Multi-omics analysis of innate and adaptive responses to BCG vaccination reveals epigenetic cell states that predict trained immunity.

Immune responses are tightly regulated yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live-attenuated vaccine induces not only an adaptive immune response against tuberculosis but also triggers innate immune activation and memory that are indicative of trained immunity. We established personal immune profiles and chromatin accessibility maps over a 90-day time course of BCG vaccination in 323 individuals. Our analysis uncovered genetic and epigenetic predictors of baseline immunity and immune response. BCG vaccination enhanced the innate immune response specifically in individuals with a dormant immune state at baseline, rather than providing a general boost of innate immunity. This study advances our understanding of BCG's heterologous immune-stimulatory effects and trained immunity in humans. Furthermore, it highlights the value of epigenetic cell states for connecting immune function with genotype and the environment.

Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients.

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Blood immune cell profiling in adults with longstanding type 1 diabetes is associated with macrovascular complications.

Aims/hypothesis: There is increasing evidence for heterogeneity in type 1 diabetes mellitus (T1D): not only the age of onset and disease progression rate differ, but also the risk of complications varies markedly. Consequently, the presence of different disease endotypes has been suggested. Impaired T and B cell responses have been established in newly diagnosed diabetes patients. We hypothesized that deciphering the immune cell profile in peripheral blood of adults with longstanding T1D may help to understand disease heterogeneity. Methods: Adult patients with longstanding T1D and healthy controls (HC) were recruited, and their blood immune cell profile was determined using multicolour flow cytometry followed by a machine-learning based elastic-net (EN) classification model. Hierarchical clustering was performed to identify patient-specific immune cell profiles. Results were compared to those obtained in matched healthy control subjects. Results: Hierarchical clustering analysis of flow cytometry data revealed three immune cell composition-based distinct subgroups of individuals: HCs, T1D-group-A and T1D-group-B. In general, T1D patients, as compared to healthy controls, showed a more active immune profile as demonstrated by a higher percentage and absolute number of neutrophils, monocytes, total B cells and activated CD4+CD25+ T cells, while the abundance of regulatory T cells (Treg) was reduced. Patients belonging to T1D-group-A, as compared to T1D-group-B, revealed a more proinflammatory phenotype characterized by a lower percentage of FOXP3+ Treg, higher proportions of CCR4 expressing CD4 and CD8 T cell subsets, monocyte subsets, a lower Treg/conventional Tcell (Tconv) ratio, an increased proinflammatory cytokine (TNFα, IFNγ) and a decreased anti-inflammatory (IL-10) producing potential. Clinically, patients in T1D-group-A had more frequent diabetes-related macrovascular complications. Conclusions: Machine-learning based classification of multiparameter flow cytometry data revealed two distinct immunological profiles in adults with longstanding type 1 diabetes; T1D-group-A and T1D-group-B. T1D-group-A is characterized by a stronger pro-inflammatory profile and is associated with a higher rate of diabetes-related (macro)vascular complications.

Alendronate modulates cytokine responses in healthy young individuals after BCG vaccination.

Bacillus Calmette-Guérin (BCG) vaccination induces memory characteristics in innate immune cells and their progenitors, a process called trained immunity mediated by epigenetic and metabolic reprogramming. Cholesterol synthesis plays an amplifying role in trained immunity through mevalonate release. Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, can inhibit cholesterol synthesis. We explored their effects on trained immunity induced by BCG in a placebo-controlled clinical study (NL74082.091.20) in young, healthy individuals. Participants receiving single-dose oral alendronate on the day of BCG vaccination had more neutrophils and plasma cells one month after treatment. Alendronate led to reduced proinflammatory cytokine production by PBMCs stimulated with heterologous bacterial and viral stimuli one month later. Furthermore, the addition of alendronate transcriptionally suppressed multiple immune response pathways in PBMCs upon stimulation. Our findings indicate that N-BPs modulate the long-lasting effects of BCG vaccination on the cytokine production capacity of innate immune cells.

Mature neutrophils and a NF-κB-to-IFN transition determine the unifying disease recovery dynamics in COVID-19.

Disease recovery dynamics are often difficult to assess, as patients display heterogeneous recovery courses. To model recovery dynamics, exemplified by severe COVID-19, we apply a computational scheme on longitudinally sampled blood transcriptomes, generating recovery states, which we then link to cellular and molecular mechanisms, presenting a framework for studying the kinetics of recovery compared with non-recovery over time and long-term effects of the disease. Specifically, a decrease in mature neutrophils is the strongest cellular effect during recovery, with direct implications on disease outcome. Furthermore, we present strong indications for global regulatory changes in gene programs, decoupled from cell compositional changes, including an early rise in T cell activation and differentiation, resulting in immune rebalancing between interferon and NF-κB activity and restoration of cell homeostasis. Overall, we present a clinically relevant computational framework for modeling disease recovery, paving the way for future studies of the recovery dynamics in other diseases and tissues.

Trio-based whole exome sequencing in patients with suspected sporadic inborn errors of immunity: A retrospective cohort study.

Background: De novo variants (DNVs) are currently not routinely evaluated as part of diagnostic whole exome sequencing (WES) analysis in patients with suspected inborn errors of immunity (IEI). Methods: This study explored the potential added value of systematic assessment of DNVs in a retrospective cohort of 123 patients with a suspected sporadic IEI that underwent patient-parent trio-based WES. Results: A (likely) molecular diagnosis for (part) of the immunological phenotype was achieved in 12 patients with the diagnostic in silico IEI WES gene panel. Systematic evaluation of rare, non-synonymous DNVs in coding or splice site regions led to the identification of 14 candidate DNVs in genes with an annotated immune function. DNVs were found in IEI genes (NLRP3 and RELA) and in potentially novel candidate genes, including PSMB10, DDX1, KMT2C, and FBXW11. The FBXW11 canonical splice site DNV was shown to lead to defective RNA splicing, increased NF-κB p65 signalling, and elevated IL-1ß production in primary immune cells extracted from the patient with autoinflammatory disease. Conclusions: Our findings in this retrospective cohort study advocate the implementation of trio-based sequencing in routine diagnostics of patients with sporadic IEI. Furthermore, we provide functional evidence supporting a causal role for FBXW11 loss-of-function mutations in autoinflammatory disease. Funding: This research was supported by grants from the European Union, ZonMW and the Radboud Institute for Molecular Life Sciences.

The Architecture of Circulating Immune Cells Is Dysregulated in People Living With HIV on Long Term Antiretroviral Treatment and Relates With Markers of the HIV-1 Reservoir, Cytomegalovirus, and Microbial Translocation.

Long-term changes in the immune system of successfully treated people living with HIV (PLHIV) remain incompletely understood. In this study, we assessed 108 white blood cell (WBC) populations in a cohort of 211 PLHIV on stable antiretroviral therapy and in 56 HIV-uninfected controls using flow cytometry. We show that marked differences exist in T cell maturation and differentiation between PLHIV and HIV-uninfected controls: PLHIV had reduced percentages of CD4+ T cells and naïve T cells and increased percentages of CD8+ T cells, effector T cells, and T helper 17 (Th17) cells, together with increased Th17/regulatory T cell (Treg) ratios. PLHIV also exhibited altered B cell maturation with reduced percentages of memory B cells and increased numbers of plasmablasts. Determinants of the T and B cell composition in PLHIV included host factors (age, sex, and smoking), markers of the HIV reservoir, and CMV serostatus. Moreover, higher circulating Th17 percentages were associated with higher plasma concentrations of interleukin (IL) 6, soluble CD14, the gut homing chemokine CCL20, and intestinal fatty acid binding protein (IFABP). The changes in circulating lymphocytes translated into functional changes with reduced interferon (IFN)- γ responses of peripheral blood mononuclear cells to stimulation with Candida albicans and Mycobacterium tuberculosis. In conclusion, this comprehensive analysis confirms the importance of persistent abnormalities in the number and function of circulating immune cells in PLHIV on stable treatment.

Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells.

The effector T-cell lineage shows great plasticity. Th17 cells are acknowledged to be instrumental in the response against microbial infection, but are also associated with autoimmune inflammatory processes. Here, we report that human regulatory T cells (CD4(pos)CD25(high)Foxp3(pos)CD127(neg)CD27(pos)) can differentiate into IL-17-producing cells, when stimulated by allogeneic antigen-presenting cells, especially monocytes, in the presence of rhIL-2/rhIL-15. These regulatory T cell (Treg)-derived IL-17-producing cells showed high expression of the Th17-related transcription factor RORgammat and were positively identified by CCR6 expression. This differentiation process was enhanced by exogenous IL-1beta, IL-23, and IL-21, whereas IL-6 or TGFbeta did not affect the emergence of IL-17-producing cells. The addition of IL-1 receptor antagonist (IL-1Ra), but not anti-IL-23 antibody, reduced IL-17-producing cell numbers. When an histone deacetylase (HDAC) inhibitor trichostatin A (TSA) was evaluated, we found a profound negative effect on the emergence of IL-17-producing cells from Tregs, implying that Treg differentiation into IL-17-producing cells depends on histone/protein deacetylase activity. Thus, the data suggest that epigenetic modification underlies the phenomenon of Treg plasticity here described.

Increased dermal expression of chromatin-associated protein HMGB1 and concomitant T-cell expression of the DNA RAGE in patients with psoriasis vulgaris.

PURPOSE: Psoriasis vulgaris (PV) is an autoimmune-related chronic inflammatory disease of the skin, with both vascular and metabolic effects. Aggravating factors have been identified that initiate and maintain inflammation, including expression of Th1-, Th17-, and Th22-cell derived cytokines. Recently, we showed that the evolutionarily ancient and highly conserved damage-associated molecular pattern molecule "high mobility group box 1 (HMGB1)" is significantly increased in the serum of PV patients with disease progression and is decreased under standard therapies. MATERIALS AND METHODS: To better understand the role of HMGB1 in the pathogenesis of PV, we recruited 22 untreated psoriatic patients with either mild or severe disease, defined by the Psoriasis Area Severity Index. We assessed HMGB1 and receptor for advanced glycation end products (RAGE) expression in the skin by immunohistochemistry and analyzed the immune-phenotype of Treg and Th17 cells by flow cytometry. RESULTS: We found increased staining for HMGB1 in the dermis of psoriatic plaques in comparison to uninvolved skin of patients with PV. In addition, the major histocompatibility complex class III-encoded DNA and HMGB1 RAGE, induced by HMGB1, were highly expressed on psoriatic CD8+ T cells and CD4+ Treg. High expression of HMGB1 in the lesional skin was associated with even higher expression of its receptor, RAGE, on the cell surface of keratino-cytes in patients with severe PV. CONCLUSION: The presence of HMGB1 and RAGE signaling may impact orchestration of chronic inflammation in PV which might have implications for Treg and Th17 cells.

Long-Term Effects of Experimental Human Endotoxemia on Immune Cell Function: Similarities and Differences With Sepsis.

Sepsis is the cause of more than 5.3 million deaths per year, and novel immunotherapeutic strategies are highly warranted. Human models that mirror sepsis immunology are instrumental to this aim. The response to endotoxin in humans during the first 24 h captures many hallmarks of the inflammatory response observed in sepsis. However, the long-term immunologic effects of human experimental endotoxemia have been sparsely studied and could be determinant for the use of this model in sepsis therapy research. In the present work, we studied the immune-composition of healthy subjects challenged with endotoxin (1 ng/kg) 4 h, 2 days, and 20 days post administration by flow cytometry to study the effects on innate and adaptive immune system, and compared it with the immune-composition in patients during the first 9 days after onset of septic shock. We found several differences and similarities between these groups. Experimental endotoxemia resulted in an increase in absolute numbers of intermediate monocytes, which also displayed lower human leucocyte antigen expression 20 days post endotoxin. These changes differed with those observed in septic shock patients. Another long-term effect of experimental endotoxemia was elevated numbers of effector CD8 cells and an increased percentage of proliferating and cytokine expressing CD8 cells, and these phenomena were also present in sepsis patients. In conclusion, despite considerable differences, experimental endotoxemia captures several long-term aspects of sepsis immunology, specifically the behavior of CD8 T cells, which may eventually aid the development of new therapies for sepsis patients.

DNA Methyltransferase Inhibition Promotes Th1 Polarization in Human CD4+CD25high FOXP3+ Regulatory T Cells but Does Not Affect Their Suppressive Capacity.

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2'-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4+CD25high FOXP3+ Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFNγ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFNγ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.

Allogeneic stimulation of naturally occurring CD4+CD25+ T cells induces strong regulatory capacity with increased donor-reactivity.

Current therapies in transplantation require continuous immunosuppression and do not result in transplantation tolerance. It is increasingly appreciated that CD4(+)CD25(+) regulatory T-cell (T(REG)) activation is pivotal for the induction and maintenance of peripheral tolerance. To optimally exploit T(REG) in allograft tolerance, we investigated how to further harness their function. In vitro, CD4(+)CD25(+)T cells were expanded by allogeneic bone-marrow derived DC or polyclonal stimulation and were compared in suppressive capacity and phenotype. In vivo, naive allogeneic CD4(+)CD25(+)T cells were analyzed in wild type hosts for proliferative capacity and suppressive capacity upon priming by alloantigen. DC of donor origin were found to potently stimulate alloreactive T(REG)in vitro. This was accompanied by a substantial enhancement of the suppressive capacity of the T(REG) population as a whole, likely due to a proportional rise of alloreactive T(REG) as indicated by CFSE analysis. In vivo analysis of infused naturally occurring allogeneic T(REG) revealed a robust proliferative capacity for T(REG) upon stimulation. Moreover, allogeneic skin transplantation resulted in enhanced capacity of the T(REG) population to suppress the response towards donor antigens. Combining, activation of alloreactive T(REG) is an intrinsic part of the regular alloimmune response and this feature can be exploited for therapeutic purposes. We propose that selectively favoring the effects of alloreactive T(REG) is a pivotal element in inducing graft acceptance.

Single CD28 stimulation induces stable and polyclonal expansion of human regulatory T cells.

CD4+FOXP3+ Treg are essential for immune tolerance. Phase-1 clinical trials of Treg-therapy to treat graft-versus-host-disease reported safety and potential therapeutic efficacy. Treg-based trials have started in organ-transplant patients. However, efficient ex vivo expansion of a stable Treg population remains a challenge and exploring novel ways for Treg expansion is a pre-requisite for successful immunotherapy. Based on the recent finding that CD28-signaling is crucial for survival and proliferation of mouse Treg, we studied single-CD28 stimulation of human Treg, without T cell receptor stimulation. Single-CD28 stimulation of human Treg in the presence of recombinant human IL-2(rhIL-2), as compared to CD3/CD28/rhIL-2 stimulation, led to higher expression levels of FOXP3. Although the single-CD28 expanded Treg population was equally suppressive to CD3/CD28 expanded Treg, pro-inflammatory cytokine (IL-17A/IFNγ) production was strongly inhibited, indicating that single-CD28 stimulation promotes Treg stability. As single-CD28 stimulation led to limited expansion rates, we examined a CD28-superagonist antibody and demonstrate a significant increased Treg expansion that was more efficient than standard anti-CD3/CD28-bead stimulation. CD28-superagonist stimulation drove both naïve and memory Treg proliferation. CD28-superagonist induction of stable Treg appeared both PI3K and mTOR dependent. Regarding efficient and stable expansion of Treg for adoptive Treg-based immunotherapy, application of CD28-superagonist stimulation is of interest.

Differential Effects of Environmental and Genetic Factors on T and B Cell Immune Traits.

Effective immunity requires a complex network of cellular and humoral components that interact with each other and are influenced by different environmental and host factors. We used a systems biology approach to comprehensively assess the impact of environmental and genetic factors on immune cell populations in peripheral blood, including associations with immunoglobulin concentrations, from ∼500 healthy volunteers from the Human Functional Genomics Project. Genetic heritability estimation showed that variations in T cell numbers are more strongly driven by genetic factors, while B cell counts are more environmentally influenced. Quantitative trait loci (QTL) mapping identified eight independent genomic loci associated with leukocyte count variation, including four associations with T and B cell subtypes. The QTLs identified were enriched among genome-wide association study (GWAS) SNPs reported to increase susceptibility to immune-mediated diseases. Our systems approach provides insights into cellular and humoral immune trait variability in humans.

Tolerizing effects of co-stimulation blockade rest on functional dominance of CD4+CD25+ regulatory T cells.

BACKGROUND: Clinical tolerance is the net result of regulatory and effector functions. In this article, the authors show that tolerance induction by co-stimulation blockade preferentially works through CD4CD25 regulatory T-cell-mediated suppression that is effectively achieved by selective reduction of the effector T-cell load. Anti-CD86 and anti-CD40L monoclonal antibody treatment during in vitro mixed lymphocyte reaction (MLR) typically results in the induction of a suppressive polyclonal T-cell population. This induced suppressive capacity was found to be dependent on the presence of CD4CD25 T cells at the start of MLR. METHODS: Using a CFSE-based strategy, the authors show that within the polyclonal T-cell population, the suppressive effect was exerted by a nondividing CD4CD25 T-cell subset. RESULTS: The cells exclusively originated from preexisting CD4CD25 regulatory T cells and proved anergic and highly suppressive on isolation. They carried the CD45RB and CD62L phenotype and expressed GITR. There was no indication of de novo induction of regulatory T cells by co-stimulation blockers. Instead, the authors observed, both in vitro and in vivo, that co-stimulation blockade shifted the ratio between alloreactive effectors and regulatory T cells in favor of the latter. CONCLUSION: The authors therefore conclude that co-stimulation blockade contributes to functional dominance of regulatory T cells by preventing expansion of alloreactive effector T cells. Tolerance-inducing protocols should ideally facilitate this phenomenon.

Targeting PKC in human T cells using sotrastaurin (AEB071) preserves regulatory T cells and prevents IL-17 production.

Regulatory T-cells (Treg) are crucial for immune homeostasis and prevention of immune pathology. Yet, Treg may lose Foxp3 and start secreting IL-17, dependent on environmental cues. Our previous data revealed that Treg from severe psoriasis patients are particularly prone to such conversion. The question of how to maintain Treg stability in the context of inflammation awaits immediate resolution. The pan-protein kinase C (PKC) inhibitor sotrastaurin has shown efficacy in clinical trials of psoriasis. Here, we show that sotrastaurin inhibited effector T-cell responses, whereas the regulatory response was enhanced. Sotrastaurin prevented TCR/CD28-induced T-cell activation and pro-inflammatory cytokine production, but preserved a stable Treg phenotype as evidenced by maintenance of suppressive capacity, high Foxp3 and CD25 expression, and lack of IL-17A and IFNγ production. Moreover, in both circulating and dermal psoriatic Treg, prone to rapid induction of IL-17, sotrastaurin enhanced Foxp3 expression and prevented IL-17A and IFNγ production even when stimulated in the presence of the helper T 17-enhancing cytokines IL-1ß or IL-23. Thus, pharmacological inhibition of PKC may serve as a powerful tool to concurrently inhibit effector T cells and to facilitate Treg, thereby showing therapeutic potential for the treatment of psoriasis.

Crosstalk between keratinocytes and T cells in a 3D microenvironment: a model to study inflammatory skin diseases.

The interaction between keratinocytes and immune cells plays a major role in the development of inflammatory skin diseases like psoriasis and atopic dermatitis. Pharmacological intervention to inhibit T cell-derived proinflammatory mediators is an effective therapy in the treatment of psoriasis. Here, we present a model to study the interaction between keratinocytes and T cells in a three-dimensional (3D) microenvironment, based on human skin equivalents populated with CD4+ T cells. T cell migration into the dermis initiated keratinocyte activation within 2 days, with hallmarks of a psoriasiform inflammation after 4 days. Expression of epidermal psoriasis marker genes was upregulated, and proinflammatory cytokines and chemokines were highly expressed. Disturbed epidermal differentiation was shown by downregulated filaggrin expression and involucrin expression in the spinous layer. These effects were mediated via soluble factors produced by the T cells. The psoriasiform inflammation was also observed using T helper type 1 (Th1)- and Th17-polarized CD4+ T cells. We validated our model by treatment with anti-inflammatory drugs that reduced the expression of proinflammatory cytokines and chemokines and suppressed the psoriasiform inflammation. We propose that our T cell-driven inflammatory skin equivalent model has potential to study the pathogenesis of inflammatory skin diseases and may serve as a preclinical screening tool for anti-inflammatory drugs.

Immunological monitoring of renal transplant recipients to predict acute allograft rejection following the discontinuation of tacrolimus.

BACKGROUND: Transplant patients would benefit from reduction of immunosuppression providing that graft rejection is prevented. We have evaluated a number of immunological markers in blood of patients in whom tacrolimus was withdrawn after renal transplantation. The alloreactive precursor frequency of CD4+ and CD8+ T cells, the frequency of T cell subsets and the functional capacity of CD4+CD25+FoxP3+ regulatory T cells (Treg) were analyzed before transplantation and before tacrolimus reduction. In a case-control design, the results were compared between patients with (n = 15) and without (n = 28) acute rejection after tacrolimus withdrawal. PRINCIPAL FINDINGS: Prior to tacrolimus reduction, the ratio between memory CD8+ T cells and Treg was higher in rejectors compared to non-rejectors. Rejectors also had a higher ratio between memory CD4+ T cells and Treg, and ratios <20 were only observed in non-rejectors. Between the time of transplantation and the start of tacrolimus withdrawal, an increase in naive T cell frequencies and a reciprocal decrease of effector T cell percentages was observed in rejectors. The proportion of Treg within the CD4+ T cells decreased after transplantation, but anti-donor regulatory capacity of Treg remained unaltered in rejectors and non-rejectors. CONCLUSIONS: Immunological monitoring revealed an association between acute rejection following the withdrawal of tacrolimus and 1) the ratio of memory T cells and Treg prior to the start of tacrolimus reduction, and 2) changes in the distribution of naive, effector and memory T cells over time. Combination of these two biomarkers allowed highly specific identification of patients in whom immunosuppression could be safely reduced.

Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27+ subset of human CD4+CD25+ regulatory T cells.

The immunosuppressive drugs rapamycin and cyclosporin A (CsA) are widely used to prevent allograft rejection. Moreover, they were shown to be instrumental in experimental models of tolerance induction. However, it remains to be elucidated whether these drugs have an effect on the CD4+ CD25+ regulatory T-cell (T(REG)) population, which plays an important role in allograft tolerance. Recently, we reported that alloantigen-driven expansion of human CD4+ CD25+ T(REG)s gives rise to a distinct highly suppressive CD27+ T(REG) subset next to a moderately suppressive CD27- T(REG) subset. In the current study we found that rapamycin and CsA do not interfere with the suppressive activity of human naturally occurring CD4+ CD25+ T cells. However, in contrast to CsA, rapamycin preserved the dominance of the potent CD27+ T(REG) subset over the CD27- T(REG) subset after alloantigen-driven expansion of CD4+ CD25+ T(REG)s in vitro. Accordingly, CD4+ CD25+ T(REG)s cultured in the presence of rapamycin displayed much stronger suppressive capacity than CD4+ CD25+ T(REG)s cultured in the presence of CsA. In addition, CD4+ CD25+ T(REG) cells cultured in the presence of rapamycin, but not CsA, were able to suppress ongoing alloimmune responses. This differential effect of rapamycin and CsA on the CD27+ T(REG) subset dominance may favor the use of rapamycin in tolerance-inducing strategies.

CTLA-4 engagement and regulatory CD4+CD25+ T cells independently control CD8+-mediated responses under costimulation blockade.

Blockade of costimulatory signals is a promising therapeutic target to prevent allograft rejection. In this study, we sought to characterize to what extent CTLA-4 engagement contributes to the development of transplantation tolerance under the cover of CD40/CD40L and CD28/CD86 blockade. In vitro, we found that inhibition of the primary alloresponse and induction of alloantigen hyporesponsiveness by costimulation blockade was abrogated by anti-CTLA-4 mAb. In addition, regulatory CD4(+)CD25(+) T cells (T(REG)) were confirmed to play a critical role in the induction of hyporesponsiveness by anti-CD40L and anti-CD86 mAb. Our data indicated that CTLA-4 engagement is not required for activation or suppressor function of T(REG). Instead, in the absence of either CTLA-4 signaling or T(REG), CD8(+) T cell division was enhanced, whereas the inhibition of CD4(+) T cell division by costimulation blockade remained largely unaffected. In vivo, the administration of additional anti-CTLA-4 mAb abrogated anti-CD40L- and anti-CD86 mAb-induced cardiac allograft survival. Correspondingly, rejection was accompanied by enhanced allograft infiltration of CD8(+) cells. We conclude that CTLA-4 signaling and T(REG) independently cooperate in the inhibition of CD8(+) T cell expansion under costimulation blockade.