SLAM/SAP signaling regulates discrete T cell developmental checkpoints and shapes the innate-like TCR repertoire

During thymic development, most T cells acquire innate-like characteristics that are critical for their function in tumor surveillance, infectious disease, and tissue repair. The mechanisms, however, that regulate T cell developmental programming remain unclear. Recently, we demonstrated that the SLAM-SAP signaling pathway regulates the development and function of multiple innate-like T cell subsets. Here, we used a single-cell proteogenomics approach to identify SAP-dependent developmental checkpoints and to define the SAP-dependent TCR repertoire. SAP deficiency resulted in both a significant loss of an immatureGzma+Blk+Etv5+Tox2+T17 precursor population, and a significant increase inCd4+Cd8+Rorc+Ptcra+Rag1+thymic T cells. SAP-dependent diversion of embryonic day 17 thymic T cell clonotypes into the T cell developmental pathway was associated with a decreased frequency of mature clonotypes in neonatal thymus, and an altered TCR repertoire in the periphery. Finally, we identify TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent V4 T1 subset. Together, the data suggest that SAP-dependent / T cell lineage commitment regulates T cell developmental programming and shapes the TCR repertoire. Single-cell CITE-Seq and TCR repertoire profiling of T cells across various developmental stages and mouse models shows the influence of SLAM/SAP signaling on discrete T cell developmental checkpoints and the shaping of the innate-like TCR repertoire. Thymus single-cell CITE-seq with TCR repertoire analysis includes embryonic day 17 (E17) B6 and B6.Sh2d1a-/- (SAP-/-) mice with four biological replicates each, along with nine-day-old (D9) neonatal B6 and B6.SAP-/- mice with three biological replicates each. Additionally, the dataset encompasses thymus samples from six-week-old B6 and B6.SAP-/-mice with three replicates per group. Following thymic analysis, single-cell RNAseq with TCR repertoire analysis of adult lung T cellsinvolved pooled cells from three 13-week-old B6 and B6.SAP-/- littermates per group. These experiments generated three libraries per experiments: RNA, VDJ, and cell surface protein (CSP). The cell surface protein (CSP) libraries encompassed both Antibody-derived tags (ADTs)and hashtag oligonucleotides (HTOs). This comprehensive approach sheds light on the molecular mechanisms underlying T cell development and the impact of SLAM/SAP signaling across different developmental stages and mouse models. --------------------------------------- ADT/HTO barcode sequence hashtagged sample ADT sample TotalSeq-C0212 anti-mouse CD24 TATATCTTTGCCGCA E17thy, D9thy, W6thy TotalSeq-C0073 anti-mouse CD44 TGGCTTCAGGTCCTA E17thy TotalSeq-C1063 anti-mouse CD45RB TTGTATCTCCCTTGG E17thy TotalSeq-C0209 anti-mouse Vg1 TCGTTTAACCAGCCT E17thy TotalSeq-C0211 anti-mouse Vg4 AAGCTGCACCGTAAT E17thy, D9thy, W6thy, Lung TotalSeq-C0930 anti-mouse SLAMF6 CGATTCTTTGCGAGT E17thy, D9thy, W6thy TotalSeq-C0203 anti-mouse SLAMF1 CAACGCCTAGAAACC E17thy, D9thy, W6thy TotalSeq-C0077 anti-mouse CD73 ACACTTAACGTCTGG E17thy, D9thy, W6thy TotalSeq-C0301 anti-mouse hashtag 1 ACCCACCAGTAAGAC E17thy_B6-1, D9thy_B6-1, W6thy_B6-1 TotalSeq-C0302 anti-mouse hashtag 2 GGTCGAGAGCATTCA E17thy_B6-2, D9thy_B6-2, W6thy_B6-2 TotalSeq-C0303 anti-mouse hashtag 3 CTTGCCGCATGTCAT E17thy_B6-3, D9thy_B6-3, W6thy_B6-3, Lung_B6 TotalSeq-C0304 anti-mouse hashtag 4 AAAGCATTCTTCACG E17thy_B6-4, D9thy_SAPKO-1, W6thy_SAPKO-1, Lung_SAPKO TotalSeq-C0305 anti-mouse hashtag 5 CTTTGTCTTTGTGAG E17thy_SAPKO-1, D9thy_SAPKO-2, W6thy_SAPKO-2 TotalSeq-C0306 anti-mouse hashtag 6 TATGCTGCCACGGTA E17thy_SAPKO-2, D9thy_SAPKO-3, W6thy_SAPKO-3 TotalSeq-C0307 anti-mouse hashtag 7 GAGTCTGCCAGTATC E17thy_SAPKO-3 TotalSeq-C0308 anti-mouse hashtag 8 TATAGAACGCCAGGC E17thy_SAPKO-4, D9thy_ADTcontrol, W6thy_ADTcontrol在胸腺发育进程中，绝大多数T细胞会获得先天样特性，这对于其在肿瘤监视、传染病防控及组织修复中的功能发挥至关重要。然而，调控T细胞发育程序的分子机制仍未明确。近期，本团队证实SLAM-SAP信号通路可调控多种先天样T细胞亚群的发育与功能。本研究采用单细胞蛋白基因组学（single-cell proteogenomics）方法，鉴定SAP依赖的发育检查点，并明确SAP依赖的T细胞受体（T cell receptor, TCR）库谱。SAP缺陷不仅会导致未成熟Gzma+Blk+Etv5+Tox2+T17前体细胞群显著减少，还会使Cd4+Cd8+Rorc+Ptcra+Rag1+胸腺T细胞群体显著增多。胚胎第17天胸腺T细胞克隆型经SAP依赖的重定向进入T细胞发育通路，会导致新生小鼠胸腺中成熟克隆型的频率降低，并使外周TCR库谱发生改变。最后，本研究鉴定出表达TRGV4/TRAV13-4(DV7)的T细胞为一类新型的、SAP依赖的V4 T1亚群。综上，研究数据表明，SAP依赖的T细胞谱系定向调控T细胞发育程序，并塑造TCR库谱。 本数据集涵盖不同发育阶段及不同小鼠模型的T细胞的单细胞CITE-Seq与TCR库谱分析数据，用以揭示SLAM/SAP信号通路对不同T细胞发育检查点及先天样TCR库谱塑造的影响。胸腺单细胞CITE-seq联合TCR库谱分析的样本包括：胚胎第17天（E17）B6及B6.Sh2d1a-/-（SAP敲除，SAP-/-）小鼠，每组各设置4个生物学重复；以及9日龄（D9）新生B6及B6.SAP-/-小鼠，每组各3个生物学重复。此外，数据集还包含6周龄（W6）B6及B6.SAP-/-小鼠的胸腺样本，每组3个重复。在胸腺分析完成后，本团队对成年肺脏T细胞开展了联合TCR库谱分析的单细胞RNA测序，其样本来自每组3只13周龄B6及B6.SAP-/-同窝小鼠的混合细胞。本实验共构建3类文库：RNA文库、VDJ文库及细胞表面蛋白（cell surface protein, CSP）文库。细胞表面蛋白文库包含抗体衍生标签（Antibody-derived tags, ADTs）与寡核苷酸标签（hashtag oligonucleotides, HTOs）两类组分。 以下为ADT/HTO条形码序列详情： 1. TotalSeq-C0212 抗小鼠CD24：条形码序列TATATCTTTGCCGCA，对应样本为E17thy、D9thy、W6thy； 2. TotalSeq-C0073 抗小鼠CD44：条形码序列TGGCTTCAGGTCCTA，对应样本为E17thy； 3. TotalSeq-C1063 抗小鼠CD45RB：条形码序列TTGTATCTCCCTTGG，对应样本为E17thy； 4. TotalSeq-C0209 抗小鼠Vg1：条形码序列TCGTTTAACCAGCCT，对应样本为E17thy； 5. TotalSeq-C0211 抗小鼠Vg4：条形码序列AAGCTGCACCGTAAT，对应样本为E17thy、D9thy、W6thy、Lung； 6. TotalSeq-C0930 抗小鼠SLAMF6：条形码序列CGATTCTTTGCGAGT，对应样本为E17thy、D9thy、W6thy； 7. TotalSeq-C0203 抗小鼠SLAMF1：条形码序列CAACGCCTAGAAACC，对应样本为E17thy、D9thy、W6thy； 8. TotalSeq-C0077 抗小鼠CD73：条形码序列ACACTTAACGTCTGG，对应样本为E17thy、D9thy、W6thy； 9. TotalSeq-C0301 抗小鼠标签1：条形码序列ACCCACCAGTAAGAC，对应样本为E17thy_B6-1、D9thy_B6-1、W6thy_B6-1； 10. TotalSeq-C0302 抗小鼠标签2：条形码序列GGTCGAGAGCATTCA，对应样本为E17thy_B6-2、D9thy_B6-2、W6thy_B6-2； 11. TotalSeq-C0303 抗小鼠标签3：条形码序列CTTGCCGCATGTCAT，对应样本为E17thy_B6-3、D9thy_B6-3、W6thy_B6-3、Lung_B6； 12. TotalSeq-C0304 抗小鼠标签4：条形码序列AAAGCATTCTTCACG，对应样本为E17thy_B6-4、D9thy_SAPKO-1、W6thy_SAPKO-1、Lung_SAPKO； 13. TotalSeq-C0305 抗小鼠标签5：条形码序列CTTTGTCTTTGTGAG，对应样本为E17thy_SAPKO-1、D9thy_SAPKO-2、W6thy_SAPKO-2； 14. TotalSeq-C0306 抗小鼠标签6：条形码序列TATGCTGCCACGGTA，对应样本为E17thy_SAPKO-2、D9thy_SAPKO-3、W6thy_SAPKO-3； 15. TotalSeq-C0307 抗小鼠标签7：条形码序列GAGTCTGCCAGTATC，对应样本为E17thy_SAPKO-3； 16. TotalSeq-C0308 抗小鼠标签8：条形码序列TATAGAACGCCAGGC，对应样本为E17thy_SAPKO-4、D9thy_ADTcontrol、W6thy_ADTcontrol； 这套全面的研究方案有助于阐明T细胞发育的分子机制，以及SLAM/SAP信号通路在不同发育阶段及小鼠模型中对T细胞发育的调控作用。