Final Report Summary - 3ITD (Impact of IL-7 and IFNa on nTreg development)
The enigma of immune regulation and Human immunodeficiency virus (HIV)-1 infection attracts scientific attention since several years. It has two principal arms - the HIV-1 infection per se, which leads to a continuous impairment of the T cell compartment in the periphery and the immune regulatory mechanisms, represented by T regulatory cells (Tregs). One of the major cross points between them is the thymus, where the new T cells become functional. Of note, during early infection the virus rapidly infects the thymus and seriously impairs its function. Two populations of Tregs have been described in the periphery - inducible and natural Tregs (iTregs and nTregs). Unlike to iTregs, that are derived from circulating mature naïve T-cells and are suspected to mainly impact on the regulation of inflammation and immune responses in the gut, nTregs are selected by high-avidity interactions in the thymus and seems to be involved in the prevention of autoimmune reactions and raise the activation threshold for all immune responses, mechanisms that are largely impaired during HIV-induced immunodeficiency. Because of the intensive research during last ten years, iTregs are already characterised phenotypically and functionally, while nTregs are less investigated. The insufficient knowledge on nTregs is due to the lack of specific markers that could permit to distinguish them from iTregs.
The project is focused on the identification of nTregs, recently emigrated from the thymus (nTreg-RTEs) with respect to their phenotype, function and how do they respond to the immune-based interventions.
During the project execution, a specific experimental design was created aimed at the precise determination of the population of interest. Three areas of investigation have been included. The first one comprises phenotypic determination of nTreg-RTEs by multicolour flow cytometry. To confirm its recent thymic origin throughout signal joint T cell excision circles (sjTRECs), an innovative real-time Polymerase chain reaction (PCR), developed by the host laboratory, was applied. An in vitro suppression assay was developed aimed to investigate function of nTreg-RTEs. Experiments were performed on both freshly isolated peripheral blood mononuclear cell (PBMC)s, as well as frozen cells of 22 healthy controls and frozen PBMCs from 17 patients with HIV-1 infection (9 under therapy and 8 therapy free).
It has been found that the population of nTreg-RTEs (CD31+FOXP3+) is present among the naïve CD4+ T cells in the peripheral blood of healthy subjects, although in a very small proportion. Their frequency in healthy subjects was higher than that, found in both patients groups. In healthy controls the frequency of nTreg-RTEs correlated well with those of CD4+RTEs, thus suggesting their common ontogenesis. The detailed phenotypic analysis revealed that it consists of two fractions, depending on the expression of the CD25 (IL-2 receptor subunit). The measurement of sjTRECs showed that both fractions are equally rich in TRECs, thus confirming their similar thymic proximity. In the control group, the CD25+ nTreg-RTEs predominated, that was not the case in either group of HIV-1 infected individuals. Further, an analysis of the CD127 expression was performed and the results obtained showed that in both healthy controls and Antiretroviral therapy (ART) treated individuals, CD127- cells predominate in nTreg-RTE subsets, thus supporting the fact that they have recently left the thymus, e.g. are RTEs.
It is shown in the literature, that regulatory T cells suppress the proliferation of conventional T cells. In order to completely describe the newly determined population, a suppression assay was performed in vitro with two-steps sorted populations of nTreg-RTEs CD25low and nTreg-RTEs CD25+. The results obtained showed that while CD25+ fraction is fully active without a preactivation, its CD25- counterpart needs to be activated before the expression of its suppressive capacity. Besides, their suppressive function was measured in two different responder cells / nTre-RTEs ratios - 1:6 and 1:32. It was found that a higher ratio results in better inhibition effect.
In conclusion:
- The population of nTreg-RTEs, defined by CD31 expression on FOXP3+ naïve CD4+ T-cells, consists of two subsets characterised by differential expression of CD25. These 2 subsets are mostly CD127-.
- Both fractions are similarly rich in sjTRECs thus suggesting a common proliferative history.
- The increased frequency of FOXP3+ within nCD4-RTEs in HIV-infected individuals suggests that their intrathymic development and/or life cycle in peripheral blood are less affected by the virus in comparison to conventional naïve T-cells.
- An inverse ratio between CD25- and CD25+ cells was found in patients as compared to healthy controls, suggesting that CD25- cells either result from an unsuccessful nTregs development or could be an independent subset nTregs, linked to the development of iTregs.
- The increased proportion of CD127- cells into two CD25 fractions in HIV-infected patients receiving therapy in parallel with the same profile in non-treated subjects, clearly shows the beneficial effect of ART on nTreg-RTEs.
The results obtained are of importance for basic science, clinical practice and scientific collaboration in the frame of European Research Area (ERA).
The contribution to the basic science is toward the better understanding of T-cell maturation process. In one animal study in Simian immunodeficiency virus (SIV) infection it has been already demonstrated that two populations naïve T cells exist in the periphery - CD25low and CD25+. Several studies demonstrated an increase in CD25lowFOXP3+ cells in peripheral blood of patients with autoimmune diseases. The results obtained showed a tendency of impaired function of this population. Animal studies indicated that the acquisition of CD25 is a result of the interaction between immature T cells and medulary epithelieal (mTECs) cells in the thymus. Therefore, it could be suggested, that the appearance of CD25low nTreg-RTEs might result from impaired T cells / mTECs interactions, which is in accordance with the strong negative influence HIV has on thymocytes. Another hypothesis might be that CD25 - Treg-RTEs represent an independent step of normal maturation process. No data exist in the literature about how these populations are represented in lymph nodes. Therefore, the results obtained provide essential clarifying information for this process and might be a platform for further investigations.
Another direction represents the investigation on the role of the thymus in the development of natural tolerance and particularly - autoimmune disorders. The ineffective maturation of the nTreg-RTEs might contribute to the impaired peripheral tolerance.
The impact of nTreg-RTEs in clinical practice consists of their application as a promising target for the development and application of immune-based therapies, aimed at restoring the balance of the immune system in HIV infection. A few years ago, the idea of immune intervention to help patients reconstituting a functional immune system emerged in the scientific community and clinical trials were built to test the efficacy of cytokines based-therapies (interleukin-2 (IL-2) and interleukin-7 (IL-7)) in the context of HIV-infection.
Despite an important increase of circulating CD4+ T-cell counts, IL-2 therapy failed to restore an efficient immune system. In contrast, the first phase I/II trials involving IL-7 treatment in HIV-infected patients demonstrated the efficacy of this molecule that, by targeting T-cell production in the thymus, certainly impacts positively on this parameter.
The results obtained might be helpful in other areas of immunologic research like reproductive immunology and primary immune deficiencies.
The 3ITD project has a significant impact on the development of scientific collaboration in the frame of ERA. The newly by the researcher obtained knowledge and organisation skills will be transferred to her home country, thus improving the present methodological base. The collaboration already established provides a platform for the development of fruitful partnership and successful participation in scientific activities of mutual interest at national and international level, like Bulgarian-French scientific programme RILA, Horizon 2020.
The project is focused on the identification of nTregs, recently emigrated from the thymus (nTreg-RTEs) with respect to their phenotype, function and how do they respond to the immune-based interventions.
During the project execution, a specific experimental design was created aimed at the precise determination of the population of interest. Three areas of investigation have been included. The first one comprises phenotypic determination of nTreg-RTEs by multicolour flow cytometry. To confirm its recent thymic origin throughout signal joint T cell excision circles (sjTRECs), an innovative real-time Polymerase chain reaction (PCR), developed by the host laboratory, was applied. An in vitro suppression assay was developed aimed to investigate function of nTreg-RTEs. Experiments were performed on both freshly isolated peripheral blood mononuclear cell (PBMC)s, as well as frozen cells of 22 healthy controls and frozen PBMCs from 17 patients with HIV-1 infection (9 under therapy and 8 therapy free).
It has been found that the population of nTreg-RTEs (CD31+FOXP3+) is present among the naïve CD4+ T cells in the peripheral blood of healthy subjects, although in a very small proportion. Their frequency in healthy subjects was higher than that, found in both patients groups. In healthy controls the frequency of nTreg-RTEs correlated well with those of CD4+RTEs, thus suggesting their common ontogenesis. The detailed phenotypic analysis revealed that it consists of two fractions, depending on the expression of the CD25 (IL-2 receptor subunit). The measurement of sjTRECs showed that both fractions are equally rich in TRECs, thus confirming their similar thymic proximity. In the control group, the CD25+ nTreg-RTEs predominated, that was not the case in either group of HIV-1 infected individuals. Further, an analysis of the CD127 expression was performed and the results obtained showed that in both healthy controls and Antiretroviral therapy (ART) treated individuals, CD127- cells predominate in nTreg-RTE subsets, thus supporting the fact that they have recently left the thymus, e.g. are RTEs.
It is shown in the literature, that regulatory T cells suppress the proliferation of conventional T cells. In order to completely describe the newly determined population, a suppression assay was performed in vitro with two-steps sorted populations of nTreg-RTEs CD25low and nTreg-RTEs CD25+. The results obtained showed that while CD25+ fraction is fully active without a preactivation, its CD25- counterpart needs to be activated before the expression of its suppressive capacity. Besides, their suppressive function was measured in two different responder cells / nTre-RTEs ratios - 1:6 and 1:32. It was found that a higher ratio results in better inhibition effect.
In conclusion:
- The population of nTreg-RTEs, defined by CD31 expression on FOXP3+ naïve CD4+ T-cells, consists of two subsets characterised by differential expression of CD25. These 2 subsets are mostly CD127-.
- Both fractions are similarly rich in sjTRECs thus suggesting a common proliferative history.
- The increased frequency of FOXP3+ within nCD4-RTEs in HIV-infected individuals suggests that their intrathymic development and/or life cycle in peripheral blood are less affected by the virus in comparison to conventional naïve T-cells.
- An inverse ratio between CD25- and CD25+ cells was found in patients as compared to healthy controls, suggesting that CD25- cells either result from an unsuccessful nTregs development or could be an independent subset nTregs, linked to the development of iTregs.
- The increased proportion of CD127- cells into two CD25 fractions in HIV-infected patients receiving therapy in parallel with the same profile in non-treated subjects, clearly shows the beneficial effect of ART on nTreg-RTEs.
The results obtained are of importance for basic science, clinical practice and scientific collaboration in the frame of European Research Area (ERA).
The contribution to the basic science is toward the better understanding of T-cell maturation process. In one animal study in Simian immunodeficiency virus (SIV) infection it has been already demonstrated that two populations naïve T cells exist in the periphery - CD25low and CD25+. Several studies demonstrated an increase in CD25lowFOXP3+ cells in peripheral blood of patients with autoimmune diseases. The results obtained showed a tendency of impaired function of this population. Animal studies indicated that the acquisition of CD25 is a result of the interaction between immature T cells and medulary epithelieal (mTECs) cells in the thymus. Therefore, it could be suggested, that the appearance of CD25low nTreg-RTEs might result from impaired T cells / mTECs interactions, which is in accordance with the strong negative influence HIV has on thymocytes. Another hypothesis might be that CD25 - Treg-RTEs represent an independent step of normal maturation process. No data exist in the literature about how these populations are represented in lymph nodes. Therefore, the results obtained provide essential clarifying information for this process and might be a platform for further investigations.
Another direction represents the investigation on the role of the thymus in the development of natural tolerance and particularly - autoimmune disorders. The ineffective maturation of the nTreg-RTEs might contribute to the impaired peripheral tolerance.
The impact of nTreg-RTEs in clinical practice consists of their application as a promising target for the development and application of immune-based therapies, aimed at restoring the balance of the immune system in HIV infection. A few years ago, the idea of immune intervention to help patients reconstituting a functional immune system emerged in the scientific community and clinical trials were built to test the efficacy of cytokines based-therapies (interleukin-2 (IL-2) and interleukin-7 (IL-7)) in the context of HIV-infection.
Despite an important increase of circulating CD4+ T-cell counts, IL-2 therapy failed to restore an efficient immune system. In contrast, the first phase I/II trials involving IL-7 treatment in HIV-infected patients demonstrated the efficacy of this molecule that, by targeting T-cell production in the thymus, certainly impacts positively on this parameter.
The results obtained might be helpful in other areas of immunologic research like reproductive immunology and primary immune deficiencies.
The 3ITD project has a significant impact on the development of scientific collaboration in the frame of ERA. The newly by the researcher obtained knowledge and organisation skills will be transferred to her home country, thus improving the present methodological base. The collaboration already established provides a platform for the development of fruitful partnership and successful participation in scientific activities of mutual interest at national and international level, like Bulgarian-French scientific programme RILA, Horizon 2020.
