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

The lineage relationships of central-memory T cells (T(CM)) cells and effector-memory T cells (T(EM)), 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 T(CM) and T(EM) populations. Repertoire studies showed that approximately two thirds of T(CM) and T(EM) clones derived from a common naive precursor, whereas the other third was distinct. Both approaches highlighted that T(CM) and T(EM) had drastically distinct behaviors in vivo, both in the absence of antigen or upon restimulation. T(CM) clones were stable in the absence of restimulation and mounted a potent and sustained recall response upon secondary challenge, giving rise to both T(CM) and T(EM), although only a fraction of T(CM) generated T(EM). In contrast, T(EM) 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.

Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.

BACKGROUND: X-linked severe combined immunodeficiency due to a mutation in the gene encoding the common gamma (gamma(c)) chain is a lethal condition that can be cured by allogeneic stem-cell transplantation. We investigated whether infusion of autologous hematopoietic stem cells that had been transduced in vitro with the gamma(c) gene can restore the immune system in patients with severe combined immunodeficiency. METHODS: CD34+ bone marrow cells from five boys with X-linked severe combined immunodeficiency were transduced ex vivo with the use of a defective retroviral vector. Integration and expression of the gamma(c) transgene and development of lymphocyte subgroups and their functions were sequentially analyzed over a period of up to 2.5 years after gene transfer. RESULTS: No adverse effects resulted from the procedure. Transduced T cells and natural killer cells appeared in the blood of four of the five patients within four months. The numbers and phenotypes of T cells, the repertoire of T-cell receptors, and the in vitro proliferative responses of T cells to several antigens after immunization were nearly normal up to two years after treatment. Thymopoiesis was documented by the presence of naive T cells and T-cell antigen-receptor episomes and the development of a normal-sized thymus gland. The frequency of transduced B cells was low, but serum immunoglobulin levels and antibody production after immunization were sufficient to avoid the need for intravenous immunoglobulin. Correction of the immunodeficiency eradicated established infections and allowed patients to have a normal life. CONCLUSIONS: Ex vivo gene therapy with gamma(c) can safely correct the immune deficiency of patients with X-linked severe combined immunodeficiency.

The repertoires of circulating human CD8(+) central and effector memory T cell subsets are largely distinct.

Memory T cells are divided into central and effector subsets with distinct functions and homing capabilities. We analyzed the composition and dynamics of the CD8(+) T cell repertoire of these subsets within the peripheral blood of four healthy individuals. Both subsets had largely distinct and autonomous TCRbeta repertoires. Their composition remained stable over a 9 month period, during which no cell passage between these subsets was detected despite important size variation of several clones. In one donor, four out of six TCRbeta clonotypes specific for the influenza A virus were detected in the central subset only, while the two others were shared. Altogether, these observations suggest that most effector memory T cells may not have derived from the central memory subset.

Gene therapy of RAG-2-/- mice: sustained correction of the immunodeficiency.

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation, which is only partially successful in the absence of an HLA genoidentical donor, thus justifying research to find an alternative therapeutic approach. To this end, RAG-2-deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2-/-Sca-1(+) selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)-based MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2-/- sublethally irradiated mice (3Gy). Two months later, T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens, allogeneic cells, and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy, a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether, this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2-/- mice, constituting a significant step toward clinical application.

Expansion and function of CD8+ T cells expressing Ly49 inhibitory receptors specific for MHC class I molecules.

MHC class I-specific Ly49 inhibitory receptors regulate NK cell activation, thereby preventing autologous damage to normal cells. Ly49 receptors are also expressed on a subset of CD8+ T cells whose origin and function remain unknown. We report here that, despite their phenotypic and cytolytic similarities, Ly49+CD8+ T cells and conventional Ly49-CD44high memory-phenotype CD8+ T cells present strikingly distinct features. First, under steady state conditions Ly49+CD8+ T cells are poor cytokine producers (TNF-alpha and IFN-gamma) upon TCR triggering. Second, Ly49+CD8+ T cells are not induced upon various settings of Ag immunization or microbial challenge. However, Ly49 can be induced on a fraction of self-specific CD8+ T cells if CD4+ T cells are present. Finally, the size of the Ly49+CD8+ T cell subset is selectively reduced in the absence of STAT1. These results indicate that Ly49 expression is associated with a differentiation program of cytolytic CD8+ T cells triggered upon chronic antigenic exposure. They further suggest that the size of the Ly49+CD8+ T cell subset marks a history of CD8+ T cell activation that might preferentially result from endogenous inducers of inflammation rather than from microbial infections.