐7 29,944 N6‐CD8 6.6293401
TRBV4‐3 TRBJ2‐7 36,285 N1‐CD8 5.84344684
TRBV12‐3 TRBJ1‐2 25,505 N8‐CD8 5.79064234
TRBV6‐5 TRBJ1‐1 25,641 N6‐CD8 5.67669348
TRBV12‐5 TRBJ2‐1 28,982 N5‐CD8 5.52497984
TRBV12‐3 TRBJ1‐1 24,064 N3‐CD8 5.10780647
TRBV30 TRBJ1‐2 45,654 M1‐CD4 13.9589493
TRBV12‐3 TRBJ2‐2 33,833 M1‐CD4 10.3446167
TRBV12‐3 TRBJ1‐1 24,993 M1‐CD4 7.64174048
TRBV6‐5 TRBJ2‐7 44,913 M6‐CD4 5.96502506
TRBV20‐1 TRBJ2‐1 39,180 M3‐CD4 5.80612177
TRBV30 TRBJ1‐1 17,997 M1‐CD4 5.50267689
TRBV30 TRBJ1‐1 281,888 M7‐CD8 40.7552844
TRBV6‐5 TRBJ2‐2 177,317 M8‐CD8 24.3322982
TRBV6‐5 TRBJ2‐7 68,199 M5‐CD8 21.2365324
TRBV12‐3 TRBJ2‐1 132,063 M7‐CD8 19.0936298
TRBV30 TRBJ2‐5 73,658 M6‐CD8 18.0772686
TRBV6‐1 TRBJ2‐7 55,800 M9‐CD8 15.5356889
TRBV4‐3 TRBJ2‐5 39,717 M3‐CD8 8.19887701
TRBV30 TRBJ2‐7 39,449 M3‐CD8 8.14355312
TRBV12‐3 TRBJ1‐6 38,230 M3‐CD8 7.89191198
TRBV6‐2 TRBJ2‐7 31,203 M2‐CD8 7.41900509
TRBV12‐3 TRBJ2‐5 43,944 M4‐CD8 7.05995759
TRBV6‐2 TRBJ2‐2 22,112 M5‐CD8 6.88547051
TRBV30 TRBJ2‐2 15,645 M1‐CD8 6.6954542
TRBV12‐3 TRBJ2‐5 27,212 M6‐CD8 6.67841418
TRBV6‐2 TRBJ2‐5 27,203 M6‐CD8 6.67620539
TRBV30 TRBJ1‐4 15,233 M1‐CD8 6.51913415
TRBV12‐3 TRBJ2‐2 30,744 M3‐CD8 6.34655877
TRBV12‐3 TRBJ2‐2 38,148 M4‐CD8 6.1287835
TRBV5‐1 TRBJ2‐1 24,851 M2‐CD8 5.90871697
TRBV2 TRBJ2‐5 12,950 M1‐CD8 5.54209855
TRBV5‐8 TRBJ1‐6 16,347 M5‐CD8 5.09030329
TRBV12‐3 TRBJ2‐7 27,534 M4‐Tissue 32.4996164
TRBV4‐3 TRBJ1‐2 73,434 M5‐Tissue 24.0886472
TRBV4‐1 TRBJ2‐6 54,498 M5‐Tissue 17.8770473
TRBV20‐1 TRBJ1‐4 27,013 M2‐Tissue 17.5660034
TRBV5‐1 TRBJ2‐7 13,014 M7‐Tissue 17.2405114
TRBV30 TRBJ1‐1 70,700 M1‐Tissue 16.0120668
TRBV4‐3 TRBJ1‐2 6,465 M6‐Tissue 14.0107925
TRBV20‐1 TRBJ2‐6 9,564 M7‐Tissue 12.6700669
TRBV20‐1 TRBJ2‐7 9,212 M7‐Tissue 12.2037491
TRBV5‐1 TRBJ1‐2 39,469 M3‐Tissue 11.3619419
TRBV6‐5 TRBJ1‐1 16,242 M2‐Tissue 10.5618416
TRBV4‐1 TRBJ2‐6 4,634 M6‐Tissue 10.0426934
TRBV5‐1 TRBJ1‐2 8,463 M4‐Tissue 9.98925886
TRBV12‐3 TRBJ1‐1 38,671 M9‐Tissue 8.83609659
TRBV30 TRBJ2‐7 29,333 M3‐Tissue 8.44409132
TRBV12‐3 TRBJ2‐2 22,954 M5‐Tissue 7.52962942
TRBV30 TRBJ2‐1 25,885 M3‐Tissue 7.45151549
TRBV12‐3 TRBJ2‐2 25,845 M3‐Tissue 7.44000069
TRBV5‐1 TRBJ2‐1 11,003 M2‐Tissue 7.15502666
TRBV30 TRBJ1‐6 30,986 M1‐Tissue 7.01767895
TRBV30 TRBJ1‐1 285 M8‐Tissue 7.00417793
TRBV30 TRBJ2‐7 285 M8‐Tissue 7.00417793
TRBV12‐3 TRBJ2‐7 29,615 M9‐Tissue 6.76685373
TRBV12‐3 TRBJ2‐7 19,908 M5‐Tissue 6.53044622
TRBV12‐3 TRBJ2‐7 4,895 M7‐Tissue 6.48473207
TRBV30 TRBJ2‐7 27,923 M9‐Tissue 6.38024166
TRBV30 TRBJ1‐4 26,309 M9‐Tissue 6.01145213
TRBV5‐1 TRBJ2‐7 2,719 M6‐Tissue 5.89255142
TRBV14 TRBJ2‐3 8,461 M2‐Tissue 5.50201587
TRBV20‐1 TRBJ1‐1 2,425 M6‐Tissue 5.25540169
TRBV5‐1 TRBJ2‐1 4,310 M4‐Tissue 5.08728651
TRBV11‐3 TRBJ2‐2 22,117 M1‐Tissue 5.00903651

In order to examine the potential contribution of specific TRBV/TRBJ combinations to disease progress, comparison of the relative frequencies of TRBV/TRBJ combinations between patients and controls was performed. There were 46 up‐regulated and 10 down‐regulated TRBV/TRBJ combinations as has been found after comparison between CD4 group of patients and controls. There were 12 up‐regulated combinations and 12 down‐regulated combinations as has been found after comparison between CD8 group of patients and controls (Figure 12). We then compared different TRBV/TRBJ combinations in CD4, CD8, tissue group of patients as shown in Figure 13.

Figure 12.

Figure 12

TRBV/TRBJ combination in groups. (a) Comparison between CD4+ cell group in patients and CD4+ cell group in controls. (b) Comparison between CD8+ cell group in patients and CD8+ cell group in controls

Figure 13.

Figure 13

TRBV/TRBJ combination in groups. (a) Comparison between CD8+ cell group in patients and CD4+ cell group in patients. (b) Comparison between tissue group in patients and CD4+ cell group in patients. (c) Comparison between CD8+ cell group in patients and tissue group in patients

4. DISCUSSION

Tuberculosis, a well‐known infectious disease, is closely related to immune reaction in its development, diagnosis and treatment process (Janis, Kaufmann, Schwartz, & Pardoll, 1989; Cooper, 2009; Andersen et al., 2000; MacMicking, Taylor, & McKinney, 2003). The immune repertoire is characterized by a complex and dynamic organization, a highly organized, dynamic and coherent structure to assist in the understanding of the generation and selection of immune TCRs. Thus, fully measuring the diversity of the T cell repertoire, which determines the flexibility and specificity in the cellular immune response, could provide new insights into the underlying disease process (Burgos, 1996; Liu et al., 2017). In 1996, Li, et al., investigated disease‐specific change in gamma‐delta T cell repertoire of pulmonary tuberculosis patients by flow cytometric analysis of blood and bronchoalveolar lavage gamma‐delta T cells (Li et al., 1996). They demonstrated the hypothesis that gamma‐delta T cells play a role in the protective immune response to Mtb infection (Li et al., 1996). In 2018, Chaofei Cheng, et al., found that the CDR3δ tended to be more polyclonal and CDR3γ tended to be longer in TB patients; the γδ T cells expressing CDR3 sequences using a Vγ9‐JγP rearrangement expanded significantly during Mtb infection by NGS study of repertoire (Cheng et al., 2018).

However, for further understanding of immune reaction, repertoire diversity and stability within tuberculosis patients and controls still needs more comprehensive studies. Here, we present a study of enormous characterization data of tuberculosis patients and comparable controls. HEC number, HEC ratio, Shannon entropy and Gini coefficient were applied to evaluate the general characteristics of the repertoires. In both patients and normal controls, the HEC number and HEC ratio showed higher frequency in tissue samples than in CD8 or CD4 samples. Besides, HEC number and HEC ratio showed higher frequency in CD8 than CD4 samples in both patients and normal controls. This suggested that a more centralized and stronger immune reaction in tissue samples than in the CD4+ or CD8+ cell samples provides potential evidence for further elucidation for the understanding of the mechanism in depth. The Shannon entropy which was previously used as an economy parameter, was introduced in this study to illustrate the multiplex of the immune system. According to the criteria, we found that the tissue group's repertoire showed lowest complexity in patients which also suggests the strong immune reaction in tuberculosis tissue than other sample type groups.

The length distribution of CDR3 in each sample was fitted to Gaussian distribution, which provides an evenly distributed data set. Consistent with the previous study, we found that there were significant differences between patients and control groups. Since, the CDR3 sequence which was commonly expressed in patient samples could provide a solid clue for disease‐specific immune reaction research, we evaluated all amino acids shared in all samples. The CD4, CD8 groups showed similar sharing of sequences which were quite different to that of tissue groups. In the analysis of TRBV and TRBJ gene usage, we found significant difference between CD4, CD8 and tissue groups of patients. These differently expressed genes showed a disease‐specific gene expression profile which provides further information for tuberculosis and control study. To find further disease‐specific CDR3 sequences, we also investigated the TRBV/TRBJ combination in patients and controls. Within same group analysis, we found highly expressed combination sequences in each certain group. And we also found differential expression level of recombination sequences in the comparison between patients’ samples and normal control samples, which revealed the important function of TRBV/TRBJ combination in the immune of tuberculosis and provide presupposition for further study of diagnosis or treatment application.

In conclusion, our study first elucidated the immune repertoire characteristics of tuberculosis patients using NGS based methods. We found that the CDR3 sequences were extremely highly expressed in tuberculosis patients’ tissue samples than other type of samples, which suggested a specific and strong immune reaction during the development of tuberculosis. We then analyzed the CDR3 sequence sharing in all samples and each group. Later, we elucidated the TRBV, TRBJ usage and TRBV/TRBJ combination in all samples. This study provides a whole spectrum and profile of tuberculosis patients and studied the specific recombination CRD3 sequences which differ between the patients and controls. Although the sample number is relatively small, we still provide a useful resource of further study on the diagnosis, prognosis and prevention of tuberculosis by understanding candidates’ immunology repertoire features.

CONFLICT OF INTEREST

The authors have no conflict of interests to declare regarding this manuscript.

AUTHOR'S CONTRIBUTIONS

YF, BL, SL and YD designed the study and drafted the manuscript. YL, MW, YY, LX, ShL and QH acquired and interpreted the data. SL, YF and YD revised the manuscript for important content. All authors read and approved the final manuscript.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All patients gave written informed consent and the present study was approved by the Medical Ethics Committee of Shenzhen People's Hospital.

CONSENT FOR PUBLICATION

Not applicable.

ACKNOWLEDGMENT

Not applicable.

Fu Y, Li B, Li Y, et al. A comprehensive immune repertoire study for patients with pulmonary tuberculosis. Mol Genet Genomic Med. 2019;7:e792 10.1002/mgg3.792

Yingyun Fu and Bo Li contributed equally to this work.

Funding information

The present study was supported by funds from the Science and Technology Commission of Shenzhen (HTP study of sarcoidosis TCRß chain CDR3 immune repertoire, JCYJ20160422142121988), Science and Technology Department of Guangdong province, (Kartagener syndrome pathogenic gene diagnosis and microarray diagnosis kit, 2017A020214016s), Science and Technology Commission of Shenzhen (identification of gene mutation sites of genetic pathogenic genes in pulmonary embolism and diagnosis panel design, JCYJ20170413093032806), Key Laboratory of respiratory diseases in Shenzhen (ZDSYS201504301616234).

Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

DATA AVAILABILITY STATEMENT

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.


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