Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor

Abstract

In this report, we have analyzed the human T cell repertoire derived in vivo from a single T cell precursor. A unique case of X-linked severe combined immunodeficiency in which a reverse mutation occurred in an early T cell precursor was analyzed to this end. It was determined that at least 1,000 T cell clones with unique T cell receptor-beta sequences were generated from this precursor. This diversity seems to be stable over time and provides protection from infections in vivo. A similar estimation was obtained in an in vitro murine model of T cell generation from a single cell precursor. Overall, our results document the large diversity potential of T cell precursors and provide a rationale for gene therapy of the block of T cell development.

Figure 1

Numerous distinct T cell clones have been generated from a single T cell progenitor. (A) cDNA prepared from the peripheral blood mononuclear cells of the patient (at 3 years of age) was amplified in multiple PCRs primed by BV- and BC-specific primers. PCR products were then subjected to run-off reactions by using a nested fluorescent primer specific for the BC segment. Labeled DNA copies were analyzed on a sequencing gel in an automated DNA sequencer. The size and the intensity of each band were recorded and analyzed with the immunoscope software. CDR3 size distribution analysis of the patient's peripheral T cells was performed for almost all BV segments and for three AV segments. (B) Altered CDR3β distribution in all BV-BJ profiles analyzed from the patient. For several BV segments, BV-BC PCR products obtained from either a healthy donor or the patient were subjected to run-off reactions by using nested fluorescent primers specific for one of the BJ segments. CDR3β size distribution profiles of the patient (black profiles) are compared with those obtained from the healthy donor (white profiles).

Figure 2

Relative stability of the patient's T cell repertoire over time. CDR3β size distribution analyses were performed from the patient's peripheral blood mononuclear cells at various time points. The figure shows the comparison of the profiles obtained at the age of 3 (black profiles) and 5 (white profiles) years. Arrows indicate the persistence of some expanded populations.

Figure 3

Phenotypic characterization of the patient's T cell subsets. Peripheral blood mononuclear cells from the patient and from the control donor were triple stained with the indicated antibodies. Profiles are gated on either CD4 or CD8 positive cells.

Figure 4

TCR diversity generated in vitro by a single murine T cell progenitor. Various number of fetal thymocytes (15, 25, or 150) were seeded in individual γ-irradiated lobes. After 2 weeks of FTOC, lobes were examined by flow cytometry for the presence of αβ-TCR positive cells. (A) Representative fluorescence-activated cell sorter profile of cells from an individual reconstituted thymic lobe. The majority of the cells expressed αβ-TCR. No cellularity was observed in nonreconstituted (negative) lobes. For each point, the percentage of negative lobes was calculated. (B) Estimation of T cell precursor frequency. According to the Poisson probability distribution, the inoculum that produces 37% negative recipients is expected to contain a single T cell progenitor. This frequency was estimated to be 1 in 26 cells. Therefore, lobes seeded with 15 cells, when reconstituted, contain the progeny of a single T cell precursor. T cell repertoire analyses were performed on these individual lobes. (C) Analysis of the T cell repertoire in lobes reconstituted with one or five T cell precursors. cDNAs prepared from individual thymic lobes were submitted to PCR by using BV10- (or BV8.2-) and BC-specific primers. Representative CDR3β size distribution profiles corresponding to an unreconstituted lobe or a lobe reconstituted with one or five T cell progenitors are shown. (D) Analysis of BV10-BJ rearrangements in lobes reconstituted with a single T cell precursor.