Longitudinal study of the frequency of cytotoxic T cell precursors in kidney allograft recipients

Clonal deletion or inactivation of donor-specific alloreactive cells are important mechanisms that are believed to account for acquired immune tolerance in allograft recipients. Serial assessment of precursor cytotoxic T lymphocyte frequencies (CTLpf) by limiting dilution analysis (LDA) provides information at the clonal level on changes in the alloimmune response of graft recipients. We performed a longitudinal study of 15 cadaveric kidney recipients before and every 3 months throughout the first year after transplantation (Tx). Pre-Tx values of donor CTLpf showed high interindividual variability without a predictive value for the clinical outcome. All patients with well functioning kidneys had decreased CTLpf at 3 months post-Tx in comparison with pre-Tx values. This decrease was donor-specific in four patients and was permanent in two cases throughout the study. Most patients presented decreased anti-donor CTLpf values from 6 to 9 months, whereas a partial recovery of donor CTLpf was observed in three patients. Reversible acute rejection was diagnosed in three patients, and it was associated with a marked increase in anti-donor CTLpf, returning to pre-Tx values by 9 months post-Tx. In addition, one patient with chronic rejection displayed a transient increase in CTLpf 6 months after Tx. The results of this sequential study indicate the establishment of a state of either hyporesponsiveness or functional clonal inactivation, transient or permanent, which could facilitate allograft acceptance.     

T-cell autoreactivity to Hsp in human transplantation may involve both proinflammatory and regulatory functions

Heat shock proteins (Hsp) are moving from the category of basically intracellular chaperone molecules to important proteins in both innate and acquired immune responses, with great potential for clinical application as immunomodulators. Both proinflammatory and regulatory Hsp-reactive T cells have been described in animal models of autoimmune diseases. To investigate the role of autoreactivity to Hsp60 and Hsp70 in human transplantation, we analyzed, sequentially, peripheral blood mononuclear cell proliferation and cytokine production before and at different time points after renal transplantation, as well as the modulation of proliferation to Hsp in the presence of exogenous cytokines. Proliferation to Hsp60 and Hsp70 in the pretransplantation (pre-Tx) period was significantly associated with rejection episodes in the first months post-Tx. In contrast, IL-4 production was significantly associated with absence of rejection. Addition of exogenous IL-4 distinctly modulated the proliferative response to Hsp60; inhibiting proliferation in 83% of patients in the early post-Tx period (0-6 months), in which rejection episodes occurred, and inducing proliferation in 62.5% of patients in the later period (>12-24 months), when no rejection was observed. Characterization of autoreactive anti-Hsp60 regulatory T cells may permit new approaches to control the proinflammatory response to the graft, as well as aggressive autoimmunity.     

Breakdown of T cell tolerance and autoimmunity in primary immunodeficiency--lessons learned from monogenic disorders in mice and men

A key feature of the immune system is the capacity to monitor and control infections from non-self pathogens while maintaining tolerance to self-antigens. Primary immunodeficiencies (PID) are characterized by an increased susceptibility to infections, often associated with aberrant inflammatory responses and a concomitant high prevalence of autoimmunity. Autoimmunity in PID raises a conundrum: How can an immune system fail to respond to non-self pathogens while reacting vigorously to self-antigens? Recent advances from studies of PID patients and related animal models have revealed the crucial role of Aire-induced expression of self-antigens for deletion of autoreactive T cells in the thymus (central tolerance). Moreover, lessons from PID have provided unequivocal evidence for the essential role of regulatory T cells in suppressing autoreactive T cells in the periphery. Finally, findings from PID have broadened our understanding of how homeostatic proliferation and increased load or decreased clearance of apoptotic cells and non-self pathogens can lead to breakdown of peripheral tolerance.     

Predominance of Type 1 cytokines and decreased number of CD4(+)CD25(+high) T regulatory cells in peripheral blood of patients with recurrent aphthous ulcerations

Recurrent aphthous ulcerations (RAU) are a chronic inflammatory disease with evidence of inappropriate immune response. Previous studies have suggested cell-mediated activation of immune response towards common micro-organisms of oral cavity in RAU. In this investigation, we explored cytokine production by peripheral blood mononuclear cells (PBMC) and T regulatory cell population in blood of active and remission RAU patients as crucial factors for maintenance of peripheral tolerance. Ten patients with minor RAU and 12 healthy individuals were selected for the study. Cytokine levels were analysed in supernatants using Cytometric Bead Array Kit for flow cytometry and ELISA. We have demonstrated increased production of Type 1 cytokines IL-2, IFN-gamma and TNF-alpha as well as IL-5, IL-6 and IL-8 by peripheral blood mononuclear cells in RAU. In contrast, IL-10 and TGF-beta anti-inflammatory cytokine production was decreased in RAU patients compared to healthy individuals. Moreover, we have found that CD4(+)CD25(+high) T regulatory cell proportion was decreased in RAU and represented 3.58+/-0.654% of CD4(+) T cells in active RAU, 4.66+/-0.561% of CD4(+) T cells in remission RAU, whereas in healthy controls CD4(+)CD25(+high) T cells represented 7.30+/-1.238% of CD4(+) T cells (p<0.001). Thus, the obtained results indicate that disproportion in cytokine production may be contributing factor in the pathogenesis of RAU. Alteration in the number of CD4(+)CD25(+high) T regulatory cells in RAU may additionally influence the development of the disease. We propose that imbalance in pro- and anti-inflammatory cytokine network may lead to the breakdown of peripheral tolerance in RAU and the excessive immune response towards harmless micro-organisms colonized oral mucosa or self-antigens.     

Mechanisms of immunological tolerance loss versus erythrocyte self-antigens and autoimmune hemolytic anemia

Recent studies on animal and human autoimmune hemolytic anemia (AIHA) suggest that immunological tolerance loss toward red blood cells (RBC) self-antigens may be originate by different, non-mutually exclusive, mechanisms. According to now available data the identified mechanisms may be: ignorance against RBC self-antigens; molecular mimicry; polyclonal T and/or B cells activation; errors in central or peripheral tolerance; immunoregulatory disorders including cytokine network alteration. In some patients with AIHA, stimulation of PMBC by synthetic Rh peptides indicate that ignorant T and/or B cell clones may recognize cryptic RBC self-antigens. AIHA associated with bacterial or viral infections seems to be produced by polyclonal T and/or B cells activation against foreign antigens which mimic protein or carbohydrate epitopes on RBC. Polyclonal activation of host B cell clones by donor alloreactive T cells causes the AIHA in chronic GVHD. As the tolerance loss is concerned, experiments on mouse lines expressing a transgene with autoantibody activity against murine RBC have shown that non-deleted peripheral B cell clones may produce RBC autoantibodies. In humans a genetic defect of Fas/FasL autoreactive lymphocytes apoptosis may be associated to AIHA. Immunoregulatory disorders due to depletion of CD4+ CD25+ T cells or Th1/Th2 cytokines imbalance may induce autoimmune diseases. In mice AIHA may be induced or improved by cytokines or anticytokine antibodies administration. In NZB/W mice and human AIHA there is an increased production of Th2 cytokines as IL4 and IL10 but INF-gamma reduced production. In addition in human AIHA has been shown a downregulation of IL12 and therefore, an IL10/IL12 immunoregulatory circuit imbalance which might facilitate the RBC autoantibodies production.     

ReviewMechanisms of Immunological Tolerance Loss Versus Erythrocyte Self-antigens and Autoimmune Hemolytic Anemia

Recent studies on animal and human autoimmune hemolytic anemia (AIHA) suggest that immunological tolerance loss toward red blood cells (RBC) self-antigens may be originate by different, non-mutually exclusive, mechanisms. According to now available data the identified mechanisms may be: ignorance against RBC self-antigens; molecular mimicry; polyclonal T and/or B cells activation; errors in central or peripheral tolerance; immunoregulatory disorders including cytokine network alteration.

In some patients with AIHA, stimulation of PMBC by synthetic Rh peptides indicate that ignorant T and/or B cell clones may recognize cryptic RBC self-antigens. AIHA associated with bacterial or viral infections seems to be produced by polyclonal T and/or B cells activation against foreign antigens which mimic protein or carbohydrate epitopes on RBC. Polyclonal activation of host B cell clones by donor alloreactive T cells causes the AIHA in chronic GVHD. As the tolerance loss is concerned, experiments on mouse lines expressing a transgene with autoantibody activity against murine RBC have shown that non-deleted peripheral B cell clones may produce RBC autoantibodies. In humans a genetic defect of Fas/FasL autoreactive lymphocytes apoptosis may be associated to AIHA. Immunoregulatory disorders due to depletion of CD4+ CD25+ T cells or Th1/Th2 cytokines imbalance may induce autoimmune diseases. In mice AIHA may be induced or improved by cytokines or anticytokine antibodies administration. In NZB/W mice and human AIHA there is an increased production of Th2 cytokines as IL4 and IL10 but INF-γ reduced production. In addition in human AIHA has been shown a downregulation of IL12 and therefore, an IL10/IL12 immunoregulatory circuit imbalance which might facilitate the RBC autoantibodies production.     

Tumor-specific immunotherapy based on dominant models of natural tolerance

The assumption that cancer immunotherapy may be based on the existence of autoreactive lymphocytes recognizing self-antigens on cancer cells, obviously opens a new opportunity. Nevertheless this analysis, relying on a recessive model of natural tolerance, limits the approach to try to activate peripheral lymphocytes, by increasing co-stimulatory signals or using modified self-antigens for immunization. Here we hypothesize that, based on emerging dominant tolerance notions in autoimmunity, it would be possible to induce a specific autoimmunity against tumor cells and arrest their growth following the removal of regulatory T cells. These immunoregulatory cells suppress available immunocompetent autoreactive cells capable of destroying tumor cells. Therefore, in order to reach a complete tumor-specific autoimmunity it is necessary to combine the T cell immunosuppression which abrogates the regulatory cells, with the cancer vaccines, which induces extensive proliferation of lymphoid cells directed towards specificities on tumor cells.     

T lymphocyte control of autoreactivity: analysis with human T cell clones and limiting dilution culture

To investigate cellular mechanisms controlling activated autoreactive T lymphocytes, a limiting dilution system was established employing cloned autoreactive major histocompatibility complex class II specific lymphocytes (a2/7) as stimulator cells for autologous peripheral blood mononuclear cells. At low responder/stimulator ratios, cytotoxic effector cells were generated capable of lysing clone a2/7. Importantly, within the population of cells mediating autocytotoxic effector function, differential specificities were found to exist. The generation of such autocytotoxic T lymphocytes appears to be inhibited by an additional population of cells circulating at lower frequency suggesting that autoreactivity is controlled at distinct levels by discrete functional cellular subsets.     

HLA-DQ-specific autoreactive T cell clone with helper and cytotoxic functions

To clarify the existence and the possible function of DQ-specific autoreactive T cells, we established a novel autoreactive T cell clone (28B8) responding to self-DQ molecules from an unimmunized normal subject. Blocking studies with monoclonal antibodies (mAbs) to HLA class II antigens and alloreactive response of the clone revealed that the clone recognized DQ molecules defined by a DR1-DQw5 haplotype. Clone 28B8 markedly induced autologous B cells to produce immunoglobulins, and was also cytotoxic against autologous lymphocytes, as well as against allogeneic lymphocytes with a DR1-DQw5 haplotype. These findings indicate that DQ antigens are involved in autoreactivity and stimulate autologous T cells that may have potent immunoregulatory functions.     

CD4 T-cell autoreactivity to the mitochondrial autoantigen PDC-E2 in AMA-negative primary biliary cirrhosis

Approximately 5% of patients with primary biliary cirrhosis (PBC) lack characteristic anti-mitochondrial antibodies (AMA). Yet clinically AMA(+) and AMA(-) patients are similar. Using both AMA(+) and AMA(-) patients, we quantitated the frequency of autoreactive T cells that respond to the major CD4 T-cell epitope, PDC-E2 163-176, using limiting dilution assays and quantitation of IFN-gamma, IL-10 and IL-4. Further, based on data that both PBC patients and healthy subjects have CD4(+) T cells that recognize PDC-E2 163-176 but with differing costimulation requirements, assays were performed using two different antigen-presenting cell (APC) systems: either autologous peripheral blood mononuclear cells (PBMC) or HLA DR53 transfected mouse fibroblast cell lines (L-DR53). When costimulation-incompetent L-DR53 were used as APCs, the PDC-E2 CD4 T-cell frequency and capacity for IFN-gamma production were equivalent in both AMA(+) and AMA(-) patients but the frequencies of such cells were significantly lower in normals, with IL-10 production similar in all three groups. Thus, in PBC there is 'universal' autoreactive CD4(+) T-cell immune responsiveness to the critical autoantigen, PDC-E2. These observations emphasize that the mitochondrial autoreactivity in PBC is a multi-lineage response and hence, AMA-negative PBC may be an anachronism that refers only to sera autoantibodies.     

TNF-alpha impairs peripheral tolerance towards beta-cells,and local costimulation by B7.1 enhances the effector function of diabetogenic T cells

Maintenance of peripheral tolerance and inactivation of autoreactive T cells is based on a delicate balance between pro-inflammatory and protective cytokines that is poorly understood. We have here addressed how the local expression of the inflammatory cytokine TNF-alpha can impair peripheral tolerance and lead to autoreactivity. After transplantation of pancreata that are immunogenic due to beta-cell expression of B7.1 and TNF-alpha, into thymectomized and euthymic syngeneic mice, we found that only euthymic mice rejected the grafts. This result suggests that under normal circumstances autoreactive T cells are functionally inactivated, and initiation of an autoreactive response requires de-novo generation of T cells. By contrast, thymectomized mice expressing TNF-alpha on the endogenous islets rejected the grafts, showing that expression of TNF-alpha prevents functional silencing of the autoreactive T cells. Thus, this study provides a mechanism by which TNF-alpha and possibly chronic inflammatory responses may promote autoimmune diseases. Furthermore, we have investigated whether B7.1 can enhance T cell responses of already activated T cells leading to islet rejection. By transplantation of wild-type and B7.1-expressing islets into overtly diabetic mice we found that only the wild-type islets could restore normoglycemia, suggesting that costimulation by B7.1 is required in the expansion or effector phase of the response.     

A molecular basis for NKT cell recognition of CD1d-self-antigen

The antigen receptor for natural killer T?cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3???resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.     

Soluble, dimeric HLA DR4-peptide chimeras: an approach for detection and immunoregulation of human type-1 diabetes

Still there are no effective methods to predict or cure type 1 diabetes (T1D) in humans. Soluble, dimeric MHC class II-peptide (DEF) chimeras have potential for both early diagnosis and immunospecific therapy. DEF chimeras prevent and reverse diabetes in mice by stimulating antigen-specific type 1 T regulatory cell (Tr1)-like cells. We also showed that diabetes could be predicted by changes in the phenotype of autoreactive CD4 T cells in peripheral blood. Herein, we demonstrated that human DEF (HLA-DR*0401/Fcgamma1) chimeras expressing peptides of beta-cell antigens stimulate Tr1-like cells in blood of patients with T1D, non-diabetic relatives, and controls. Furthermore, the specific and stable binding of DEF chimeras to cognate TCR and CD4 coreceptor allowed quantification and phenotyping of autoreactive CD4 T cells in non-stimulated blood by FACS. Our results indicate that (1) autoreactive CD4 T cells to GAD65 autoantigen are commonly present in humans expressing diabetes-susceptible HLA-DR*0401 molecules; (2) these autoreactive T cells undergo avidity maturation upon encountering the self antigen early in life; (3) the disease is associated with an imbalance between autoreactive CD4+CD25+ and CD4+CD69+ T cells specific for GAD65. Based on this, we propose a model to explain the kinetics of autoreactive CD4 T cells in blood during the natural history of T1D.     

Pre-existing autoimmunity determines type 1 diabetes outcome after Flt3-ligand treatment

Redirection of immune responses by manipulation of antigen-presenting cells is an emerging strategy for immunosuppressive treatment of autoimmune diseases. In vivo expansion of dendritic cells (DC) by Fms-like tyrosine kinase-3 (Flt3)-Ligand (FL) treatment was shown to delay diabetes onset in the NOD model of autoimmune diabetes. However, we show here that Flt3 stimulation actually accelerates autoimmunity when autoreactive CD8 T cells are detectable in blood prior to treatment. With autoreactive CD8 cells present, the capacity of FL to expand DCs and induce Treg remained intact, but both numbers and the functional response of islet-specific CD8s were boosted. Also, the inhibitory receptor PD-1 on (autoreactive) CD8 T cells and its ligand PD-L1 on Treg were no longer upregulated. These data highlight the need to pre-screen for T cell autoreactivity prior to generalized DC expansion and illustrate how accelerated disease can occur when the intended initiation of regulatory mechanisms is impaired later in diabetogenesis.     

Nonself-antigens are the cognate specificities of Foxp3+ regulatory T cells

The majority of regulatory Foxp3+CD4+ T cells naturally arises in the thymus. It has been proposed that T cell receptors (TCRs) on these cells recognize self-MHC class II-peptide complexes with high or higher affinity and that their specificities mirror specificities of autoreactive T cells. Here, we analyzed hundreds of TCRs derived from regulatory or nonregulatory T cells and found little evidence that the former population preferably recognizes self-antigens as agonists. Instead, these cells recognized foreign MHC-peptide complexes as often as nonregulatory T cells. Our results show that high-affinity, autoreactive TCRs are rare on all CD4+ T cells and suggest that selecting self-peptide is different from the peptide that activates the same regulatory T cells in the periphery.     

Increased numbers of circulating donor-specific T helper lymphocytes after human heart valve transplantation

Implantation of cryopreserved human donor heart valves for either congenital or acquired cardiac disease has been performed since the last three decades. Although the clinical outcome is good, long-term valve degeneration resulting in dysfunction has been observed. A specific immunological response of the recipient against the allograft has been proposed as one of the factors involved in this process. Helper T lymphocytes play an important intermediate role in cellular and humoral immune response. Increasing numbers of circulating donor-specific helper T lymphocytes precursors (HTLp) correlate with graft rejection after organ transplantation. To investigate whether cryopreserved human donor heart valves are able to induce a donor-specific T helper response, we monitored the HTLp frequencies (HTLpf) in peripheral blood samples of 13 patients after valve allograft transplantation by use of a limiting dilution assay followed by an interleukin-2 bioassay. Prior to transplantation, HTLpf specific for donor and third-party antigens showed individual baseline levels. After allografting, the antidonor frequencies significantly increased in 11 of the 13 patients (P = 0.02). This was not found for stimulation with third-party spleen cells (P = 0.68), which indicates a donor-specific response. Maximal donor-specific HTLpf were already found at 1--2 months after operation. Valve allograft transplantation induces an increase in the numbers of donor-specific HTLp in peripheral blood of the patients. Analogous to organ transplantation, these HTLp may play a crucial role in events that lead to valve damage. Therefore, monitoring of HTLp in peripheral blood samples might be informative for donor valve degeneration (rejection) and subsequently valve allograft failure.     

Regulatory T cell therapy as individualized medicine for asthma and allergy

PURPOSE OF REVIEW: Regulatory T cells have been identified as key players in the maintenance of peripheral tolerance, which prevents inappropriate immune responses to both self-antigens and innocuous allergens. This review aims to provide an update on our current understanding of the therapeutic potential of naturally occurring and adaptive regulatory T cell subsets in allergic and asthmatic disease. RECENT FINDINGS: Evidence is emerging that regulatory T cells control aberrant immune responses to allergens in health and exhibit impaired function in active disease. These data provide a rationale for developing therapeutic strategies that promote regulatory T cell numbers or function in patients. SUMMARY: Preclinical studies of adoptive transfer of antigen-specific regulatory T cells into mouse models of allergic airway disease ameliorate the inflammatory response and in some studies airway hyperresponsiveness. Although these studies are encouraging this remains an invasive and expensive therapeutic protocol for the treatment of allergic disease in humans and a number of concerns relating to safety and efficacy exist. Existing therapies, both allergen specific immunotherapy and nonspecific treatments such as glucocorticoids, induce IL-10 secreting T regulatory populations in patients. Strategies to combine allergen immunotherapy with add-on treatments or adjuvants with the potential to boost regulatory T cells, safety and efficacy remain a major research focus.     

Natural regulatory T cells: number and function are normal in the majority of patients with lupus nephritis

CD4(+) CD25(+) regulatory T cells have been shown to be a vital component of the mechanisms that prevent autoreactivity in mice and also in humans. Previous studies have examined CD4(+) CD25(hi) regulatory T cell frequency and function in patients with systemic lupus erythematosus (SLE) with mixed results. We investigated frequency, phenotype and function in 21 patients with SLE and six with inactive disease. We found no reduction in frequency of the CD25(hi) subset, although active disease was associated with an increased proportion of CD4(+) CD25(+) T cells. When examining function, in the majority of individuals suppression was comparable with controls, although cells isolated from one patient with active disease failed to suppress proliferation. On testing the effect of CD25(hi) depletion on the responses of whole peripheral blood mononuclear cells to nucleosomes we found that, where a response was detectable from patients, depletion augmented interferon-gamma secretion, demonstrating intact suppression of responses implicated in the pathogenesis of SLE. Our results did not confirm an association of failure in CD4(+) CD25(hi) regulatory T cell function or a reduction in their frequency with active disease. Instead, perturbations in the CD4(+) CD25(hi) regulatory T cell population may play a role in disease in only a minority of the patients afflicted by the diverse syndromes of SLE.     

Autoreactive T cells in rheumatic disease (1). Analysis of growth frequencies and autoreactivity of T cells in patients with rheumatoid arthritis and Lyme disease

A limiting dilution system was established in order to estimate frequencies of interleukin-2 (IL-2)-responsive, autoreactive and alloreactive T cells in samples of peripheral blood (PBL) and synovial fluid lymphocytes (SFL), from patients with rheumatoid arthritis (RA) and lyme disease, as well as from healthy donors and a patient with osteoarthrosis. The frequencies of IL-2-dependent T-cell colony formation were significantly higher in patients with RA and lyme disease (median: 1/287) as compared to controls (median: 1/1,313) indicating a preactivation of T cells in these patients in vivo. Autoreactivity was measured by the proliferative response of T-cell lines to autologous irradiated PBL as stimulating cells. The frequencies of autoreactive T cells in blood were significantly higher in patients (median: 1/2,615) as compared to controls (median: 1/19,607). There was no significant difference in autoreactive T-cell frequencies between the patients' SFL (median: 1/3,185) and PBL (median: 1/2,615). In every case the frequency of alloreactive T cells exceeded the frequency of autoreactive T cells. Most autoreactive T-cell lines were also alloreactive and were shown to be MHC Class II-restricted. There is evidence of a down regulation of autoreactive T cells by suppressor cells in peripheral blood in two cases with elevated autoreactive T-cell frequencies (one RA patient and one control patient suffering from a viral infection). In contrast, no suppression of autoreactive T cells was observed in the RA patients' SFL or in PBL and SFL from patients with lyme disease. These results suggest that the chronic inflammation observed in RA and lyme disease may be supported by an elevated number of autoreactive T cells in the absence of suppressive mechanisms.