Search

Hemophilia A (HA) is a blood clotting disorder that is caused by various genetic deficiencies in the factor VIII (FVIII)-encoding F8gene. Patients receiving FVIII-replacement therapy are at risk for developing neutralizing antibodies (FVIII inhibitors), rendering the FVIII-replacement therapy ineffective. Immunological tolerance toward FVIII can be achieved through immune tolerance induction protocols in some patients, but this is a lengthy and costly desensitization program. Long-term eradication of inhibitors in patients with HA could be achieved by antigen-specific immunotherapy targeting CD4+T-cells, because formation of FVIII inhibitors is T-cell dependent. Here, we report a peptide-based antigen-specific immunotherapy that is designed to specifically reestablish immune tolerance to FVIII through the development of antigen-processing-independent epitopes (apitopes). We identified 2 FVIII immunodominant peptides in immunized HLA-DRA*0101/DRB1*1501 transgenic (HLA-DR2tg) mice that were optimized for tolerogenicity. These modified peptide analogs were initially screened for recognition using FVIII-specific T-cell hybridoma clones from FVIII-immunized HLA-DR2tg mice. The FVIII apitopes were promiscuous and bound common human HLA-DRB1* allelic variants. The combination of these 2 FVIII apitopes (ATX-F8-117), administered according to a dose-escalation protocol, promoted T-cell tolerance toward FVIII in HLA-DR2tg mice. Furthermore, treatment with ATX-F8-117 significantly reduced FVIII inhibitor formation. ATX-F8-117 regulates anti-FVIII T-cell and B-cell responses, specifically the generation of FVIII inhibitors, revealing peptide-based antigen-specific immunotherapy as a promising approach to suppress and treat inhibitor formation in susceptible patients with HA.

Hemophilia A (HA) is a blood clotting disorder that is caused by various genetic deficiencies in the factor VIII (FVIII)-encoding F8 gene. Patients receiving FVIII-replacement therapy are at risk for developing neutralizing antibodies (FVIII inhibitors), rendering the FVIII-replacement therapy ineffective. Immunological tolerance toward FVIII can be achieved through immune tolerance induction protocols in some patients, but this is a lengthy and costly desensitization program. Long-term eradication of inhibitors in patients with HA could be achieved by antigen-specific immunotherapy targeting CD4+ T-cells, because formation of FVIII inhibitors is T-cell dependent. Here, we report a peptide-based antigen-specific immunotherapy that is designed to specifically reestablish immune tolerance to FVIII through the development of antigen-processing-independent epitopes (apitopes). We identified 2 FVIII immunodominant peptides in immunized HLA-DRA*0101/DRB1*1501 transgenic (HLA-DR2tg) mice that were optimized for tolerogenicity. These modified peptide analogs were initially screened for recognition using FVIII-specific T-cell hybridoma clones from FVIII-immunized HLA-DR2tg mice. The FVIII apitopes were promiscuous and bound common human HLA-DRB1* allelic variants. The combination of these 2 FVIII apitopes (ATX-F8-117), administered according to a dose-escalation protocol, promoted T-cell tolerance toward FVIII in HLA-DR2tg mice. Furthermore, treatment with ATX-F8-117 significantly reduced FVIII inhibitor formation. ATX-F8-117 regulates anti-FVIII T-cell and B-cell responses, specifically the generation of FVIII inhibitors, revealing peptide-based antigen-specific immunotherapy as a promising approach to suppress and treat inhibitor formation in susceptible patients with HA.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with a range of persistent symptoms impacting everyday functioning, known as post-COVID-19 condition or long COVID. We undertook a retrospective matched cohort study using a UK-based primary care database, Clinical Practice Research Datalink Aurum, to determine symptoms that are associated with confirmed SARS-CoV-2 infection beyond 12 weeks in non-hospitalized adults and the risk factors associated with developing persistent symptoms. We selected 486,149 adults with confirmed SARS-CoV-2 infection and 1,944,580 propensity score-matched adults with no recorded evidence of SARS-CoV-2 infection. Outcomes included 115 individual symptoms, as well as long COVID, defined as a composite outcome of 33 symptoms by the World Health Organization clinical case definition. Cox proportional hazards models were used to estimate adjusted hazard ratios (aHRs) for the outcomes. A total of 62 symptoms were significantly associated with SARS-CoV-2 infection after 12 weeks. The largest aHRs were for anosmia (aHR 6.49, 95% CI 5.02–8.39), hair loss (3.99, 3.63–4.39), sneezing (2.77, 1.40–5.50), ejaculation difficulty (2.63, 1.61–4.28) and reduced libido (2.36, 1.61–3.47). Among the cohort of patients infected with SARS-CoV-2, risk factors for long COVID included female sex, belonging to an ethnic minority, socioeconomic deprivation, smoking, obesity and a wide range of comorbidities. The risk of developing long COVID was also found to be increased along a gradient of decreasing age. SARS-CoV-2 infection is associated with a plethora of symptoms that are associated with a range of sociodemographic and clinical risk factors.

ObjectiveTo determine the rates of asymptomatic viral carriage and seroprevalence of SARS-CoV-2 antibodies in healthcare workers.DesignA cross-sectional study of asymptomatic healthcare workers undertaken on 24/25 April 2020.SettingUniversity Hospitals Birmingham NHS Foundation Trust (UHBFT), UK.Participants545 asymptomatic healthcare workers were recruited while at work. Participants were invited to participate via the UHBFT social media. Exclusion criteria included current symptoms consistent with COVID-19. No potential participants were excluded.InterventionParticipants volunteered a nasopharyngeal swab and a venous blood sample that were tested for SARS-CoV-2 RNA and anti-SARS-CoV-2 spike glycoprotein antibodies, respectively. Results were interpreted in the context of prior illnesses and the hospital departments in which participants worked.Main outcome measureProportion of participants demonstrating infection and positive SARS-CoV-2 serology.ResultsThe point prevalence of SARS-CoV-2 viral carriage was 2.4% (n=13/545). The overall seroprevalence of SARS-CoV-2 antibodies was 24.4% (n=126/516). Participants who reported prior symptomatic illness had higher seroprevalence (37.5% vs 17.1%, χ2=21.1034, p<0.0001) and quantitatively greater antibody responses than those who had remained asymptomatic. Seroprevalence was greatest among those working in housekeeping (34.5%), acute medicine (33.3%) and general internal medicine (30.3%), with lower rates observed in participants working in intensive care (14.8%). BAME (Black, Asian and minority ethnic) ethnicity was associated with a significantly increased risk of seropositivity (OR: 1.92, 95% CI 1.14 to 3.23, p=0.01). Working on the intensive care unit was associated with a significantly lower risk of seropositivity compared with working in other areas of the hospital (OR: 0.28, 95% CI 0.09 to 0.78, p=0.02).Conclusions and relevanceWe identify differences in the occupational risk of exposure to SARS-CoV-2 between hospital departments and confirm asymptomatic seroconversion occurs in healthcare workers. Further investigation of these observations is required to inform future infection control and occupational health practices.

We have combined major histocompatibility complex-binding assays with immunization and tolerance induction experiments in HLA-DR3 transgenic mice to design apitopes (antigen-processing independent epitopes) derived from thyrotropin receptor (TSHR) for treatment of patients with Graves' disease (GD). A challenge model was created by using an adenovirus-expressing part of the extracellular domain of the thyrotropin receptor (TSHR289). This model was used to test whether current drug treatments for GD would have an impact on effective antigen-specific immunotherapy using the apitope approach. Furthermore, selected peptides were assessed for their antigenicity using peripheral blood mononuclear cell samples from patients with GD. A mixture of two immunodominant apitopes was sufficient to suppress both the T-cell and antibody response to TSHR when administered in soluble form to HLA-DR transgenic mice. Tolerance induction was not disrupted by current drug treatments. These results demonstrate that antigen-specific immunotherapy with apitopes from TSHR is a suitable approach for treatment of GD.

Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-cell epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Today it is generally accepted that B cells require cognate interactions with CD4+T cells to develop high-affinity antibodies against proteins. CD4+T cells recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+T cells that can bind to the MHC class II–peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+T-cell epitopes presented by HLA-DRB1*1501 to CD4+T cells during immune responses against FVIII. CD4+T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays.

Recent advances in immunology have greatly increased our understanding of immunological tolerance. In particular, there has been a resurgence of interest in mechanisms of immune regulation. Immune regulation refers to the phenomenon, previously known as immune suppression, by which excessive responses to infectious agents and hypersensitivities to otherwise innocuous antigens such as self antigens and allergens are avoided. We now appreciate that various distinct cell types mediate immune suppression and that some of these may be induced by appropriate administration of antigens, synthetic peptides and drugs of various types. The induction of antigen specific immunotherapy for treatment of autoimmune and allergic diseases remains the 'holy grail' for treatment of these diseases. This goal comes ever closer as understanding of the mechanisms of immune suppression and in particular antigen specific immunotherapy increases. Here we review evidence that immune suppression is mediated by various different subsets of CD4 T cells.

Most autoantigens implicated in multiple sclerosis (MS) are expressed not only in the central nervous system (CNS) but also in the thymus and the periphery. Nevertheless, these autoantigens might induce a strong autoimmune response leading to severe destruction within the CNS. To investigate the influence of a dominantly presented autoantigen on experimental autoimmune encephalomyelitis (EAE), we generated transgenic mice expressing the autoantigenic peptide MBP1–10 covalently bound to the MHC class II molecule I-Au. These mice were crossed either with B10.PL or with TCR-transgenic Tg4 mice, specific for the transgenic peptide–MHC combination. In double transgenic mice we found strong thymic deletion and residual peripheral T cells were refractory to antigen stimulation in vitro. Residual peripheral CD4+ T cells expressed activation markers and a high proportion was CD25 positive. Transfer of both CD25-negative and CD25-positive CD4+ T cells from double transgenic animals into B10.PL mice strongly inhibited the progression of EAE. Despite this thorough tolerance induction, some double transgenic mice developed severe signs of EAE after an extended period of time. Our data show that in the circumstances where autoantigenic priming persists, and where the number of antigen-specific T cells is high enough, autoimmunity may prevail over very potent tolerance-inducing mechanisms.

The major target of the pathogenic red blood cell (RBC) autoantibodies in New Zealand black (NZB) mice is the anion channel protein band 3, and CD4+ T cells from NZB mice respond to band 3. Here, we demonstrate that a band 3 peptide 861-875, which is the predominant sequence recognized by NZB T cells in vitro, bears a dominant helper epitope able to modulate the autoimmune hemolyic anemia in vivo. The development of RBC-bound autoantibodies and anemia was accelerated in NZB mice injected with peptide 861-874, which is relatively insoluble, and inhalation of the peptide primed T cells for both peptide 861-874 and band 3 responses. By contrast, inhalation of a soluble analog (Glu861, Lys875) of peptide 861-874 deviated the autoimmune response toward a T helper-2 (Th2) profile, with marked increases in the ratio of interleukin-4 to interferon-γ produced by splenic T cells responding in vitro to either peptide 861-874 or band 3. Moreover, in mice that had received such treatment, the proportion of RBC-bound immunoglobulin G (IgG) molecules that were of the Th2-associated IgG1 isotype was also increased, and anemia was less severe. It is concluded that NZB autoimmune hemolytic anemia is helper dependent and that nasal administration of different peptides containing the dominant T-cell epitope can have potentially detrimental or beneficial effects on the disease. (Blood. 2003; 102:3800-3806)

The major target of the pathogenic red blood cell (RBC) autoantibodies in New Zealand black (NZB) mice is the anion channel protein band 3, and CD4+T cells from NZB mice respond to band 3. Here, we demonstrate that a band 3 peptide 861-875, which is the predominant sequence recognized by NZB T cells in vitro, bears a dominant helper epitope able to modulate the autoimmune hemolyic anemia in vivo. The development of RBC-bound autoantibodies and anemia was accelerated in NZB mice injected with peptide 861-874, which is relatively insoluble, and inhalation of the peptide primed T cells for both peptide 861-874 and band 3 responses. By contrast, inhalation of a soluble analog (Glu861, Lys875) of peptide 861-874 deviated the autoimmune response toward a T helper-2 (Th2) profile, with marked increases in the ratio of interleukin-4 to interferon-γ produced by splenic T cells responding in vitro to either peptide 861-874 or band 3. Moreover, in mice that had received such treatment, the proportion of RBC-bound immunoglobulin G (IgG) molecules that were of the Th2-associated IgG1 isotype was also increased, and anemia was less severe. It is concluded that NZB autoimmune hemolytic anemia is helper dependent and that nasal administration of different peptides containing the dominant T-cell epitope can have potentially detrimental or beneficial effects on the disease. (Blood. 2003; 102:3800-3806)

Cytokines direct the differentiation of naive CD4(+) T cells into either IFN-gamma-producing T(h)1 cells or IL-4-producing T(h)2 cells. In this study, we analyzed the activation of signal transducer and activator of transcription (STAT)1, STAT3 and STAT5 (together with STAT4 and STAT6), and the expression of the recently identified suppressor of cytokine signalling (SOCS) proteins, in differentiated T(h)1 and T(h)2 cells, both before and after re-stimulation with anti-CD3 and anti-CD28. In addition to the polarized activation of STAT4 in T(h)1 cells and STAT6 in T(h)2 cells, we found that STAT3 and STAT5 are selectively activated in T(h)1 cells after differentiation. This activation of STAT3 and STAT5 was maintained after TCR re-stimulation. The selective activation of STAT3 and STAT5 in T(h)1 cells was associated with differential induction of SOCS molecules. After re-stimulation, SOCS1 expression was significantly increased in T(h)2 cells, but not in T(h)1 and non-polarized 'T(h)' cells. Additionally, the level of CIS was higher in T(h)2 cells compared with T(h)1 and T(h) cells. In contrast, the expression of SOCS3 was higher in T(h)1 cells. The differential induction of SOCS proteins was paralleled by the differential expression of cytokines in re-stimulated T(h)1 and T(h)2 cells (IFN-gamma and IL-4/IL-13 respectively). Our results suggests that STAT3 and STAT5, possibly regulated by the SOCS proteins, may play a role in the differentiation of T(h) cells, and in the maintenance of the T(h)1 and T(h)2 phenotype.

The onset of autoimmune diseases such as type I diabetes and multiple sclerosis is often thought to be associated with infection. This has led to studies of molecular mimicry between infectious agents and the self-antigens associated with autoimmunity. Despite many claims, however, a single causative infectious agent for autoimmunity has not been found. An alternative possibility is that many infectious agents are capable of non-specifically enhancing the likelihood of an autoimmune attack. Here we show how infectious agents may activate antigen-presenting cells leading to the activation of autoreactive T cells by otherwise innocuous antigens. The mechanism of activation involves upregulation of co-stimulatory molecules on the antigen-presenting cell resulting in a lowering of the threshold required for activation. These results help explain how diverse infectious agents could cause autoimmune disease in susceptible individuals.

Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen–major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 Au-restricted, acetylated NH2-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for Au. In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

The use of altered peptide ligands (APL) with TCR antagonist properties holds promise as an antigen-specific therapy for autoimmune disorders. We are investigating the therapeutic potential of APL in experimental autoimmune encephalomyelitis (EAE) using the Ac1 – 9 peptide of myelin basic protein in H-2^u mice. Encephalitogenic T cells recognize Ac1 – 9 using residues 3Gln and 6Pro as the major TCR contact sites. Use of position 6 APL is compromised by the heterogeneous nature of the Ac1 – 9-specific repertoire. Here we identify two position 3 APL that act as TCR antagonists on transgenic T cells expressing Ac1 – 9-specific TCR and that inhibit EAE in H-2^u mice. However, the therapeutic capacity of these two APL correlated directly with the ability to maximally inhibit activation of a heterogeneous T cell pool. The implications of these findings for the requirements for EAE induction, the relative contribution of a given T cell subpopulation to pathology and the mechanism underlying EAE inhibition in this model are discussed.

Nasal administration of peptide antigens has been shown to induce T cell tolerance. We have investigated the potential for peptide therapy of the autoimmune response to myelin antigens in experimental autoimmune encephalomyelitis (EAE). Three major encephalitogenic epitopes were studied for their ability to induce nasal tolerance to myelin antigens. These included epitopes Ac1 – 9 and 89 – 101 of myelin basic protein (MBP) and 139 – 151 from proteolipid protein (PLP). Peptide Ac1 – 9 from MBP effectively suppressed responses to both MBP epitopes, following immunization with whole myelin (linked suppression). The N-terminal epitope failed, however, to modify the response to epitope 139 – 151 of PLP. The second MBP epitope (89 – 101) was poorly tolerogenic for the immune response to any naturally processed myelin epitope. By contrast, PLP[139 – 151] was able to induce bystander suppression of T cells responsive to both itself and the two epitopes from MBP. Furthermore, this epitope suppressed EAE induced with peptides derived from MBP and was capable of treating ongoing disease. The mechanism of bystander suppression, mediated by PLP[139 – 151], did not correlate with an overt switch from the T helper 1 to the T helper 2 phenotype. These results demonstrate how a complex autoimmune disease may be controlled by treatment with a single peptide epitope and reveal a hierarchy in the suppressive properties of different myelin antigens.

The indirect pathway of allorecognition involves the processing and presentation of donor molecules by recipient antigen-presenting cells to alloreactive CD4+T cells. Our objective was to assess the occurrence and significance of the indirect presentation of allogeneic major histocompatibility complex molecules in the rejection of major histocompatibility complex class I-disparate skin.

K6, an influenza-specific cytotoxic T (Tc)-cell clone derived from C3H/He mice, was Dk-restricted. Further analysis showed that K6 also recognized Ddand Dqas allotargets. The clonal nature of the three recognition specificities was confirmed by cold-target cell inhibition. After extensive in vitro selection, K6 lost the ability to lyse Ddcells while retaining both other specificities. The role of the Lyt-2 molecule in antigen-specific self-H-2-restricted vs allospecific recognition was investigated by inhibition with the Lyt-2-specific monoclonal antibody 53-6.7. Both activities were proportionately inhibited, indicating that the Lyt-2 molecule is equally important for both recognition specificities of this Tc-cell clone.

In conclusion, rapid advances in our understanding of the molecular basis of T cell recognition have revealed a number of approaches for immune intervention in autoimmune disease. Each procedure has been tried and tested in experimental models. Characterization of T cell recognition in human autoimmune disease is a step behind. However, their success in experimental models indicates that MHC-blocking peptides will find use both in identification of restriction elements for disease and in the prevention and treatment of human autoimmune conditions.

Nr4areceptors are activated by T cell receptor (TCR) signaling and play key roles in T cell differentiation. Which TCR signaling pathways regulate Nr4a receptors and their sensitivities to TCR signal strength and duration remains unclear. Using Nr4a1/Nur77-GFP and Nr4a3-Timer of cell kinetics and activity (Tocky) mice, we elucidate the signaling pathways governing Nr4a receptor expression. We reveal that Nr4a1–Nr4a3are Src family kinase dependent. Moreover, Nr4a2and Nr4a3are attenuated by calcineurin inhibitors and bind nuclear factor of activated T cells 1 (NFAT1), highlighting a necessary and sufficient role for NFAT1 in the control of Nr4a2and Nr4a3,but redundancy for Nr4a1. Nr4a1-GFP is activated by tonic and cognate signals during T cell development, whereas Nr4a3-Tocky requires cognate peptide:major histocompatibility complex (MHC) interactions for expression. Compared to Nr4a3-Tocky, Nr4a1-GFP is approximately 2- to 3-fold more sensitive to TCR signaling and is detectable by shorter periods of TCR signaling. These findings suggest that TCR signal duration may be an underappreciated aspect influencing the developmental fate of T cells in vivo.

Immunological homeostasis in T cells is maintained by a tightly regulated signaling and transcriptional network. Full engagement of effector T cells occurs only when signaling exceeds a critical threshold that enables induction of immune response genes carrying an epigenetic memory of prior activation. Here we investigate the underlying mechanisms causing the suppression of normal immune responses when T cells are rendered anergic by tolerance induction. By performing an integrated analysis of signaling, epigenetic modifications, and gene expression, we demonstrate that immunological tolerance is established when both signaling to and chromatin priming of immune response genes are weakened. In parallel, chromatin priming of immune-repressive genes becomes boosted, rendering them sensitive to low levels of signaling below the threshold needed to activate immune response genes. Our study reveals how repeated exposure to antigens causes an altered epigenetic state leading to T cell anergy and tolerance, representing a basis for treating auto-immune diseases.

Mesenchymal stem cells (MSCs) can abrogate the animal model ofmultiple sclerosis, experimental autoimmune encephalomyelitis (EAE), but whether this therapeutic effect occurs entirely through systemic immune modulation and whether CNS infiltration occurs after peripheral delivery are uncertain. We studied the clinical and neuropathologic effects of intravenously administered human MSCs (hMSCs) in C57BL/6 mice with EAE. Human MSCs significantly reduced the clinical disease severity, particularly in later disease. Large numbers of hMSCs migrated into gray and white matter at all levels of the spinal cord in both naive mice and mice with EAE. In the latter, hMSCs accumulated over time in demyelinated areas. There were 2 distinct morphological appearances of the hMSCs in the tissue, that is, rounded and less numerous process-bearing forms; very few expressed neural markers. The number of spinal cord white matter lesions and areas of white matter demyelination were reduced after hMSC treatment compared with control treatment. These findings show that central nervous system infiltration occurs after peripheral delivery of hMSCs, that they accumulate where there is myelin damage, and that they are associated with a reduced extent ofdemyelination. These data support a potential role for hMSCs in autologous cell therapy in multiple sclerosis.

There is extensive evidence from murine models supporting a role for CD4+CD25+ regulatory T cells in suppressing alloreactivity. However, clinical evidence regarding the role of regulatory T cells in human alloresponses is conflicting and may reflect the difficulty in defining and isolating naturally occurring human CD4+CD25+ regulatory T cells. Moreover, the mechanism of action of Foxp3+ regulatory T cells remains somewhat controversial with a number of proposed modes of suppression described, including cell contact-dependent inhibition, cytokine mediated suppression and cytokine consumption. We have previously used peripheral blood monocyte-derived dendritic cells (DCs) as antigen presenting cells (APCs) in allogeneic mixed lymphocyte reactions (MDCLRs), to assess the in vitro suppressive function of human CD4+CD25+ T cells. Healthy volunteer donor MACS isolated CD4+CD25+ regulatory T cells demonstrated anergy and significant dose-dependent suppression of responder cell proliferation, to physiological frequencies, in allogeneic MDCLRs (p<0.005). In this study we investigated the in vitro mechanism of action of human CD4+CD25+ T cells in suppressing alloreactivity in MDCLRs. Co-culture of CD4+CD25+ regulatory T cells with autologous CD4+ responder cells in allogeneic MDCLRs, at a ratio of 1:4, resulted in suppression of proliferation by 68%. Peak suppression of CD4+ responder cell proliferation, observed on day five of co-culture, was accompanied by significant suppression of co-culture supernatant IL-5 concentration (p<0.05), and preceded by inhibition of IL-2. Intracellular cytokine staining confirmed that CD4+ responder cell intracellular IL-2 was reduced by 60% on co-culturing with CD4+CD25+ regulatory T cells in the allogeneic MDCLR. CD4+CD25+ regulatory T cells mediated suppression of proliferation and cytokine responses across transwell membranes, demonstrating a cell contact-independent mechanism and a potential soluble factor in the mode of action of CD4+CD25+ regulatory T cells in suppressing human alloresponses. This was confirmed by suppression of responder cell proliferation and cytokine responses by supernatant transfer from DCs co-cultured with allogeneic CD4+CD25+ regulatory cells. We surveyed possible candidate molecules responsible for mediating the cell contact-independent suppression; in our hands, neither IL-10 nor TGF-β was identified as the soluble factor. Next, we examined the effect of CD4+CD25+ regulatory T cells on APCs in the allogeneic MDCLR. Immunophenotypic characterization of DCs recovered from MDCLRs in the presence of CD4+CD25+ regulatory T cells showed down-regulation of HLA-DR, CD83, and co-stimulatory molecules, CD80 and CD86, compared with DCs cultured with CD4+CD25− T cells or cytokines, IL-4 and GM-CSF, alone. The suppression of DC activation by CD4+CD25+ regulatory T cells was not mediated across transwell membranes, demonstrating a cell contact-requirement. Moreover, downregulation of DC activation markers was not accompanied by IL-12 suppression, implying no role for IL-10 in mediating the suppressive effect of the regulatory cells on the APCs. Our results demonstrate both a cell contact-independent mode of action in suppressing CD4+ responder cell proliferation, Th1 and Th2 cytokine responses, independent of IL-10 and TGF-β, and a cell contact-requirement in the suppression of allogeneic DC maturation and activation. In summary, our findings suggest that multiple mechanisms of action contribute to the in vitro suppressive effects of human CD4+CD25+ regulatory T cells on alloreactivity.

There is extensive evidence from murine models supporting a role for CD4+CD25+regulatory T cells in suppressing alloreactivity. However, clinical evidence regarding the role of regulatory T cells in human alloresponses is conflicting and may reflect the difficulty in defining and isolating naturally occurring human CD4+CD25+regulatory T cells. Moreover, the mechanism of action of Foxp3+regulatory T cells remains somewhat controversial with a number of proposed modes of suppression described, including cell contact-dependent inhibition, cytokine mediated suppression and cytokine consumption. We have previously used peripheral blood monocyte-derived dendritic cells (DCs) as antigen presenting cells (APCs) in allogeneic mixed lymphocyte reactions (MDCLRs), to assess the in vitrosuppressive function of human CD4+CD25+T cells. Healthy volunteer donor MACS isolated CD4+CD25+regulatory T cells demonstrated anergy and significant dose-dependent suppression of responder cell proliferation, to physiological frequencies, in allogeneic MDCLRs (p<0.005). In this study we investigated the in vitromechanism of action of human CD4+CD25+T cells in suppressing alloreactivity in MDCLRs. Co-culture of CD4+CD25+regulatory T cells with autologous CD4+responder cells in allogeneic MDCLRs, at a ratio of 1:4, resulted in suppression of proliferation by 68%. Peak suppression of CD4+responder cell proliferation, observed on day five of co-culture, was accompanied by significant suppression of co-culture supernatant IL-5 concentration (p<0.05), and preceded by inhibition of IL-2. Intracellular cytokine staining confirmed that CD4+responder cell intracellular IL-2 was reduced by 60% on co-culturing with CD4+CD25+regulatory T cells in the allogeneic MDCLR. CD4+CD25+regulatory T cells mediated suppression of proliferation and cytokine responses across transwell membranes, demonstrating a cell contact-independent mechanism and a potential soluble factor in the mode of action of CD4+CD25+regulatory T cells in suppressing human alloresponses. This was confirmed by suppression of responder cell proliferation and cytokine responses by supernatant transfer from DCs co-cultured with allogeneic CD4+CD25+regulatory cells. We surveyed possible candidate molecules responsible for mediating the cell contact-independent suppression; in our hands, neither IL-10 nor TGF-β was identified as the soluble factor. Next, we examined the effect of CD4+CD25+regulatory T cells on APCs in the allogeneic MDCLR. Immunophenotypic characterization of DCs recovered from MDCLRs in the presence of CD4+CD25+regulatory T cells showed down-regulation of HLA-DR, CD83, and co-stimulatory molecules, CD80 and CD86, compared with DCs cultured with CD4+CD25−T cells or cytokines, IL-4 and GM-CSF, alone. The suppression of DC activation by CD4+CD25+regulatory T cells was not mediated across transwell membranes, demonstrating a cell contact-requirement. Moreover, downregulation of DC activation markers was not accompanied by IL-12 suppression, implying no role for IL-10 in mediating the suppressive effect of the regulatory cells on the APCs. Our results demonstrate both a cell contact-independent mode of action in suppressing CD4+responder cell proliferation, Th1 and Th2 cytokine responses, independent of IL-10 and TGF-β, and a cell contact-requirement in the suppression of allogeneic DC maturation and activation. In summary, our findings suggest that multiple mechanisms of action contribute to the in vitrosuppressive effects of human CD4+CD25+regulatory T cells on alloreactivity.

Graft versus host disease (GVHD) is the primary cause of transplant related morbidity and mortality, limiting the widespread application of haemopoietic stem cell transplantation (HSCT). Evidence from murine models supports the role of CD4+CD25+ regulatory T cells in the suppression of GVHD. Human evidence regarding the role of regulatory T cells in alloresponses is conflicting and may reflect the difficulty in defining and isolating the regulatory T cell population in humans. We have investigated the use of peripheral blood monocyte-derived dendritic cells (DCs) as stimulator cells in allogeneic mixed lymphocyte reactions (MLRs), as a means of assessing the in vitro suppressive function of regulatory T cells in human alloresponses. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult volunteers. Magnetically isolated CD4+CD25+ T cells were combined with 50×103 autologous PBMCs or 20×103 autologous CD4+ T cells as responders, and 5×103 allogeneic irradiated DCs. Proliferation was assessed by tritiated thymidine incorporation. The CD4+CD25+ cells were anergic and demonstrated dose-dependent suppression of responder cell proliferation in the DC-driven allogeneic MLR. Greater than 50% suppression was seen with CD4+CD25+ T cells co-cultured with responder PBMCs at ratios of 1:4 to 1:32. Furthermore, depletion of CD4+CD25+ T cells from whole CD4+ responder cells resulted in enhanced proliferation and an increase in the amplitude of the MLR. Flow cytometry indicated that the majority of the magnetically isolated CD4+CD25+ T cells were FoxP3+ on intracellular staining and demonstrated down-regulation of cell surface expression of the IL-7 receptor (CD127). The potent suppression demonstrated here by CD4+CD25+CD127- T cells at ratios of 1:32 responder cells, suggests that these cells have a potential role for suppressing alloresponses at physiological levels. Moreover, this assay provides the basis for future investigation into regulatory T cell function in patients post-HSCT.

Graft versus host disease (GVHD) is the primary cause of transplant related morbidity and mortality, limiting the widespread application of haemopoietic stem cell transplantation (HSCT). Evidence from murine models supports the role of CD4+CD25+regulatory T cells in the suppression of GVHD. Human evidence regarding the role of regulatory T cells in alloresponses is conflicting and may reflect the difficulty in defining and isolating the regulatory T cell population in humans. We have investigated the use of peripheral blood monocyte-derived dendritic cells (DCs) as stimulator cells in allogeneic mixed lymphocyte reactions (MLRs), as a means of assessing the in vitrosuppressive function of regulatory T cells in human alloresponses. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy adult volunteers. Magnetically isolated CD4+CD25+T cells were combined with 50×103autologous PBMCs or 20×103autologous CD4+T cells as responders, and 5×103allogeneic irradiated DCs. Proliferation was assessed by tritiated thymidine incorporation. The CD4+CD25+cells were anergic and demonstrated dose-dependent suppression of responder cell proliferation in the DC-driven allogeneic MLR. Greater than 50% suppression was seen with CD4+CD25+T cells co-cultured with responder PBMCs at ratios of 1:4 to 1:32. Furthermore, depletion of CD4+CD25+T cells from whole CD4+responder cells resulted in enhanced proliferation and an increase in the amplitude of the MLR. Flow cytometry indicated that the majority of the magnetically isolated CD4+CD25+T cells were FoxP3+on intracellular staining and demonstrated down-regulation of cell surface expression of the IL-7 receptor (CD127). The potent suppression demonstrated here by CD4+CD25+CD127−T cells at ratios of 1:32 responder cells, suggests that these cells have a potential role for suppressing alloresponses at physiological levels. Moreover, this assay provides the basis for future investigation into regulatory T cell function in patients post-HSCT.