For understanding the rules and laws of adaptive immunity, high-throughput profiling of T-cell receptor (TCR) repertoires becomes a powerful tool. The structure of TCR repertoires is instructive even before the antigen specificity of each particular receptor becomes available. It embodies information about the thymic and peripheral selection of T cells; the readiness of an adaptive immunity to withstand new challenges; the character, magnitude and memory of immune responses; and the aetiological and functional proximity of T-cell subsets. Here, we describe our current analytical approaches for the comparative analysis of murine TCR repertoires, and show several examples of how these approaches can be applied for particular experimental settings. We analyse the efficiency of different metrics used for estimation of repertoire diversity, repertoire overlap, V-gene and J-gene segments usage similarity, and amino acid composition of CDR3. We discuss basic differences of these metrics and their advantages and limitations in different experimental models, and we provide guidelines for choosing an efficient way to lead a comparative analysis of TCR repertoires. Applied to the various known and newly developed mouse models, such analysis should allow us to disentangle multiple sophisticated puzzles in adaptive immunity.
Immunosuppressive drugs are widely used to treat undesirable immune reaction, however their clinical use is often limited by harmful side effects. The combined application of immunosuppressive agents with mesenchymal stem cells (MSCs) offers a promising alternative approach that enables the reduction of immunosuppressive agent doses and simultaneously maintains or improves the outcome of therapy. The present study aimed to determinate the effects of immunosuppressants on individual T cell subpopulations and to investigate the efficacy of MSC-based treatment combined with immunosuppressive drugs. We tested the effect of five widely used immunosuppressants with different action mechanisms: cyclosporine A, mycophenolate mofetil, rapamycin, and two glucocorticoids - prednisone and dexamethasone in combination with MSCs on mouse CD4+ and CD8+ lymphocyte viability and activation, Th17 (RORγt+), Th1 (T-bet+), Th2 (GATA-3+) and Treg (Foxp3+) cell proportion and on the production of corresponding key cytokines (IL-17, IFNγ, IL-4 and IL-10). We showed that MSCs modulate the actions of immunosuppressants and in combination with immunosuppressive drugs display distinct effect on cell activation and balance among different T lymphocytes subpopulations and exert a suppressive effect on proinflammatory T cell subsets while promoting the functions of anti-inflammatory Treg lymphocytes. The results indicated that MSC-based therapy could be a powerful strategy to attenuate the negative effects of immunosuppressive drugs on the immune system.
- MeSH
- aktivace lymfocytů účinky léků imunologie MeSH
- cyklosporin farmakologie MeSH
- cytokiny metabolismus MeSH
- dexamethason farmakologie MeSH
- glukokortikoidy farmakologie MeSH
- imunosupresiva farmakologie MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- kyselina mykofenolová farmakologie MeSH
- mezenchymální kmenové buňky cytologie imunologie metabolismus MeSH
- myši inbrední BALB C MeSH
- prednison farmakologie MeSH
- proliferace buněk účinky léků MeSH
- průtoková cytometrie MeSH
- sirolimus farmakologie MeSH
- T-lymfocyty - podskupiny cytologie účinky léků imunologie MeSH
- transplantace mezenchymálních kmenových buněk metody MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-β (IL-2Rβ):IL-2Rγ heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2Rα (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2Rβ and IL-2:IL-2Rγ interactions, but also allosterically lowered the IL-2:IL-2Rα affinity through a "triggered exchange" mechanism favoring IL-2Rα(hi) Treg cells, creating a positive feedback loop for IL-2Rα(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2Rα interaction, while also conformationally stabilizing the IL-2:IL-2Rβ interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2Rβ(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies.
- MeSH
- autoimunitní nemoci imunologie MeSH
- interleukin-2 chemie genetika imunologie metabolismus MeSH
- kompetitivní vazba účinky léků MeSH
- lidé MeSH
- molekulární modely * MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- progrese nemoci MeSH
- proliferace buněk účinky léků MeSH
- protilátky chemie imunologie farmakologie MeSH
- průtoková cytometrie MeSH
- regulace genové exprese imunologie MeSH
- regulační T-lymfocyty cytologie účinky léků imunologie MeSH
- signální transdukce účinky léků MeSH
- T-lymfocyty - podskupiny cytologie účinky léků imunologie MeSH
- terciární struktura proteinů MeSH
- vazba proteinů účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Porcine γδ T cells have two levels of TCRγδ expression. Whereas TCRγδ(med) cells are mostly CD2(+)CD8(-) and CD2(+)CD8(+), TCRγδ(hi) cells are highly enriched for CD2(-)CD8(-). This distribution is independent of bacterial colonization and it is already established in the thymus prior to export of γδ cells to the periphery. Sorting and cultivation experiments revealed that CD2(-)CD8(-) γδ cells are unable to acquire CD2 and CD8, whereas CD2(+) subsets can gain or loose CD8. There is also differential susceptibility for proliferation between CD2(+) and CD2(-) γδ cells. Although CD2(-)CD8(-) almost do not proliferate, proliferation of CD2(+)CD8(-) and CD2(+)CD8(+) is substantial. Population of CD2(-) γδ cells is also absent in CD1(+) immature thymocytes. Additionally, subpopulations of CD2(+) and CD2(-) γδ cells in the thymus differ in expression of auxiliary surface molecules such as CD25, CD45RA/RC, and MHC class II. Moreover, TCRγδ(hi) cells can generate TCRγδ(med) cells but never the opposite. The only exception is the thymus, where a few TCRγδ(med) cells can be induced to TCRγδ(hi) but only under IL-2 influence. The repertoire of TCRδ is polyclonal in all subsets, indicating that there is the same extent of diversification and equal capability of immune responses. Results collectively indicate that CD2 expression determines two lineages of γδ cells that differ in many aspects. Because CD2(-) γδ cells are missing in the blood of humans and mice but are obvious in other members of γδ-high species such as ruminants and birds, our findings support the idea that circulating CD2(-) γδ T cells are a specific lineage.
- MeSH
- antigeny CD1 genetika imunologie MeSH
- antigeny CD2 genetika imunologie MeSH
- antigeny CD8 genetika imunologie MeSH
- buněčná diferenciace imunologie MeSH
- buněčný rodokmen imunologie MeSH
- exprese genu MeSH
- interleukin-2 imunologie MeSH
- lidé MeSH
- myši MeSH
- prasata MeSH
- proliferace buněk MeSH
- receptory antigenů T-buněk gama-delta genetika imunologie MeSH
- T-lymfocyty - podskupiny cytologie imunologie MeSH
- T-lymfocyty cytologie imunologie MeSH
- thymocyty cytologie imunologie MeSH
- thymus cytologie imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The expression of selected molecules was chosen to study porcine γδ lymphocytes and their CD2/CD8 subsets in different lymphoid organs in vivo and in vitro. Results indicate that many γδ T cells can constitutively express CD25 and MHC-II and that the frequency of γδ T cells positive for CD25, CD11b, SWC1 and SWC7 can be increased by stimulation. A diversified TCRδ repertoire was found inside CD25(+), CD11b(+), SWC1(-) and CD45RA(-) cells. Ontogenetic studies revealed various age and/or colonization dependency for expression of all studied molecules except of SWC7. Findings generally indicate that CD25 represent an activation molecule that probably marks a functionally distinct subsets, expression of CD11b is perhaps connected to early functions of naive γδ T cells in the periphery, SWC1 is lineage specific marker, SWC7 may represent an activation molecule with intrinsic or transient expression, and the expression of CD45RA/RC most likely defines naive and terminally differentiated cells.
- MeSH
- antigeny CD45 metabolismus MeSH
- CD antigeny metabolismus MeSH
- prasata imunologie MeSH
- receptor interleukinu-2 - alfa-podjednotka metabolismus MeSH
- T-lymfocyty - podskupiny cytologie imunologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Antiviral prophylaxis against cytomegalovirus has been associated with reduced risk of allograft rejection and improved allograft survival after renal transplantation. This phenomenon might not be fully explained by preventing the indirect effects of cytomegalovirus. The effect of antiviral agents on lymphocyte function in patients treated with modern immunosuppression has not been studied to date. METHODS: Adult renal transplant recipients were assigned to 3-month prophylaxis with either valganciclovir (900 mg once daily; n=19) or valacyclovir (2 g four times daily; n=17) as part of an ongoing randomized trial. Subsets of lymphocytes, lymphocyte proliferation and/or cytokine production after in vitro mitogen stimulation were evaluated at the end of prophylaxis and 1 month after withdrawal of antiviral drugs. RESULTS: Lymphocyte proliferation was significantly decreased both after phytohemagglutinine (25% ±15% versus 32% ±18%; P=0.025) and concanavalin A stimulation (17% ±9% versus 25% ±16%; P=0.011) during valganciclovir, but not valacyclovir therapy. Moreover, a lower activated T-cell count (CD3(+)HLA-DR(+) cells) was noted in valganciclovir-treated patients (13% ±10% versus 17% ±12% of total CD3(+) T-cells; P=0.005). CONCLUSIONS: Valganciclovir suppresses lymphocyte proliferation and activation in patients after renal transplantation.
- MeSH
- acyklovir aplikace a dávkování analogy a deriváty terapeutické užití MeSH
- aktivace lymfocytů účinky léků MeSH
- antivirové látky aplikace a dávkování terapeutické užití MeSH
- cytomegalovirové infekce farmakoterapie etiologie prevence a kontrola virologie MeSH
- Cytomegalovirus účinky léků imunologie MeSH
- dospělí MeSH
- fytohemaglutininy farmakologie MeSH
- ganciklovir aplikace a dávkování analogy a deriváty terapeutické užití MeSH
- imunosupresiva aplikace a dávkování škodlivé účinky MeSH
- imunosupresivní léčba škodlivé účinky MeSH
- konkanavalin A farmakologie MeSH
- kultivované buňky MeSH
- lidé středního věku MeSH
- lidé MeSH
- počet lymfocytů MeSH
- přežívání štěpu účinky léků MeSH
- proliferace buněk účinky léků MeSH
- rejekce štěpu prevence a kontrola MeSH
- T-lymfocyty - podskupiny cytologie účinky léků imunologie MeSH
- transplantace ledvin imunologie MeSH
- valin aplikace a dávkování analogy a deriváty terapeutické užití MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Type 1 diabetes mellitus (T1D) and multiple sclerosis (MS) are organ-specific autoimmune diseases leading to an attack of auto-aggressive lymphocytes against the pancreatic beta-cells and central nervous system, respectively. Using four-colour flow cytometry, T-lymphocyte populations having an important function in autoimmune processes were analyzed. T-regulatory cells (Treg) CD4(+)CD25(+)CD127(low), T-suppressor cells (Ts) CD8(+)CD28(-), activated helper CD4(+)CD25(+)CD127(+) and cytotoxic CD8(+)CD25(+) T-cells and also naive CD4(+)CD45RA(+) and memory T-cells CD4(+)CD45RO(+) were compared in the group of patients with T1D (n=30), MS (n=31) and in the group of healthy controls (n=29). Significant differences in Ts cells, activated helper and cytotoxic cells and also memory T-cells were recognized in the group of T1D patients compared to healthy controls. Ts population was significantly lowered in MS patients as well. However, no significant differences were noticed in Treg population. The observed data demonstrate significant differences among patients with T1D and MS in comparison to healthy individuals.
- MeSH
- antigeny CD28 imunologie MeSH
- CD8-pozitivní T-lymfocyty cytologie imunologie MeSH
- diabetes mellitus 1. typu imunologie MeSH
- dospělí MeSH
- imunologická paměť imunologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- počet lymfocytů MeSH
- průtoková cytometrie metody MeSH
- relabující-remitující roztroušená skleróza imunologie MeSH
- T-lymfocyty - podskupiny cytologie imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The developing porcine fetus offers an excellent opportunity for the study of lymphocyte development. Studies on B cell, alphabeta T cells and gammadelta T cells in the last decade have expanded our knowledge of lymphocyte development in pigs. These studies have revealed several interesting differences between swine, mice and humans. For example, porcine peripheral lymphocytes include CD4+CD8+ alphabeta T cells and an abundance of gammadelta T cells that may even prevail over the alphabeta population. There are numerous CD2- gammadelta T cells in the blood and a large number of CD8alphaalpha-bearing cells that include NK cells, conventional gammadelta and alphabeta T cells. All porcine B lymphocytes are CD25(lo) and sIgM+ B cells may differ in the expression of CD2 antigen. Unlike mice, porcine B cells appear approximately 2 weeks before T cells and progenitors undergo VDJH rearrangement at 20th day of gestation (DG20) in the yolk sac and DG30 in the fetal liver before consummating high level lymphogenesis in the bone marrow after DG45. Early B cells show an unexpectedly high proportion of in-frame rearrangements, undergo switch recombination in thymus on DG60 and use N-region insertion from the time of the earliest VDJ rearrangement. The genomic repertoire of VH, DH and JH genes is small compared to mice and humans and swine appear to depend on junctional diversity for the majority of their repertoire. The limited VH repertoire of swine contrasts sharply with the porcine TCRbeta repertoire, which is extensive, extraordinarily conserved and nearly identical to that in humans. Therefore, swine present an example of two highly related receptor systems that have diverged in the same species.
- MeSH
- B-lymfocyty cytologie imunologie MeSH
- buněčná diferenciace MeSH
- financování organizované MeSH
- lidé MeSH
- lymfocyty cytologie imunologie MeSH
- lymfopoéza MeSH
- myši MeSH
- prasata embryologie imunologie MeSH
- T-lymfocyty - podskupiny cytologie imunologie MeSH
- těhotenství MeSH
- vývoj plodu imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
