Tregitopes and impaired antigen presentation: Drivers of the immunomodulatory effects of IVIg?

INTRODUCTION: Although intravenous immunoglobulin (IVIg) is commonly used in the clinic to treat various autoimmune and severe inflammatory diseases, the mode of action is not fully elucidated. This work investigates two proposed mechanisms: (1) the potential role of regulatory T-cell epitopes (Tregitopes) from the constant domain of IgG in the immunosuppressive function of IVIg; and (2) a potential impact of IVIg on the ability of antigen presenting cells (APCs) to present peptides. METHODS AND RESULTS: Investigation of the HLA class II peptide repertoire from IVIg-loaded dendritic cells (DCs) via MHC-associated peptide proteomics (MAPPs) revealed that numerous IgG-derived peptides were strongly presented along the antibody sequence. Surprisingly, Tregitopes 167 and 289 did not show efficient natural presentation although they both bound to HLA class II when directly loaded as "naked" peptides on human DCs. In addition, both Tregitopes could not reproduce the inhibitory effect of IVIg in a human in vitro T-cell proliferation assay as well as in vivo in mice. MAPPs data demonstrate that presentation of peptides from several antigens remained unchanged even when competed with high doses of IVIg, in both human and mouse. CONCLUSION: These data suggest that the effects mediated by IVIg are not caused by Tregitopes nor by impaired antigen presentation.

Affinity for self antigen selects Treg cells with distinct functional properties.

The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

Coreceptor scanning by the T cell receptor provides a mechanism for T cell tolerance.

In the thymus, high-affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen-engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck; this is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (0.2 s) to initiate negative selection compared to MHCI-restricted TCRs (0.9 s) because more CD4 coreceptors are Lck-loaded compared to CD8. We generated a model (Lck come&stay/signal duration) that accurately predicts the observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance.

The transcription factor T-bet is induced by IL-15 and thymic agonist selection and controls CD8αα(+) intraepithelial lymphocyte development.

CD8αα(+) intraepithelial lymphocytes (IELs) are instrumental in maintaining the epithelial barrier in the intestine. Similar to natural killer cells and other innate lymphoid cells, CD8αα(+) IELs constitutively express the T-box transcription factor T-bet. However, the precise role of T-bet for the differentiation or function of IELs is unknown. Here we show that mice genetically deficient for T-bet lacked both TCRαß(+) and TCRγδ(+) CD8αα(+) IELs and thus are more susceptible to chemically induced colitis. Although T-bet was induced in thymic IEL precursors (IELPs) as a result of agonist selection and interleukin-15 (IL-15) receptor signaling, it was dispensable for the generation of IELPs. Subsequently, T-bet was required for the IL-15-dependent activation, differentiation, and expansion of IELPs in the periphery. Our study reveals a function of T-bet as a central transcriptional regulator linking agonist selection and IL-15 signaling with the emergence of CD8αα(+) IELs.

Antigen recognition by autoreactive CD4⁺ thymocytes drives homeostasis of the thymic medulla.

The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4⁺ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4⁺ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4⁺ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.

Elevation of intracellular cyclic AMP in alloreactive CD4(+) T Cells induces alloantigen-specific tolerance that can prevent GVHD lethality in vivo.

Cyclic AMP (cAMP) is an important negative regulator of T cell activation, and an increased level of cAMP is associated with T cell hyporesponsiveness in vitro. We sought to determine whether elevating intracellular cAMP levels ex vivo in alloreactive T cells during primary mixed lymphocyte reactions (MLR) is sufficient to induce alloantigen-specific tolerance and prevent graft-versus-host disease (GVHD). Primary MLRs were treated with exogenous (8)Br-cAMP and IBMX, a compound that increases intracellular cAMP levels by inhibition of phosphodiesterases. T cell proliferation and IL-2 responsiveness in the treated primary MLR cultures were greatly reduced, and viable T cells recovered on day 8 also had impaired responses to restimulation with alloantigen compared to control-treated cells, but without an impairment to nonspecific mitogens. Labeling experiments showed that cAMP/IBMX inhibited alloreactive T cell proliferation by limiting the number of cell divisions, increasing susceptibility to apoptosis, and rendering nondeleted alloreactive T cells hyporesponsive to alloantigen restimulation. cAMP/IBMX-treated CD4(+) T cells had a markedly reduced capacity for GVHD lethality in major histocompatibility complex class II disparate recipients, but maintained the capacity to mediate other CD4(+) T cell responses in vivo. Thus, our results provide the first preclinical evidence of using cAMP-elevating pharmaceutical reagents to achieve long-term alloantigen-specific T cell tolerance that is sufficient to prevent GVHD.

Lck regulates the threshold of activation in primary T cells, while both Lck and Fyn contribute to the magnitude of the extracellular signal-related kinase response.

The src family kinases p56lck (Lck) and p59fyn (Fyn) are the most proximal signaling molecules to be activated downstream of the T-cell receptor. Using an inducible transgenic model, we can regulate the expression of Lck in primary T cells and ask how the signaling cascade and differentiation potential are affected by the absence or the presence of reduced levels of Lck. We show that in naïve T cells, Lck controls the threshold of activation by preferentially regulating multiple signaling pathways that result in the mobilization of Ca2+ through activation of phospholipase C-gamma and protein kinase C as well as activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. Fyn is also able to stimulate the ERK/MAPK pathway in primary T cells but has little influence on the mobilization of Ca2+. Only Lck efficiently stimulates production of diacylglycerol and therefore RasGRP1 recruitment to the plasma membrane and phosphorylation of Shc, suggesting that Fyn activates ERK via a different upstream signaling route. Finally, we show that signals through Lck are essential for the development of T-cell-effector potential, particularly for effective cytokine transcription.

The CD3epsilon proline-rich sequence, and its interaction with Nck, is not required for T cell development and function.

The CD3epsilon proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3epsilon PRS, we have generated mice that lack this motif (CD3epsilon.PRS(M)). Pull-down experiments demonstrated the inability of Nck to bind to the CD3epsilon PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3epsilon.PRS(M) T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3epsilon PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.

Distinct temporal programming of naive CD4+ T cells for cell division versus TCR-dependent death susceptibility by antigen-presenting macrophages.

Naive T cells become programmed for clonal expansion and contraction during the early hours of antigenic signaling. Recent studies support an 'autopilot' model, wherein the commitment to proliferate and the magnitude of the proliferative response are simultaneously determined during a single, brief period of antigen exposure. Here, we have examined whether the proliferation of naive CD4+ T cells must occur on 'autopilot', or whether extended periods of antigenic signaling can impact primary proliferative responses to antigen-presenting macrophages (macrophage APC). We found that a single exposure to antigen (18 h) simultaneously committed T cells to (1) up-regulate surface TCR above the level expressed on naive T cells, (2) undergo minimal cell division, and (3) acquire susceptibility to TCR-dependent activation-induced cell death. However, continued antigenic signaling between 18 and 72 h was required to amplify the number of daughter cells derived from the already committed T cells. Thus, a discrete commitment time was followed by a 'tuning' period, where extended antigenic signaling determined the volume of the proliferative response. We conclude that T cell commitment to full clonal expansion versus TCR-dependent death susceptibility represent two separate programming events whose timing can be segregated by macrophage APC.

T cell receptor engagement by peptide-MHC ligands induces a conformational change in the CD3 complex of thymocytes.

The T cell receptor (TCR) can recognize a variety of cognate peptide/major histocompatibility complex (pMHC) ligands and translate their affinity into distinct cellular responses. To achieve this, the nonsignaling alphabeta heterodimer communicates ligand recognition to the CD3 signaling subunits by an unknown mechanism. In thymocytes, we found that both positive- and negative-selecting pMHC ligands expose a cryptic epitope in the CD3 complex upon TCR engagement. This conformational change is induced in vivo and requires the expression of cognate MHC. We conclude that TCR engagement with a cognate pMHC ligand induces a conformational change in the CD3 complex of thymocytes and propose that this marks an initial event during thymic selection that signals the recognition of self-antigen.

T cell division and death are segregated by mutation of TCRbeta chain constant domains.

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.

Mutational analysis of conserved amino acids in the T cell receptor alpha-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression.

Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.

The beta1 and beta3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation.

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.

CD3 delta establishes a functional link between the T cell receptor and CD8.

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.

Inability to induce tolerance through direct antigen presentation.

Both the direct and indirect antigen presentation pathways are important mechanisms for T cell-mediated allograft rejection. Studies using knockout mice and monoclonal antibodies have demonstrated that CD4+ T cells are both necessary and sufficient for the rejection of allogeneic tissues, including skin, heart, and islet. Furthermore, combined blockade of the CD28/B7 and CD154/CD40 costimulatory pathways induces tolerance in multiple CD4+ T-cell dependent allograft models. In this study, we addressed the T-cell requirement for costimulation in direct antigen presentation. We demonstrated that class II-specific alloreactive T-cell receptor transgenic T cells were sufficient to mediate allograft rejection independent of costimulatory blockade. Analysis of the costimulatory capacity of different antigen presenting cell (APC) populations demonstrated that APCs resident within the donor skin, Langerhans cells, are potent stimulators not requiring CD28- or CD154-dependent costimulation for direct major histocompatibility complex (MHC) antigen presentation. These results complement previous work examining the role of costimulation on CD8+ T cells, supporting a model in which the effectiveness of costimulatory blockade in the setting of transplantation may be selective for the indirect pathway of MHC alloantigen presentation.