Final Report Summary - TREG-IN-GVHD (Longitudinal analysis of human CD4+FOXP3+ regulatory T cell subpopulations in acute graft-versus-host disease)
Rationale
CD4+FOXP3+ regulatory T cells (Treg) are essential in maintaining peripheral self-tolerance and preventing inflammatory disease. The perspective to harness the potent immunosuppressive activity of Treg for clinical applications, such as prevention or treatment of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation, improved tolerance after solid organ transplantation and treatment of autoimmune diseases, has sparked numerous studies that revealed that Treg are not a phenotypically or functionally homogeneous population. Distinct human Treg subsets have been described by analysing cell surface or intracellular markers such as ICOS, CD45RA, Helios, HLA-DR and chemokine receptors. It is unclear how the Treg subsets so defined relate to each other, and which subset may be more suitable for therapeutic applications.
A further concern comes from studies showing that Treg may have the potential to convert into CD4+ effector T cells with proinflammatory functions, in particular in an inflammatory environment. However, the notion of Treg reprogramming remains controversial, with a study demonstrating a remarkable stability of Treg even under highly inflammatory conditions.
Objective
The main goal of this project was to promote the fundamental knowledge of human Treg subpopulations, and to address their dynamics in human GVHD.
Results
Heterogeneity of human CD4+FOXP3+ T cells
Phenotypic analysis of the CD4+FOXP3+ T cell compartment revealed three distinct Treg populations (CD45RA+HLADR-, CD45RA-HLA-DR- and CD45RA-HLA-DR+) present at different frequencies in peripheral blood, thymus and cord blood. The majority of cord blood Treg exhibits a 'naïve' phenotype (RA+DR-), while higher percentages of RA-DR- and RA-DR+ Treg are found in adult peripheral blood. The frequency of total CD4+FOXP3+ T cells is not significantly different in neonates and adults. The Treg subpopulations vary for the expression of regulatory T cell markers, the proliferation state, and the expression of Helios, a transcription factor primarily expressed by Treg of thymic origin. In the thymus, the three Treg subpopulations show weak proliferative activity, while in cord blood and peripheral blood Treg proliferation is observed, mainly in the RA-DR+ subpopulation. This subpopulation has also the highest suppressive activity in vitro, consistent with higher expression levels of molecules associated with Treg function. We also observed that RA-DR+ Treg, as well as RA-DR- Treg express CCR6 but not CCR7, suggesting homing to mucosal or inflamed tissues. In contrast, RA+DR- Treg express high levels of CCR7 but lack expression of CCR6, indicating that these cells may preferentially home to secondary lymphoid organs.
Single-cell gene expression profiling reveals heterogeneity within Treg subpopulations
The expression patterns of Helios, CCR6 and CCR7 suggested that Treg display marked heterogeneity also within the subpopulations defined by CD45RA and HLA-DR. To examine heterogeneity at the single-cell level within each subset, we selected genes from the Treg signatures and analyzed their expression in single TCR-activated Tconv and Treg using a microfluidic qPCR platform. Hierarchical clustering analysis of gene expression shows that the signatures defined at the population level segregate each Treg subpopulation at the single-cell level and reveals a strong heterogeneity of gene expression within each subpopulation. Conversely, expression of the house-keeping gene B2M fluctuated considerably less. Consistent with the flow cytometry data, expression of Treg cell markers FOXP3 and CTLA4 is higher in the RA-DR+ Treg subpopulation, and CCR6 expression is restricted to the RA-DR- and RA-DR+ Treg populations. These results demonstrate a yet unappreciated heterogeneity of the human Treg compartment at the single-cell level, reminiscent of the heterogeneity of effector CD4+ T cell populations.
Cytokine-secreting CD4+FOXP3+ T cells share features of Treg and effector T cells
Our microarray analysis revealed an unexpected enrichment of transcripts encoding cytokines within the RA-DR- Treg population. qPCR analysis confirmed that effector cytokine genes (IFNG, IL17, IL22, IL4) and the anti-inflammatory cytokine IL10 were expressed by RA-DR- Treg, but not by naive Tconv or RA+DR- and RA-DR+ Treg. Intracellular staining shows that a small fraction of RA-DR- Treg expresses either IFN-&# 947; or IL-17A. The frequency of IFN-&# 947;-secreting cells is significantly lower in Treg compared to Tconv, however the frequency of IL-17A-producing cells is similar in RA-DR- Treg and Tconv. To test if IFN-&# 947; and IL-17-production by Treg is caused by lineage-reprogramming of human Treg into Th1 or Th17 effector cells, we cultured the three Treg populations and CD4+CD45RA- Tconv for two weeks in the presence of Th1 or Th17-inducing cytokines. We did not observe a potent induction of cytokine transcripts in any Treg population in these conditions.
To determine if cytokine-producing CD4+FOXP3+ T cells maintain the molecular profiles of Treg, we analysed whether a single Treg cell can co-express Treg marker genes and effector cytokines. We found that IFN-&# 947; and IL-17A-expressing Treg maintain the expression of Treg markers such as FOXP3 and CTLA4. A substantial fraction of IFN-? and IL-17A-secreting Treg express IKZF2. In addition, cytokine-producing Treg express the Th1-specific transcription factor TBX21 or the Th17-specific factor RORC, together with effector cytokines, however they do not up-regulate IL2 expression.
However, the mRNA levels of TBX21/IFNG and RORC/IL17A are lower in Treg compared to Th1 and Th17 cells. Hierarchical clustering based on genes from the Treg signature and genes associated with Th1 or Th17 cells indicates that Th17 cells are more closely related to cytokine-producing Treg than to Th1 cells. Cytokine-secreting Treg express lower levels of the Treg markers FOXP3, IKZF2 and FAS compared to Treg that do not produce IFN-&# 947; or IL-17A. These data demonstrate the co-existence within the same cell of transcriptional programs controlling Treg and Th1 or Th17 cell functions, and provide further evidence for the functional diversity of the human CD4+FOXP3+ T cell compartment.
Conclusions
Our single-cell analysis of the human regulatory T cell compartment revealed an unexpected level of complexity and functional heterogeneity of human Treg. We found that genes belonging to our Treg signatures showed, at the single-cell level, a highly variegated gene expression pattern that could not be appreciated by the analysis of bulk populations or by flow cytometry. Similar to CD4+ FOXP3- effector T cells, the mosaic-like expression of transcription factors, signalling molecules and homing receptors is likely to endow individual Treg with specific functions and migratory properties. Despite the remarkable qualitative and quantitative differences in gene expression from cell to cell, we also noted two invariable features shared by all human Treg. Single-cell analysis revealed that all Treg express FOXP3 and lack IL-2 expression emphasising their key role in regulating Treg function. Consistent with flow cytometry analysis, we noted that the expression level of FOXP3 and CTLA4 and other genes associated with the function of Treg varies by 2-3 orders of magnitude from cell to cell, indicating that these molecules may act as important rheostats to tune the immunosuppressive function of individual cells. An important question that remains to be solved concerns the mechanisms accounting for the remarkably strong variation in the expression of FOXP3 and other Treg signature genes from cell to cell. In particular, it will be important to understand the relative contributions of stochasticity in gene expression on the one hand, and environmental factors such as TCR signal strength, costimulatory signals and cytokines on the other.
We have characterised at the single-cell level a very small population of IFN-&# 947; and IL-17A-producing Treg by combining the isolation of viable cytokine-secreting cells with single-cell gene profiling. This analysis revealed that IFN-&# 947; and IL-17A-producing Treg are characterised by an overlap within the same cell of gene expression signatures of Th1 or Th17 cells and of Treg. The lack of IL-2 expression as well as the expression of high levels of CTLA-4 and other molecules typically associated with Treg functions suggests immunoregulatory properties for these cells. We noted that IL-17A and IFN-&# 947; transcripts are expressed at lower levels in cytokine-secreting Treg than in conventional CD4+ effector T cells, which may indicate a lower pro-inflammatory potential. Interestingly, IL-17A and IFN-&# 947;-secreting Treg expressed lower levels of FOXP3 than Treg that do not produce these cytokines. These data suggest that FOXP3 controls not only the immunosuppressive activity of Treg, but may also suppress their 'inflammatory' functions.
Our work demonstrates that multi-parameter single-cell analysis techniques not only uncover previously unappreciated levels of heterogeneity within cell 'subsets', but can also provide valuable information about transcriptional networks in single cells.
CD4+FOXP3+ regulatory T cells (Treg) are essential in maintaining peripheral self-tolerance and preventing inflammatory disease. The perspective to harness the potent immunosuppressive activity of Treg for clinical applications, such as prevention or treatment of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation, improved tolerance after solid organ transplantation and treatment of autoimmune diseases, has sparked numerous studies that revealed that Treg are not a phenotypically or functionally homogeneous population. Distinct human Treg subsets have been described by analysing cell surface or intracellular markers such as ICOS, CD45RA, Helios, HLA-DR and chemokine receptors. It is unclear how the Treg subsets so defined relate to each other, and which subset may be more suitable for therapeutic applications.
A further concern comes from studies showing that Treg may have the potential to convert into CD4+ effector T cells with proinflammatory functions, in particular in an inflammatory environment. However, the notion of Treg reprogramming remains controversial, with a study demonstrating a remarkable stability of Treg even under highly inflammatory conditions.
Objective
The main goal of this project was to promote the fundamental knowledge of human Treg subpopulations, and to address their dynamics in human GVHD.
Results
Heterogeneity of human CD4+FOXP3+ T cells
Phenotypic analysis of the CD4+FOXP3+ T cell compartment revealed three distinct Treg populations (CD45RA+HLADR-, CD45RA-HLA-DR- and CD45RA-HLA-DR+) present at different frequencies in peripheral blood, thymus and cord blood. The majority of cord blood Treg exhibits a 'naïve' phenotype (RA+DR-), while higher percentages of RA-DR- and RA-DR+ Treg are found in adult peripheral blood. The frequency of total CD4+FOXP3+ T cells is not significantly different in neonates and adults. The Treg subpopulations vary for the expression of regulatory T cell markers, the proliferation state, and the expression of Helios, a transcription factor primarily expressed by Treg of thymic origin. In the thymus, the three Treg subpopulations show weak proliferative activity, while in cord blood and peripheral blood Treg proliferation is observed, mainly in the RA-DR+ subpopulation. This subpopulation has also the highest suppressive activity in vitro, consistent with higher expression levels of molecules associated with Treg function. We also observed that RA-DR+ Treg, as well as RA-DR- Treg express CCR6 but not CCR7, suggesting homing to mucosal or inflamed tissues. In contrast, RA+DR- Treg express high levels of CCR7 but lack expression of CCR6, indicating that these cells may preferentially home to secondary lymphoid organs.
Single-cell gene expression profiling reveals heterogeneity within Treg subpopulations
The expression patterns of Helios, CCR6 and CCR7 suggested that Treg display marked heterogeneity also within the subpopulations defined by CD45RA and HLA-DR. To examine heterogeneity at the single-cell level within each subset, we selected genes from the Treg signatures and analyzed their expression in single TCR-activated Tconv and Treg using a microfluidic qPCR platform. Hierarchical clustering analysis of gene expression shows that the signatures defined at the population level segregate each Treg subpopulation at the single-cell level and reveals a strong heterogeneity of gene expression within each subpopulation. Conversely, expression of the house-keeping gene B2M fluctuated considerably less. Consistent with the flow cytometry data, expression of Treg cell markers FOXP3 and CTLA4 is higher in the RA-DR+ Treg subpopulation, and CCR6 expression is restricted to the RA-DR- and RA-DR+ Treg populations. These results demonstrate a yet unappreciated heterogeneity of the human Treg compartment at the single-cell level, reminiscent of the heterogeneity of effector CD4+ T cell populations.
Cytokine-secreting CD4+FOXP3+ T cells share features of Treg and effector T cells
Our microarray analysis revealed an unexpected enrichment of transcripts encoding cytokines within the RA-DR- Treg population. qPCR analysis confirmed that effector cytokine genes (IFNG, IL17, IL22, IL4) and the anti-inflammatory cytokine IL10 were expressed by RA-DR- Treg, but not by naive Tconv or RA+DR- and RA-DR+ Treg. Intracellular staining shows that a small fraction of RA-DR- Treg expresses either IFN-&# 947; or IL-17A. The frequency of IFN-&# 947;-secreting cells is significantly lower in Treg compared to Tconv, however the frequency of IL-17A-producing cells is similar in RA-DR- Treg and Tconv. To test if IFN-&# 947; and IL-17-production by Treg is caused by lineage-reprogramming of human Treg into Th1 or Th17 effector cells, we cultured the three Treg populations and CD4+CD45RA- Tconv for two weeks in the presence of Th1 or Th17-inducing cytokines. We did not observe a potent induction of cytokine transcripts in any Treg population in these conditions.
To determine if cytokine-producing CD4+FOXP3+ T cells maintain the molecular profiles of Treg, we analysed whether a single Treg cell can co-express Treg marker genes and effector cytokines. We found that IFN-&# 947; and IL-17A-expressing Treg maintain the expression of Treg markers such as FOXP3 and CTLA4. A substantial fraction of IFN-? and IL-17A-secreting Treg express IKZF2. In addition, cytokine-producing Treg express the Th1-specific transcription factor TBX21 or the Th17-specific factor RORC, together with effector cytokines, however they do not up-regulate IL2 expression.
However, the mRNA levels of TBX21/IFNG and RORC/IL17A are lower in Treg compared to Th1 and Th17 cells. Hierarchical clustering based on genes from the Treg signature and genes associated with Th1 or Th17 cells indicates that Th17 cells are more closely related to cytokine-producing Treg than to Th1 cells. Cytokine-secreting Treg express lower levels of the Treg markers FOXP3, IKZF2 and FAS compared to Treg that do not produce IFN-&# 947; or IL-17A. These data demonstrate the co-existence within the same cell of transcriptional programs controlling Treg and Th1 or Th17 cell functions, and provide further evidence for the functional diversity of the human CD4+FOXP3+ T cell compartment.
Conclusions
Our single-cell analysis of the human regulatory T cell compartment revealed an unexpected level of complexity and functional heterogeneity of human Treg. We found that genes belonging to our Treg signatures showed, at the single-cell level, a highly variegated gene expression pattern that could not be appreciated by the analysis of bulk populations or by flow cytometry. Similar to CD4+ FOXP3- effector T cells, the mosaic-like expression of transcription factors, signalling molecules and homing receptors is likely to endow individual Treg with specific functions and migratory properties. Despite the remarkable qualitative and quantitative differences in gene expression from cell to cell, we also noted two invariable features shared by all human Treg. Single-cell analysis revealed that all Treg express FOXP3 and lack IL-2 expression emphasising their key role in regulating Treg function. Consistent with flow cytometry analysis, we noted that the expression level of FOXP3 and CTLA4 and other genes associated with the function of Treg varies by 2-3 orders of magnitude from cell to cell, indicating that these molecules may act as important rheostats to tune the immunosuppressive function of individual cells. An important question that remains to be solved concerns the mechanisms accounting for the remarkably strong variation in the expression of FOXP3 and other Treg signature genes from cell to cell. In particular, it will be important to understand the relative contributions of stochasticity in gene expression on the one hand, and environmental factors such as TCR signal strength, costimulatory signals and cytokines on the other.
We have characterised at the single-cell level a very small population of IFN-&# 947; and IL-17A-producing Treg by combining the isolation of viable cytokine-secreting cells with single-cell gene profiling. This analysis revealed that IFN-&# 947; and IL-17A-producing Treg are characterised by an overlap within the same cell of gene expression signatures of Th1 or Th17 cells and of Treg. The lack of IL-2 expression as well as the expression of high levels of CTLA-4 and other molecules typically associated with Treg functions suggests immunoregulatory properties for these cells. We noted that IL-17A and IFN-&# 947; transcripts are expressed at lower levels in cytokine-secreting Treg than in conventional CD4+ effector T cells, which may indicate a lower pro-inflammatory potential. Interestingly, IL-17A and IFN-&# 947;-secreting Treg expressed lower levels of FOXP3 than Treg that do not produce these cytokines. These data suggest that FOXP3 controls not only the immunosuppressive activity of Treg, but may also suppress their 'inflammatory' functions.
Our work demonstrates that multi-parameter single-cell analysis techniques not only uncover previously unappreciated levels of heterogeneity within cell 'subsets', but can also provide valuable information about transcriptional networks in single cells.