The immunopathological landscape of human pre-TCRα deficiency: from rare to common variants

We report humans with biallelic loss-of-function PTCRA rare variants impairing pre-TCR-⍺ expression. Low naive ⍺β T cell blood counts at birth persist over time with normal memory ⍺β and high γδ T cell counts. Early productive TCR-δ or TCR-⍺ rearrangements can rescue ⍺β T cell differentiation by stabilizing low levels of cell surface CD3. Only a minority of them are sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that ~1/4,000 individuals from the Middle East and South Asia are homozygous for a hypomorphic PTCRA common variant. They have normal naive ⍺β T cell but high γδ T cell blood counts. Residual expression of their pre-TCR-⍺ enables differentiation of more ⍺β T cells. However, these patients have autoimmune conditions more commonly than the general population. Cryopreserved peripheral blood mononuclear cells (PBMC) in R10 medium (RPMI 1640, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) were thawed and immediately centrifuged to obtain a cell pellet. CD3+ T cells were sorted by flow cytometry from the PBMCs. Cells were then labeled with oligonucleotide-linked hashing antibody and stained by incubation with antibody cocktail diluted in Brilliant Stain Buffer. 12,000 T cells (CD3+CD14−CD19−CD56−Live/Dead−) were sorted from each sample with a BD FACSymphony S6 Cell Sorter running BD FACSDiva Software version 9.5.1. Sorted cells were pooled four by four, and each pool was loaded in a different lane of the 10x Genomics Chromium Chip for sequencing. For the sequencing of single-cell V(D)J repertoires for sorted T cells, the cell suspension was loaded on the 10x Genomics Chromium Instrument according to the manufacturer’s protocol for the Next GEM Single-Cell 5′ Kit v1.1 to generate gel bead-in-emulsions, and to perform GEM-RT and the amplification of total cDNA. Following purification with SPRIselect beads, specific TCR targets were amplified from the cDNA with the PTCR1 primer (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC-3′) and constant region primers: TRB (5′-TGCTTCTGATGGCTCAAACACAGCGACCT-3′), TRA (5′-TCTCAGCTGGTACACGGCAGGGTCAGGGT-3′), TRG (5′-GAAGGAAGAAAAATAGTGGGCTTGGGGGAAAC-3′), or TRD (5′-CACCAGACAAGCGACATTTGTTCC-3′) with a barcode and the P7 sequence added to the constant region primers. The Illumina-ready libraries were sequenced by paired-end MiSeq with 2×300 base-pair reads to obtain VDJ sequences. 10X single-cell transcriptome libraries were processed with Cellranger (v6.1.1) and Seurat (v4.0.4). The TCR-⍺β and TCR-γδ libraries were demultiplexed and cell barcodes were assigned with Minnn (v10.1). TCR libraries were annotated with MiXCR (v3.0.13) and then separated by subject. The numbers of αβ or γδ TCRs for the patients and controls were calculated by counting the numbers of cells expressing both TRA and TRB V-J genes and both TRG and TRD V-J genes. The final counts corresponded to the intersection of cells expressing combinations of TRA, TRB, TRG or TRD genes, accounting for 10365, 21755, 2233 and 1440 cells, respectively, for the controls (n=11) and 5963, 11416, 2095 and 529 cells, respectively, for the patients (n=5). The diversity of α, β, γ and δ TCRs was estimated by calculating Shannon’s entropy (H) index. Entropy was calculated by summing the frequencies of each clone (CDR3 amino-acid sequence) and multiplying by the base 2 logarithm of the same frequency over all cells expressing TRA, TRB, TRG or TRD V-J genes. Higher H index values indicate a more diverse distribution of CDR3 clones. Contributors: Marie Materna, Ottavia M. Delmonte, Marita Bosticardo, Mana Momenilandi, Peyton E. Conrey, Bénédicte Charmeteau-De Muylder, Clotilde Bravetti, Rebecca Bellworthy, Axel Cederholm, Frederik Staels, Christian A. Ganoza, Samuel Darko, Samir Sayed, Corentin Le Floc’h, Masato Ogishi, Darawan Rinchai, Andrea Guenoun, Alexandre Bolze, Taushif Khan, Adrian Gervais, Renate Krüger, Mirjam Völler, Boaz Palterer, Mahnaz Sadeghi-Shabestari, Anne Langlois de Septenville, Chaim A. Schramm, Sanjana Shah, John J. Tello-Cajiao, Francesca Pala, Kayla Amini, Jose S. Campos, Noemia Santana Lima, Daniel Eriksson, Romain Lévy, Yoann Seeleuthner, Soma Jyonouchi, Manar Ata, Fatima Al Ali, Caroline Deswarte, Anaïs Pereira, Jérôme Mégret, Tom Le Voyer, Paul Bastard, Laureline Berteloot, Michaël Dussiot, Natasha Vladikine, Paula P. Cardenas, Emmanuelle Jouanguy, Mashael Alqahtani, Amal Hasan, Thangavel Alphonse Thanaraj, Jérémie Rosain, Fahd Al Qureshah, Vito Sabato, Marie Alexandra Alyanakian, Marianne Leruez-Ville, Flore Rosenberg, Elie Haddad, Jose R. Regueiro, Maria L. Toribio, Judith R. Kelsen, Mansoor Salehi, Shahram Nasiri, Mehdi Torabizadeh, Hassan Rokni-Zadeh, Majid Changi-Ashtiani, Nasimeh Vatandoost, Hossein Moravej, Seyed Mohammad Akrami, Mohsen Mazloomrezaei, Aurélie Cobat, Isabelle Meyts, Toyofuku Etsushi, Madoka Nishimura, Kunihiko Moriya, Tomoyuki Mizukami, Kohsuke Imai, Laurent Abel, Bernard Malissen, Fahd Al-Mulla, Fowzan Sami Alkuraya, Nima Parvaneh, Horst von Bernuth, Christian Beetz, Frédéric Davi, Daniel C. Douek, Rémi Cheynier, David Langlais, Nils Landegren, Nico Marr, Tomohiro Morio, Mohammad Shahrooei, Rik Schrijvers, Sarah E. Henrickson, Hervé Luche, Luigi D. Notarangelo, Jean-Laurent Casanova, Vivien Béziat