Final Report Summary - INVIVO TCELL IMAGING (Twophoton microscopy analysis of phosphoinositide-3 kinase function during primary and secondary T cell activation in vivo)
The project entitled 'Twophoton microscopy analysis of phosphoinositide-3 kinase function during primary and secondary activation in vivo' started on 01.04.2011. The project goal was to elucidate the contribution of an isoform of the phosphoinositide-3-kinase (PI3K) family of enzymes on T cell activation. The best-characterized PI3K isoform involved in T-cell receptor signaling is the PI3K δ, or p110δ. As models, we employed mice carrying a target mutation in the catalytic domain of p110δ. In these mice, p110δ lipid kinase activity was completely abrogated, with no alteration in kinase activities of p110α and p110β.
The interactions between dendritic cells (DC) and T cells play a central role during the induction of an immune response and immunological tolerance. DC are highly specialized cells, which capture antigen in peripheral tissues and migrate to draining peripheral lymph nodes (PLN), where they present peptide-MHC (pMHC) complexes to naive T cells. Accordingly to the grant proposal, we performed an in vivo imaging-based approach applied to lymphoid and non-lymphoid tissue, in combination with inflammatory models, flow cytometry and other techniques, to address two fundamental questions on the role of p110δ on CD4+ T cell activation:
-First, we characterized the contribution of p110δ in the regulation of CD4+ T cell -DC interactions during the priming phase of T cells;
- Second, we investigated the function of the p110δ isoform during the secondary activation of T cells in a mouse model of antigen-induced arthritis.
Initially, we standardized the use two-photon microscopy (2PM) imaging of adoptively transferred control and p110δ mutant-expressing OT-II TCR transgenic CD4+ T cells recognizing the ovalbumine (OVA)323-339 peptide inside lymphoid tissue. DCs presenting varying amounts of OVA peptide-loaded pMHC complexes on their surface were injected into the footpad of mice. Eighteen hours after transferring DCs, T cells were transferred and 2PM was performed in time intervals from 0-48 h. In our set-up three different cell populations needed to be visualized, therefore, labeling protocols using CellTracker dyes had to be optimized.
The cell velocity inside the lymph node was similar between control and p110δ mutant CD4 T cells (8.354±3.13µm/min for control versus 8.592±3.62µm/min for p110δ mutant T CD4 cells). There was no difference between both populations regarding interaction time (4.865±6.2min for control versus 3.678±5.1min for p110δ mutant T CD4 cells). Next, we adoptively transferred T cells and DCs into recipient mice to carry out a flow cytometry analysis of T cell activation markers (CD44, CD69, CD62L and CD25) at varying time points. A minor defect in up-regulating CD44 was observed 24 hours after transferring p110δ mutant T cells. However, such defect appeared to be transient once it was not observed at 48 or 72 hours. The results for other activation markers (CD69, CD62L and CD25) did not point differences between control and p110 δ mutant T CD4 cells. These data suggested relatively similar parameters of T cell -DC interactions.
Despite this apparent similarity in primary interactions parameters, we sought to investigate further downstream effects of T cells activated in the presence or absence of functional p110δ. We therefore decided to analyze a specific form of delayed type of hypersensitivity, in an arthritis model. In order to perform experiments with the murine model of antigen-induced arthritis (AIA), we standardized a new protocol in which T cells recognizing OVA peptide would be activated. Therefore, animals were immunized by subcutaneous injection with two proteins (methylated bovine serum albumin (mBSA) and OVA), emulsified in complete Freund's adjuvant. Arthritis was induced on day 0 by injecting mBSA and OVA into the left knee joint, while 20 µl PBS was injected into the right knee joint as a control. Animals were analyzed at various time points after the induction of arthritis (day one, day three, day fourteen). The histology results showed a massive cell infiltrate into the joints of mice and such inflammation persevered in presence of wild type T cells. AIA was induced and peri-articular tissue was removed and analyzed by flow-cytometry. Our data showed a massive neutrophil recruitment already at day one, with monocytes arriving at day three and persisting up to day fourteen. Interestingly, we observed that, during arthritis onset, the p110δ mutant T cell population was completely eradicated from the lymph nodes, as well as from the peri-articular tissue.
The CD4 control population percentage was 3.705±0.641 versus 0.029±0.018 in the absence of functional p110δ at the inflammatory site. Then, we hypothesized that impairment in proliferation could explain our findings. However, there was no difference between both populations regarding proliferation rates. The percentage of CD4 cells that proliferate in the control situation was 50.6±0.28 versus 55.85±1.2 in the absence of functional p110δ. Overall, the arthritis results could not be explained by an in vivo proliferation defect. We are currently pursuing the hypothesis that impairment in T follicular helper differentiation and a possible impairment in the immunological synapse could explain our results.
In sum, we performed a comprehensive molecular analysis of the inflammatory action of p110 δ and we are at the moment preparing figures for the manuscript. Overall by the end of the grant period (01.04.2013) I consider that the project developed as expected. The transfer of skills and knowledge of the 2PM surgery, as well as the arthritis induced model and knee imaging, has been very successful. Our findings are promising and we look forward in publishing them.
At the beginning of the fellowship, we experienced a minor delay in obtaining mice with the p110δ mutant mice genotype, delaying for 4 months the first experiments to be done. During this time I engaged a 2PM based project. In this project, we analyzed the role of G-protein coupled receptors (GPCRs) during B cell migration within the T and B cell areas of PLNs, using multichannel 2PM of lymphoid tissue of live, anesthetized mice. Altogether, our data provides an overview on the contribution of prototype GPCRs and integrins during B cell migration within lymphoid tissue, and sheds light on the local availability of promigratory factors. The article, in which I am first author, has been recently accepted in Blood Journal. Taken together, the grant period has achieved more data and knowledge than anticipated.
The interactions between dendritic cells (DC) and T cells play a central role during the induction of an immune response and immunological tolerance. DC are highly specialized cells, which capture antigen in peripheral tissues and migrate to draining peripheral lymph nodes (PLN), where they present peptide-MHC (pMHC) complexes to naive T cells. Accordingly to the grant proposal, we performed an in vivo imaging-based approach applied to lymphoid and non-lymphoid tissue, in combination with inflammatory models, flow cytometry and other techniques, to address two fundamental questions on the role of p110δ on CD4+ T cell activation:
-First, we characterized the contribution of p110δ in the regulation of CD4+ T cell -DC interactions during the priming phase of T cells;
- Second, we investigated the function of the p110δ isoform during the secondary activation of T cells in a mouse model of antigen-induced arthritis.
Initially, we standardized the use two-photon microscopy (2PM) imaging of adoptively transferred control and p110δ mutant-expressing OT-II TCR transgenic CD4+ T cells recognizing the ovalbumine (OVA)323-339 peptide inside lymphoid tissue. DCs presenting varying amounts of OVA peptide-loaded pMHC complexes on their surface were injected into the footpad of mice. Eighteen hours after transferring DCs, T cells were transferred and 2PM was performed in time intervals from 0-48 h. In our set-up three different cell populations needed to be visualized, therefore, labeling protocols using CellTracker dyes had to be optimized.
The cell velocity inside the lymph node was similar between control and p110δ mutant CD4 T cells (8.354±3.13µm/min for control versus 8.592±3.62µm/min for p110δ mutant T CD4 cells). There was no difference between both populations regarding interaction time (4.865±6.2min for control versus 3.678±5.1min for p110δ mutant T CD4 cells). Next, we adoptively transferred T cells and DCs into recipient mice to carry out a flow cytometry analysis of T cell activation markers (CD44, CD69, CD62L and CD25) at varying time points. A minor defect in up-regulating CD44 was observed 24 hours after transferring p110δ mutant T cells. However, such defect appeared to be transient once it was not observed at 48 or 72 hours. The results for other activation markers (CD69, CD62L and CD25) did not point differences between control and p110 δ mutant T CD4 cells. These data suggested relatively similar parameters of T cell -DC interactions.
Despite this apparent similarity in primary interactions parameters, we sought to investigate further downstream effects of T cells activated in the presence or absence of functional p110δ. We therefore decided to analyze a specific form of delayed type of hypersensitivity, in an arthritis model. In order to perform experiments with the murine model of antigen-induced arthritis (AIA), we standardized a new protocol in which T cells recognizing OVA peptide would be activated. Therefore, animals were immunized by subcutaneous injection with two proteins (methylated bovine serum albumin (mBSA) and OVA), emulsified in complete Freund's adjuvant. Arthritis was induced on day 0 by injecting mBSA and OVA into the left knee joint, while 20 µl PBS was injected into the right knee joint as a control. Animals were analyzed at various time points after the induction of arthritis (day one, day three, day fourteen). The histology results showed a massive cell infiltrate into the joints of mice and such inflammation persevered in presence of wild type T cells. AIA was induced and peri-articular tissue was removed and analyzed by flow-cytometry. Our data showed a massive neutrophil recruitment already at day one, with monocytes arriving at day three and persisting up to day fourteen. Interestingly, we observed that, during arthritis onset, the p110δ mutant T cell population was completely eradicated from the lymph nodes, as well as from the peri-articular tissue.
The CD4 control population percentage was 3.705±0.641 versus 0.029±0.018 in the absence of functional p110δ at the inflammatory site. Then, we hypothesized that impairment in proliferation could explain our findings. However, there was no difference between both populations regarding proliferation rates. The percentage of CD4 cells that proliferate in the control situation was 50.6±0.28 versus 55.85±1.2 in the absence of functional p110δ. Overall, the arthritis results could not be explained by an in vivo proliferation defect. We are currently pursuing the hypothesis that impairment in T follicular helper differentiation and a possible impairment in the immunological synapse could explain our results.
In sum, we performed a comprehensive molecular analysis of the inflammatory action of p110 δ and we are at the moment preparing figures for the manuscript. Overall by the end of the grant period (01.04.2013) I consider that the project developed as expected. The transfer of skills and knowledge of the 2PM surgery, as well as the arthritis induced model and knee imaging, has been very successful. Our findings are promising and we look forward in publishing them.
At the beginning of the fellowship, we experienced a minor delay in obtaining mice with the p110δ mutant mice genotype, delaying for 4 months the first experiments to be done. During this time I engaged a 2PM based project. In this project, we analyzed the role of G-protein coupled receptors (GPCRs) during B cell migration within the T and B cell areas of PLNs, using multichannel 2PM of lymphoid tissue of live, anesthetized mice. Altogether, our data provides an overview on the contribution of prototype GPCRs and integrins during B cell migration within lymphoid tissue, and sheds light on the local availability of promigratory factors. The article, in which I am first author, has been recently accepted in Blood Journal. Taken together, the grant period has achieved more data and knowledge than anticipated.