Christophe Pannetier

T-cell repertoire diversity and clonal expansions in normal and clinical samples

Improved polymerase chain reaction (PCR)-based methods now permit a more in-depth analysis of the repertoire of T cells recovered in biological samples from mice and humans. At a certain level of resolution, the diversity of the T-cell repertoire can be readily estimated and clonal expansions become easily detectable. As discussed here by Christophe Pannetier, Jos Even and Philippe Kourilsky, these improvements allow a better appreciation of the degree of reproducibility of immune responses, both in mice and humans, and should have a significant impact on clinical investigations.

Size estimate of the alpha beta TCR repertoire of naive mouse splenocytes.

The diversity of the T cell repertoire of mature T splenocytes is generated, in the thymus, by pairing of α and β variable domains of the αβ TCR and by the rearrangements of various gene segments encoding these domains. In the periphery, it results from competition between various T cell subpopulations including recent thymic migrants and long-lived T cells. Quantitative data on the actual size of the T cell repertoire are lacking. Using PCR methods and extensive sequencing, we have measured for the first time the size of the TCR-αβ repertoire of naive mouse T splenocytes. There are 5–8 × 105 different nucleotide sequences of BV chains in the whole spleen of young adult mice. We have also determined the size of the BV repertoire in a subpopulation of AV2+ T splenocytes, which allows us to provide a minimum estimate of the αβ repertoire. We find that the mouse spleen harbors about 2 × 106 clones of about 10 cells each. This figure, although orders of magnitude smaller than the maximum theoretical diversity (estimated up to 1015), is still large enough to maintain a high functional diversity.

Partial T and B lymphocyte immunodeficiency and predisposition to lymphoma in patients with hypomorphic mutations in Artemis

We have previously described the identification of Artemis, a factor involved in the nonhomologous end joining (NHEJ) phase of V(D)J recombination of T and B cell receptor genes. Null mutations of the Artemis gene result in a complete absence of T and B lymphocytes that is associated with increased cell radiosensitivity, causing the radiosensitive T(-)B(-) SCID (RS-SCID) condition. We presently report the occurrence of hypomorphic mutations of the Artemis gene in four patients from two kindreds. Partially preserved in vivo activity of Artemis is associated with the presence of polyclonal T and B lymphocyte populations, albeit in reduced numbers, along with chromosomal instability and development of EBV-associated lymphoma in two of four patients. This syndrome emphasizes the role of Artemis in the NHEJ pathway of DNA repair and suggests that other, yet ill-defined, conditions associating immunodeficiency and lymphoma could be caused by mutations in genes encoding NHEJ factors.

Lineage relationships, homeostasis, and recall capacities of central– and effector–memory CD8 T cells in vivo

The lineage relationships of central–memory T cells (TCM) cells and effector–memory T cells (TEM), as well as their homeostasis and recall capacities, are still controversial. We investigated these issues in a murine model using two complementary approaches: T cell receptor repertoire analysis and adoptive transfer experiments of purified H-Y–specific TCM and TEM populations. Repertoire studies showed that approximately two thirds of TCM and TEM clones derived from a common naive precursor, whereas the other third was distinct. Both approaches highlighted that TCM and TEM had drastically distinct behaviors in vivo, both in the absence of antigen or upon restimulation. TCM clones were stable in the absence of restimulation and mounted a potent and sustained recall response upon secondary challenge, giving rise to both TCM and TEM, although only a fraction of TCM generated TEM. In contrast, TEM persisted for only a short time in the absence of antigen and, although a fraction of them were able to express CD62L, they were unable to mount a proliferative response upon secondary challenge in this model.

Graft-infiltrating T helper cells, CD45RC phenotype, and Th1/Th2-related cytokines in donor-specific transfusion-induced tolerance in adult rats.

Specific tolerance to LEW.1W (RT1u) heart allografts can be induced in adult LEW.1A (RT1a) rats by donor-specific blood transfusion (DST). We have previously shown that both rejected and tolerated grafts are heavily infiltrated by T lymphocytes, and that in both cases these T cells are capable of developing similar cytotoxic responses against donor cells in vitro; tolerance is therefore not due to the deletion of alloreactive T cells. At the same time, we found that the accumulation of IL-2 and IFN-gamma mRNA was decreased in tolerated grafts compared with rejected grafts. These results suggested that the induction of allograft tolerance in DST-treated animals could be mediated by anergy or suppression of graft-infiltrating Th1 cells. Although Th1 and Th2 clones have not yet been characterized in the rat, peripheral CD4+ rat T cells can be divided into two populations, based on their expression of the isoform RC of the CD45 molecule. Upon activation, CD45RChigh CD4+ T cells produce IL-2 and IFN-gamma and responsible for the induction of the graft-versus-host reaction, whereas CD45RClow CD4+ T cells produce IL-4 in vitro and provide B cell help. In the present study, we show that heart allografts from both DST-treated and untreated rats were infiltrated by equivalent numbers of leukocytes, of which CD4+ T cells also made up similar percentages. Among these CD4+ T cells, we observed that in allografts from DST-treated recipients the CD45RChigh population on day 5 was very significantly smaller (P = 0.004) than in the untreated group, while CD45RClow populations remained comparable. Moreover, using a new quantitative RT-PCR method, we found a dramatic reduction in the accumulation of IL-2, IFN-gamma, IL-10, IL-4, and IL-13 mRNA in hearts from DST-treated recipients compared with those of untreated recipients during the week following transplantation. These results show that in heart allografts from DST-treated recipients, despite phenotypic changes suggesting Th1 inhibition by Th2 imbalance, T helper function was inhibited as a whole, and that in vivo the phenotype CD4+ CD45RClow does not always correlate with Th2-related cytokine-producing cells.

In TH2 cells the Il4 gene has a series of accessibility states associated with distinctive probabilities of IL-4 production

TH2 clones may produce very variable amounts of IL-4. Among six TH2 clones prepared from homozygous or heterozygous mice in which Gfp replaced the first exon of Il4, a range of patterns of CpG methylation in the Il4/Il13 locus was observed correlating with the degree of expression of IL-4 or green fluorescence protein. Patterns of histone acetylation also showed differences between “high” and “low” TH2 clones. These results indicate that in TH2 cells the Il4 locus may display variable patterns of chromatin accessibility associated with distinct degrees of IL-4 expression. This finding suggests a regulation of IL-4 expression keyed to the function of this cytokine in cell/cell interactions and in the regulation of threshold responses.

MHC GENE Q8/9D OF THE BALB/CJ MOUSE STRAIN CANNOT ENCODE A QA-2,3 CLASS I ANTIGEN

We have determined the nucleotide sequence of Q8/9d gene of the BALB/c strain of mice, isolated from Steinmetzs cosmid library. As for all other class I genes of the Qa region, the Q8/9d gene spans approximately 4.7 kilobases (kb) and consists of seven exons and six introns. A seven bases deletion in exon 3 results in the occurrence of an early termination codon. Thus the Q8/9d gene cannot encode a normal class I protein. Comparison of the nucleotide sequence of the Q8/9d gene with that of other class I MHC genes revealed a stronger homology to Q7 and Q8 than to K, D, L, TL, and other Q genes. However, the gene cannot originate from a mere fusion between Q8 and Q9 genes except if the ancestor to putative Q8d was markedly different from the present Q8b gene. Using polymerase chain reaction (PCR) technology, we have confirmed the presence of a Q8/9 gene, identical to that present in cosmid 46.1, in the genome of BALB/cJ (Qa-2low). Finally, it has been reported that cDNA clone 94-A, which codes for a Qa-2 antigen, could derive from a transcript of gene Q8/9d. The nucleotide sequences of gene Q8/9d and of cDNA clone 94-A are distinctly different in their 5′ regions, in spite of an almost perfect matching in their 3′ regions. Thus, clone 94-A cannot derive from an mRNA transcribed from the Q8/9d gene.