CTLA-4 suppresses the pathogenicity of self antigen-specific T cells by cell-intrinsic and cell-extrinsic mechanisms.

The inhibitory immunoregulatory receptor CTLA-4 is critical in maintaining self-tolerance, but the mechanisms of its actions have remained controversial. Here we examined the antigen specificity of tissue-infiltrating CD4(+) T cells in Ctla4(-/-) mice. After adoptive transfer, T cells isolated from tissues of Ctla4(-/-) mice showed T cell antigen receptor (TCR)-dependent accumulation in the tissues from which they were derived, which suggested reactivity to tissue-specific antigens. We identified the pancreas-specific enzyme PDIA2 as an autoantigen in Ctla4(-/-) mice. CTLA-4 expressed either on PDIA2-specific effector cells or on regulatory T cells was sufficient to control tissue destruction mediated by PDIA2-specific T cells. Our results demonstrate that both cell-intrinsic and non-cell-autonomous actions of CTLA-4 operate to maintain T cell tolerance to a self antigen.

The interplay between citrullination and HLA-DRB1 polymorphism in shaping peptide binding hierarchies in rheumatoid arthritis.

The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated self-peptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/non-citrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarization assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the ß-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structural determination of eight HLA-DR4-self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA ß-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated self-peptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide-HLA-DR4 binding affinities in RA.

Engineering of a novel anti-CD40L domain antibody for treatment of autoimmune diseases.

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

Diversity and Clonality of T Cell Receptor Repertoire and Antigen Specificities in Small Joints of Early Rheumatoid Arthritis.

OBJECTIVE: CD4+ T cells are implicated in rheumatoid arthritis (RA) pathology from the strong association between RA and certain HLA class II gene variants. This study was undertaken to examine the synovial T cell receptor (TCR) repertoire, T cell phenotypes, and T cell specificities in small joints of RA patients at time of diagnosis before therapeutic intervention. METHODS: Sixteen patients, of whom 11 patients were anti-citrullinated protein antibody (ACPA)-positive and 5 patients were ACPA-, underwent ultrasound-guided synovial biopsy of a small joint (n = 13) or arthroscopic synovial biopsy of a large joint (n = 3), followed by direct sorting of single T cells for paired sequencing of the αß TCR together with flow cytometry analysis. TCRs from expanded CD4+ T cell clones of 4 patients carrying an HLA-DRB1*04:01 allele were artificially reexpressed to study antigen specificity. RESULTS: T cell analysis demonstrated CD4+ dominance and the presence of peripheral helper T-like cells in both patient groups. We identified >4,000 unique TCR sequences, as well as 225 clonal expansions. Additionally, T cells with double α-chains were a recurring feature. We identified a biased gene usage of the Vß chain segment TRBV20-1 in CD4+ cells from ACPA+ patients. In vitro stimulation of T cell lines expressing selected TCRs with an extensive panel of citrullinated and viral peptides identified several different virus-specific TCRs (e.g., human cytomegalovirus and human herpesvirus 2). Still, the majority of clones remained orphans with unknown specificity. CONCLUSION: Minimally invasive biopsies of the RA synovium allow for single-cell TCR sequencing and phenotyping. Clonally expanded, viral-reactive T cells account for part of the diverse CD4+ T cell repertoire. TRBV20-1 bias in ACPA+ patients suggests recognition of common antigens.

Do the peptide-binding properties of diabetogenic class II molecules explain autoreactivity?

One seminal aspect in autoimmune diabetes is antigen presentation of beta cell antigens by the diabetes-propensity class II histocompatibility molecules. The binding properties of I-Ag7 molecules are reviewed here and an emphasis is placed on their selection of peptides with a highly specific sequence motif, in which one or more acidic amino acids are found at the carboxy end interacting at the P9 anchoring site of I-Ag7. The reasons for the central role of I-Ag7 in the autoimmune response are analyzed. The insulin B chain segment 9-23 is a hot spot for T cell selection and a striking example of a weak MHC binding peptide that triggers autoreactivity.

IL-12 enhances CTL synapse formation and induces self-reactivity.

Immunological synapse formation between T cells and target cells can affect the functional outcome of TCR ligation by a given MHC-peptide complex. Although synapse formation is usually induced by TCR signaling, it is not clear whether other factors can affect the efficiency of synapse formation. Here, we tested whether cytokines could influence synapse formation between murine CTLs and target cells. We found that IL-12 enhanced synapse formation, whereas TGFbeta decreased synapse formation. The enhanced synapse formation induced by IL-12 appeared to be functional, given that IL-12-treated cells could respond to weak peptides, including self-peptides, to which the T cells were normally unresponsive. These responses correlated with expression of functionally higher avidity LFA-1 on IL-12-treated CTLs. These findings have implications for the function of IL-12 in T cell-mediated autoimmunity.

Dendritic cells in islets of Langerhans constitutively present beta cell-derived peptides bound to their class II MHC molecules.

Islets of Langerhans from normal mice contained dendritic cells (DCs) in the range of 8-10 per islet. DCs were found in several mouse strains, including those from lymphocyte-deficient mice. DCs were absent in islets from colony stimulating factor-1 deficient mice and this absence correlated with small size islets. Most DCs were found next to blood vessels and resided in islets for several days. Some DCs contained insulin-like granules, and most expressed peptide-MHC complexes derived from beta cell proteins. Islet DCs were highly effective in presenting beta cell antigens to CD4 T cells ex vivo. Presentation of beta cell-derived peptide-MHC complexes by DCs neither depended on islet inflammation nor correlated with the extent of spontaneous beta cell death. Periislet stroma DCs did not contain beta cell peptide-MHC complexes; however, 50% of DCs in pancreatic node were positive. Hence, presentation of high levels of beta cell antigens normally takes place by islet DCs, a finding that has to be placed in the perspective of autoimmune diabetes.

Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

The shared susceptibility epitope of HLA-DR4 binds citrullinated self-antigens and the TCR.

Individuals expressing HLA-DR4 bearing the shared susceptibility epitope (SE) have an increased risk of developing rheumatoid arthritis (RA). Posttranslational modification of self-proteins via citrullination leads to the formation of neoantigens that can be presented by HLA-DR4 SE allomorphs. However, in T cell-mediated autoimmunity, the interplay between the HLA molecule, posttranslationally modified epitope(s), and the responding T cell repertoire remains unclear. In HLA-DR4 transgenic mice, we show that immunization with a Fibß-74cit69-81 peptide led to a population of HLA-DR4Fibß-74cit69-81 tetramer+ T cells that exhibited biased T cell receptor (TCR) ß chain usage, which was attributable to selective clonal expansion from the preimmune repertoire. Crystal structures of pre- and postimmune TCRs showed that the SE of HLA-DR4 represented a main TCR contact zone. Immunization with a double citrullinated epitope (Fibß-72,74cit69-81) altered the responding HLA-DR4 tetramer+ T cell repertoire, which was due to the P2-citrulline residue interacting with the TCR itself. We show that the SE of HLA-DR4 has dual functionality, namely, presentation and a direct TCR recognition determinant. Analogous biased TCR ß chain usage toward the Fibß-74cit69-81 peptide was observed in healthy HLA-DR4+ individuals and patients with HLA-DR4+ RA, thereby suggesting a link to human RA.

IFN-gamma-dependent regulatory circuits in immune inflammation highlighted in diabetes.

We demonstrate diverse roles of IFN-gamma in the induction and regulation of immune-mediated inflammation using a transfer model of autoimmune diabetes. The diabetogenic CD4(+)BDC2.5 (BDC) T cell clone upon transfer into NOD.scid mice induced destruction of islets of Langerhans leading to diabetes. Administration of a neutralizing Ab to IFN-gamma (H22) resulted in long-term protection (LTP) from diabetes, with inflammation but persistence of a significant, albeit decreased, number of beta cells. BDC T cells were a mixture of cells expressing high, intermediate, and low levels of the TCR. Clonotype(low) BDC T cells were required for LTP. Furthermore, islet-infiltrating leukocytes in the LTP mice contained Foxp3(+)CD4 T cells. Islet inflammation in both diabetic and LTP mice was characterized by heavy infiltration of macrophages. Gene expression profiles indicated that macrophages in diabetic mice were M1 type, while LTP mice contained M2 differentiated. The LTP was abolished if mice were treated with either Ab-depleting CD4 T cells or a neutralizing Ab to CTLA-4, in this case, only at a late stage. Neutralization of IL-10, TGF-beta, glucocorticoid-induced TNF receptor (GITR), or CD25 had no effect. Transfer of only clonotype(high)-expressing BDC T cells induced diabetes; in contrast, H22 Abs did not inhibit diabetes. While clonotype(high) T cells induced diabetes even when IFN-gamma was neutralized, paradoxically there was reduced inflammation and no diabetes if host myeloid cells lacked IFN-gamma receptor. Hence, using monoclonal CD4 T cells, IFN-gamma can have a wide diversity of roles, depending on the setting of the immune process.

CUE-101, a Novel E7-pHLA-IL2-Fc Fusion Protein, Enhances Tumor Antigen-Specific T-Cell Activation for the Treatment of HPV16-Driven Malignancies.

PURPOSE: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. EXPERIMENTAL DESIGN: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

The emerging role of TRPV1 in diabetes and obesity.

The capsaicin receptor transient receptor potential vanilloid subfamily member 1 (TRPV1) is highly expressed on sensory nerve fibers innervating the pancreas. Indeed, the role of TRPV1 in mediating pain during pancreatitis is well established. The initial excitation of these nerves by capsaicin is followed by a reversible refractory state (desensitization) or, under certain conditions such as neonatal treatment, neurotoxicity. Interestingly, ablation of TRPV1-positive fibers by subcutaneous capsaicin treatment not only ameliorates pancreatitis pain but also diminishes aging-associated weight gain and improves glucose tolerance both in mice on a high-fat diet and in rat models of type 2 diabetes. New evidence implies an unexpected, pivotal role for TRPV1 in type 1 (autoimmune) diabetes. Non-obese diabetic (NOD) mice carry a hypofunctional TRPV1 mutant. Ablation of nerves carrying this mutant TRPV1 by capsaicin prevents immune-mediated destruction of islet beta cells despite the persistence of diabetogenic T cells. Collectively, these findings establish a crucial link among sensory nerves, obesity and diabetes and identify pharmacological TRPV1 blockade as a novel therapeutic approach for diabetes prevention and weight control.

The wide diversity and complexity of peptides bound to class II MHC molecules.

The identification and quantitation of peptides selected by class II MHC molecules during natural processing of proteins is of key importance in understanding the repertoire and distribution of T cells. The examination of peptides selected by class II MHC molecules has depended greatly on mass spectrometry, a powerful technique that identifies and sequences peptides in complex mixtures with great sensitivity and precision. Such analysis has resulted in the identification of several factors, including the repertoire of peptides selected by MHC molecules during natural processing of proteins, motifs important for selection of processed peptides, conformational isomers of peptide-MHC complexes, and post-translational changes to the peptides.

Natural peptides selected by diabetogenic DQ8 and murine I-A(g7) molecules show common sequence specificity.

In this study, a large number of naturally processed peptides was isolated and identified from the human diabetes-susceptible class II MHC molecules HLA-DQ8 (DQA1*0301,DQB1*0302) and from murine I-A species, both of which are expressed in genetically identical APC lines. The peptides presented during the processing of autologous proteins were highly selective in showing sequence specificity, mainly consisting of 1 or more acidic residues at their C terminus. Testing for binding to the MHC molecules revealed that the position 9 (P9) acidic residues of the peptides contributed decisively to binding. For HLA-DQ8, the P1 residue, which was also an acidic amino acid, influenced binding positively. Both HLA-DQ8 and I-A(g7) selected for common peptides that bound in the same register. There was no evidence for selection of peptides having nonspecific or promiscuous binding. Thus, diabetogenic class II MHC molecules are highly selective in terms of the peptides presented by their APCs, and this is governed by the features of their P9 anchor pocket. These results are in striking contrast to those from studies examining synthetic peptide or phage display libraries, in which many peptides were shown to bind.

Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications.

Absence of lymph nodes in NOD mice treated with lymphotoxin-beta receptor immunoglobulin protects from diabetes.

Pregnant nonobese diabetic (NOD) mice were treated with lymphotoxin-beta receptor immunoglobulin fusion protein (LTbetaR-Ig) or control human immunoglobulin on days embryonic day 11 (E11) and E14, and offspring were followed for the development of anti-beta-cell antibodies, islet pathology, and hyperglycemia. The development of anti-beta-cell surface antibodies was abrogated in treated mice compared with controls. Autopsy examination of the mice at 30 weeks of age revealed normal development of secondary lymphoid structures in the control animals; however, mice treated with LTbetaR-Ig had no axillary, inguinal, popliteal, or peripancreatic lymph nodes. Histological examination of the pancreata of the control mice revealed a severe and destructive mononuclear cellular infiltrate in the islets, whereas the islets of the LTbetaR-Ig-treated mice were devoid of any insulitis. None of the LTbetaR-Ig-treated mice (n = 22) developed diabetes; in contrast, 80% of the control mice (n = 46) developed diabetes at 1 year of age. The LTbetaR-Ig-treated mice did not contain diabetogenic T-cells. However, the treated mice developed diabetes upon inoculation with diabetogenic T-cells. In this model of spontaneous autoimmune diabetes, secondary lymphoid structures, most likely the peripancreatic lymph nodes, were essential for the development of pathologic anti-beta-cell autoimmunity.

Antigen presentation: lysoyme, autoimmune diabetes, and Listeria--what do they have in common?

We discuss three areas of antigen presentation and macrophage biology being investigated in the laboratory. Using hen egg-white lysozyme as a protein antigen, all the segments of the molecules selected by the class II histocompatibility molecule I-A(k) were identified and characterized. The display of each family of peptides was explained biochemically and quantitated. Conformational isomers of a peptide-major histocompatibility complex (MHC) complex were identified. The relationship between the amounts of peptide-MHC displayed by the antigen-presenting cells and two biologic responses, central thymic selection and T-cell responses after immunization in adjuvant, were examined. The class II MHC molecule of the nonobese diabetic I-Ag7 is being examined for its properties of peptide selection. The objective is to identify the diabetogenic peptides, as well as the repertoire of protein antigens from beta-cells that trigger autoantibodies. The I-Ag7 molecule selects peptides that show very distinctive sequence motifs: one or more acidic residues at the carboxy terminus that interact at the P9 pocket of the binding groove. Finally, the investigations in listeriosis examined the early events in immune induction. More important, we found that Listeria causes marked apoptosis of lymphocytes around infective foci resulting from the apoptogenic properties of the pore-forming molecule Listeriolysin O.

Pellet digestion: a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma.

BACKGROUND: There is a need for a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma. RESULTS: The sample preparation technique presented here is based upon trypsin digestion of the pellet obtained following precipitation of the protein analyte from plasma. The pellet digestion technique was shown to facilitate efficient digestion of large therapeutic proteins, with concomitant removal of a substantial amount of potentially problematic plasma phospholipids. The technique was successfully applied to a pharmacokinetic study of a large therapeutic protein. CONCLUSION: This simple sample preparation approach will be beneficial to bioanalytical laboratories engaged in the LC-MS/MS quantification of large therapeutic proteins in biological matrices.

Targeting lymphocyte co-stimulation: from bench to bedside.

T and B lymphocytes are central regulators and effectors of immune responses and are believed to have a key role in many autoimmune diseases. Targeting the activation or effector function of lymphocytes is a potentially effective approach to treat autoimmunity. Typically, T-cell activation occurs after engagement of the T-cell receptor with its cognate peptide-major histocompatibility complex (signal 1) and subsequent engagement of co-stimulatory molecules (signal 2). This "second signal" contributes to T-cell activation by promoting proliferation, survival, and effector function. In general, activation in the absence of co-stimulation leads to a reduced immune response, anergy, or even tolerance. B-cell activation similarly requires co-stimulation for the development of complete effector function. The most potent co-stimulatory molecules identified to date are CD28 for T-cells and CD40 for B-cells. Both molecules are recognized for their potential as immune modulators; however, thus far neither molecule has been successfully targeted directly for the treatment of autoimmune disease. The only current therapy to target either of these pathways is cytotoxic T-lymphocyte antigen-4 (CTLA-4-Ig), which indirectly blocks CD28 signaling and has proven efficacy in rheumatoid arthritis and juvenile idiopathic arthritis patients. In addition to CD28 and CD40, an array of other co-stimulatory as well as inhibitory pathways has recently been identified and scientists are just beginning to understand how these different signaling pathways interact to regulate lymphocyte activation. In the more than two decades since the discovery of the first co-stimulatory molecule, the full clinical potential of these pathways is yet to be realized. In this review, we will primarily focus on CD28 and CD40 which are the most clinically validated co-stimulatory pathways, and briefly summarize and discuss some of the other T-cell co-stimulatory molecules.

Weak proinsulin peptide-major histocompatibility complexes are targeted in autoimmune diabetes in mice.

OBJECTIVE: Weak major histocompatibility complex (MHC) binding of self-peptides has been proposed as a mechanism that may contribute to autoimmunity by allowing for escape of autoreactive T-cells from the thymus. We examined the relationship between the MHC-binding characteristics of a beta-cell antigen epitope and T-cell autoreactivity in a model of autoimmune diabetes. RESEARCH DESIGN AND METHODS: The binding of a proinsulin epitope, proinsulin-1(47-64) (PI-1[47-64]), to the MHC class II molecules I-A(g7) and I-A(k) was measured using purified class II molecules. T-cell reactivity to the proinsulin epitope was examined in I-A(g7+) and I-A(k+) mice. RESULTS: C-peptide epitopes bound very weakly to I-A(g7) molecules. However, C-peptide-reactive T-cells were induced after immunization in I-A(g7)-bearing mice (NOD and B6.g7) but not in I-A(k)-bearing mice (B10.BR and NOD.h4). T-cells reactive with the PI-1(47-64) peptide were found spontaneously in the peripancreatic lymph nodes of pre-diabetic NOD mice. These T-cells were activated by freshly isolated beta-cells in the presence of antigen-presenting cells and caused diabetes when transferred into NOD.scid mice. CONCLUSIONS: These data demonstrate an inverse relationship between self-peptide-MHC binding and T-cell autoreactivity for the PI-1(47-64) epitope in autoimmune diabetes.