Intrinsic transgene immunogenicity gears CD8(+) T-cell priming after rAAV-mediated muscle gene transfer.

Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.

Extrathymic induction of Foxp3⁺ regulatory T cells declines with age in a T-cell intrinsic manner.

Extrathymically induced Foxp3⁺ regulatory T (Treg) cells contribute to the pool of Treg cells and are implicated in the maintenance of immune tolerance at environmental interfaces. The impact of T-cell senescence on their generation and function is, however, poorly characterized. We report here that steady-state induction of Foxp3 is impaired in aged T cells in vivo. In vitro assays further revealed that this defective generation of Treg cells was independent from the strength of TCR stimulation and arose before T-cell proliferation. Importantly, they also revealed that this impairment of Foxp3 induction is unrelated to known age-related T-cell defects, such as IL-2 secretion impairment, accumulation of activated T-cell populations, or narrowing of the T-cell repertoire. Finally, a loss of extrathymic induction of Foxp3 and tolerance to minor-mismatched skin graft were observed in aged mice treated by nondepleting anti-CD4 antibody. The T-cell intrinsic impairment of Treg-cell generation revealed here highlights age as a key factor to be considered in immune tolerance induction.

Antigen-specific Treg impair CD8(+) T-cell priming by blocking early T-cell expansion.

Foxp3(+) Treg are crucial for the maintenance of self-tolerance and have been shown to control CD8(+) T-cell effector functions. In addition, Treg are thought to control the priming of CD8(+) T cells, which recognize the same antigens as Treg. Taking advantage of our model of peripheral tolerance induction to influenza hemagglutinin (HA) after HA gene transfer, we found that HA-specific Treg suppress antigen-linked CTL responses through early blockade of CD8(+) T-cell expansion. Confronted with their cognate antigen, Treg expand more rapidly than CD8(+) T cells and are highly suppressive only during the initial stages of immune priming. They nullify HA-specific CD8(+) T-cell responses, local inflammatory responses and rejection of HA transduced cells. When HA gene transfer is performed with extensive tissue inflammation, HA-specific Treg are less effective but still reduce the frequency of newly primed HA-specific CD8(+) T cells and the ensuing frequency of memory CD8(+) T cells. Our results demonstrate that Treg control CTL priming in an antigen-specific manner at the level of T-cell expansion, highlighting how self-reactive Treg could prevent the induction of autoimmune responses through selective blockade of autoreactive T-cell proliferation.

Foxp3+ Regulatory and Conventional CD4+ T Cells Display Similarly High Frequencies of Alloantigen-Reactive Cells.

Foxp3+ regulatory T cells (Tregs) play a major role in acquired immune tolerance to allogenic transplants. Their suppressive activity is thought to require T cell receptor (TCR)-driven antigen recognition; little, however, is known about the fraction of Tregs able to recognize alloantigens within this T cell subset primarily educated against self-antigens. Performing transfer experiments of Tregs or conventional T cells (Tconv) into both lymphoreplete and lymphopenic mice, we observed a similarly high proportion of cells signaling through their TCR and proliferating in allogenic hosts. Furthermore, using an in vivo proliferation assay with limited T cell numbers infused into lymphopenic mice, we found that the overall frequency of alloreactive Tregs was similar if not higher to that of alloreactive Tconv. Overall our study highlights a noticeably high level of alloreactive Foxp3+ regulatory T cells accounting for their predominant role in transplantation tolerance.