Final Activity Report Summary - CD8 DC THERA (The generation of CD8 t cell memory)
The current notions propose that CD8 cells get fully programmed upon brief antigen stimulation and that CD8 differentiation is a synchronised process leading to the generation of cohorts of uniform cells that upon in vitro restimulation synthesise a fixed set of cytokines. In our laboratory, we have designed a new RT-PCR multiplex method that allows us to quantify up to 20 genes in a single CD8 cell without the need for in vitro restimulation. This approach was applied to measure CD8 differentiation during various immune responses: viral and bacterial infections, and a response to male HY antigen.
Our results have challenged the previously established notions. In the response to HY and LM-OVA, instead of expressing cytotoxic effector genes in a coordinated way, CD8 T cells exhibited an apparently random initiation of gene expression at early phases of the response, resulting in the appearance of cells that expressed either none or several cytotoxic genes. Gene expression culminated at the peak of CD8 expansion with the maximal co-expression of several cytotoxic molecules by the same cell, while memory cells were marked by a significant loss of effector gene co-expression but maintained cytotoxic gene expression of selected genes. To investigate if this cell behaviour was a peculiarity of TCR-Tg systems, we studied the response to LCMV. We compared the behaviour of GP33-specific P14 TCR Tg cells, and endogenous cells recognising the same or different LCMV epitopes. All LCMV-specific cells tested, i.e. two dominant endogenous populations (NP396- and GP33-specific), subdominant GP276-specific and adoptively transferred P14 TCR Tg cells were similar to each other regardless of differences in their precursor numbers or the kinetics of their expansion / contraction. However, a most prominent finding was that CD8 T cells responding to LCMV, Listeria-OVA, Influenza and male antigen exhibited completely distinct gene expression patterns. The differences in the selection of the genes expressed were first observed at the acute phase of the response and were further emphasised and became stabilised in the memory phase of the response. Moreover, we did not observe any differential segregation of cytotoxic traits in the subpopulations of cells with disparate homing capacities (based on CD62L or Ccr7 expression).
While it is generally accepted that the innate environment has an orchestrating role in directing CD4 helper immune responses, the fact that CD8 CTL responses are delicately tailored to respond to individual pathogens comes as a novelty. We demonstrated that T cell intrinsic features such as clone avidity or even their scarcity / abundance and therefore putative intra- and inter-clonal competition do not have repercussions on the quality of individual effector and memory cells within a given immune response. The cohort of responding cells harboured several subtypes based on their gene expression profiles but the same subtypes were found in all tested populations, regardless of their avidity and/or size. The findings that environmental factors rather than precursor frequency determine the quality of the responding population should have major implications in vaccine development to sub-optimally immunogenic epitopes. Finally, the fact that CD8 responses are heterogeneous opens up possibilities for amelioration of inadequate CD8 responses (such as chronic infections). The database of gene expression patterns in different infectious/immunisation models that we are building, will be valuable for predicting the outcomes of different vaccination protocols, as well as for designing plans on how to tune CD8 cells to a desired gene expression profile that is best fit to confront the specific challenge.
Our results have challenged the previously established notions. In the response to HY and LM-OVA, instead of expressing cytotoxic effector genes in a coordinated way, CD8 T cells exhibited an apparently random initiation of gene expression at early phases of the response, resulting in the appearance of cells that expressed either none or several cytotoxic genes. Gene expression culminated at the peak of CD8 expansion with the maximal co-expression of several cytotoxic molecules by the same cell, while memory cells were marked by a significant loss of effector gene co-expression but maintained cytotoxic gene expression of selected genes. To investigate if this cell behaviour was a peculiarity of TCR-Tg systems, we studied the response to LCMV. We compared the behaviour of GP33-specific P14 TCR Tg cells, and endogenous cells recognising the same or different LCMV epitopes. All LCMV-specific cells tested, i.e. two dominant endogenous populations (NP396- and GP33-specific), subdominant GP276-specific and adoptively transferred P14 TCR Tg cells were similar to each other regardless of differences in their precursor numbers or the kinetics of their expansion / contraction. However, a most prominent finding was that CD8 T cells responding to LCMV, Listeria-OVA, Influenza and male antigen exhibited completely distinct gene expression patterns. The differences in the selection of the genes expressed were first observed at the acute phase of the response and were further emphasised and became stabilised in the memory phase of the response. Moreover, we did not observe any differential segregation of cytotoxic traits in the subpopulations of cells with disparate homing capacities (based on CD62L or Ccr7 expression).
While it is generally accepted that the innate environment has an orchestrating role in directing CD4 helper immune responses, the fact that CD8 CTL responses are delicately tailored to respond to individual pathogens comes as a novelty. We demonstrated that T cell intrinsic features such as clone avidity or even their scarcity / abundance and therefore putative intra- and inter-clonal competition do not have repercussions on the quality of individual effector and memory cells within a given immune response. The cohort of responding cells harboured several subtypes based on their gene expression profiles but the same subtypes were found in all tested populations, regardless of their avidity and/or size. The findings that environmental factors rather than precursor frequency determine the quality of the responding population should have major implications in vaccine development to sub-optimally immunogenic epitopes. Finally, the fact that CD8 responses are heterogeneous opens up possibilities for amelioration of inadequate CD8 responses (such as chronic infections). The database of gene expression patterns in different infectious/immunisation models that we are building, will be valuable for predicting the outcomes of different vaccination protocols, as well as for designing plans on how to tune CD8 cells to a desired gene expression profile that is best fit to confront the specific challenge.