Long-term robustness of a T-cell system emerging from somatic rescue of a genetic block in T-cell development

Abstract

Backgound: The potential of a single progenitor cell to establish and maintain long-term protective T-cell immunity in humans is unknown. For genetic disorders disabling T-cell immunity, somatic reversion was shown to support limited T-cell development attenuating the clinical phenotype. However, the cases reported so far deteriorated over time leaving unanswered the important question of long-term activity of revertant precursors and the robustness of the resulting T-cell system.

Methods: We applied TCRβ-CDR3 sequencing and mass cytometry on serial samples of a now 18 year-old SCIDX1 patient with somatic reversion to analyse the longitudinal diversification and stability of a T-cell system emerging from somatic gene rescue.

Findings: We detected close to 10⁵ individual CDR3β sequences in the patient. Blood samples of equal size contained about 10-fold fewer unique CDR3β sequences compared to healthy donors, indicating a surprisingly broad repertoire. Despite dramatic expansions and contractions of individual clonotypes representing up to 30% of the repertoire, stable diversity indices revealed that these transient clonal distortions did not cause long-term repertoire imbalance. Phenotypically, the T-cell system did not show evidence for progressive exhaustion. Combined with immunoglobulin substitution, the limited T-cell system in this patient supported an unremarkable clinical course over 18 years.

Interpretation: Genetic correction in the appropriate cell type, in our patient most likely in a T-cell biased self-renewing hematopoietic progenitor, can yield a diverse T-cell system that provides long-term repertoire stability, does not show evidence for progressive exhaustion and is capable of providing protective and regulated T-cell immunity for at least two decades.

Funding: DFG EH 145/9-1, DFG SCHW 432/4-1 and the German Research Foundation under Germany's Excellence Strategy-EXC-2189-Project ID: 390939984.

Keywords: Immunodeficiency; T cells; T-cell development; TCR diversity.

Fig. 1

Somatic gene rescue provides a longitudinally stable T-cell system in a SCIDX1 patient. (a) Sanger sequencing of sorted lymphocyte subsets. CD3+TCRγδ+, CD3+TCRαβ+ T cells and CD19+ B cells were isolated by FACS. The pellet of Ficoll density centrifugation was the source of the granulocyte fraction. These results were validated by nanopore sequencing (data not shown). (b) Scheme of the hematopoietic system in P2. SCIDX1 patients are characterized by a complete block in NK and T-cell development with normal development of B cells. In P2, somatic genetic rescue allowed development of both αβ and γδ T cells while not sufficiently reconstituting NK cells. Peripheral blood counts for CD4+ (c), CD8+ (d) and TCRγδ+ (e) T cells and differentiation pattern of CD4+ (f) and CD8+ (g) T cells based on surface expression of CD27 and CD45RA. EM=effector memory, CM=central memory, TEMRA=effector memory T cells re-expressing CD45RA. Right graphs show published reference values . (h) Peripheral blood counts for CD4+CD45RA+CD31+ Recent Thymic Emigrant T cells. (i) Number of T cell receptor excision circles (TRECs) per 100.000 T cells as determined by digital droplet PCR. NTC = no template control. BJAB = Burkitt lymphoma B cell line (negative control), DGS = patient with severe DiGeorge Syndrome. Dotted line represents mean background measurement in the TREC-negative BJAB cell line. For BJAB and DGS three independent measurements of the same sample are shown. For P2, samples from three time points are shown, for one of which three independent replicates were performed. For HD (23-25y) 2 replicates each of 3 donors are shown.

Fig. 2

No evidence for continuous exhaustion of the T-cell pool over 7 years. (a) PBMCs analysed by mass cytometry and projected after multi-dimensional reduction with UMAP. Cells were gated on CD3+ T cells. 6 longitudinal samples of P2 and samples of 6 HD obtained at single time points are included in the analysis. CD4+ T cells, CD8+ T cells and TCRγδ+ T cells are circled. Clustering analysis was performed using FlowSOM and overlaid in respective colour. (b) Dendrogram and expression levels of indicated markers on clusters identified by FlowSOM. Arcsinhyp normalized mean intensities are displayed. (c) Abundance of cluster 2 and cluster 9 in longitudinal samples of P2 and individual samples of HDs. (d) Longitudinal expression of selected markers upregulated on exhausted T cells for cluster 2 and cluster 9 of P2. Rows are scaled to have a mean of 0 and SD of 1.

Fig. 3

Somatic gene rescue provides a surprisingly broad, but reduced T-cell repertoire. (a) Abundance of lymphocyte subsets in peripheral blood of CID patients analysed for TCRαβ repertoire diversity. Reference are values taken from . Vβ-usage analysed by flow cytometry for CD8+ (b) and CD4+ (c) T cells. Data are depicted as fold change over mean reference values. Grey bars indicate standard deviation of reference values. (d) Analysis of V and J segment usage by CDR3β sequencing. For analysis of CDR3β sequencing data, all samples were downsampled to contain sequences from equal amounts of TCRαβ+ T cells. Colour code represents square root normalized frequency among productive CDR3β rearrangements. Mutual information was calculated based on unique (e) and total (f) sequences for each donor. (g) Number of unique CDR3β regions. Dashed line represents total number of templates after downsampling. Frequency of top 10, top 100, top 500 clonotypes among productive rearrangements (h) and clonality score (i) were calculated for each donor. (j/k) Representative power law slopes for P2 and 1 HD. Expanded clonotypes (grey) were excluded for calculation of the slope as described . Red line indicates linear regression for clone number plotted against clone frequency of non-expanded (black) clonotypes. (l) Slopes of power law distribution were calculated for all donors. In e-l, age at analysis is given in parentheses. DGS = DiGeorge syndrome. OS = Omenn Syndrome.

Fig. 4

Increased dynamics of top clones across an overall stable T-cell repertoire. Longitudinal samples from one healthy donor (HD), P2 and DGS were analysed by flow cytometry and CDR3β sequencing. The number of unique CDR3β regions (a), clonality score (b) and slopes of power law distributions (c) were analysed in longitudinal samples. Longitudinal analysis of Vβ-family usage by flow cytometry in CD4+ (d/g/j) and CD8+ (e/h/k) T cells and productive frequency of the top 100 CDR3β clonotypes by sequencing are depicted (f/i/l). For P2, the most drastically expanded CD8+ T cell clones with matching Vβ-family usage are highlighted in blue and red respectively.

Fig. 5

Increased overall CDR3β population dynamics is determined top clones. The numbers of significantly expanding (a) and contracting (b) clonotypes between longitudinal samples were calculated as previously described [23,24] (c) BUB overlap index was calculated on longitudinal samples for 1 HD and P2. Morisita Horn (MH) Index was calculated considering all clonotypes (d) and excluding the top 2 clonotypes for P2 (e; highlighted in red and blue in Fig. 4I). MH index visualized against time between blood draws including all productive rearrangements (f) and excluding the top2 clonotypes for P2 (g). Solid blue lines show linear regression for P2 (F: R²=0.2713; p=0.0091 (F-test); G: R²= 0.8680; p < 0.0001 (F-test)).