T-FINDER: A highly sensitive, pan-HLA platform for functional T cell receptor and ligand discovery.

Effective, unbiased, high-throughput methods to functionally identify both class II and class I HLA-presented T cell epitopes and their cognate T cell receptors (TCRs) are essential for and prerequisite to diagnostic and therapeutic applications, yet remain underdeveloped. Here, we present T-FINDER [T cell Functional Identification and (Neo)-antigen Discovery of Epitopes and Receptors], a system to rapidly deconvolute CD4 and CD8 TCRs and targets physiologically processed and presented by an individual's unmanipulated, complete human leukocyte antigen (HLA) haplotype. Combining a highly sensitive TCR signaling reporter with an antigen processing system to overcome previously undescribed limitations to target expression, T-FINDER both robustly identifies unknown peptide:HLA ligands from antigen libraries and rapidly screens and functionally validates the specificity of large TCR libraries against known or predicted targets. To demonstrate its capabilities, we apply the platform to multiple TCR-based applications, including diffuse midline glioma, celiac disease, and rheumatoid arthritis, providing unique biological insights and showcasing T-FINDER's potency and versatility.

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.

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.

An endogenous peptide positively selects and augments the activation and survival of peripheral CD4+ T cells.

Although CD4(+) and CD8(+) T cells differ in the strength of their positively selecting signal, endogenous positively selecting ligands have been identified only for major histocompatibility complex (MHC) class I-restricted T cell antigen receptors (TCRs). Here we screened for ligands able to positively select MHC class II-restricted TCRs using thymocytes from four I-E(k)-restricted TCR-transgenic mice and a large panel of self peptides. One peptide, gp250, induced positive selection of AND CD4(+) T cells, had no homology with the AND TCR agonist ligand and was recognized with a high degree of specificity. The gp250 peptide acted as a coagonist to initiate the activation and enhance the survival of peripheral AND CD4(+) T cells. Thus, positively selecting ligands are critical in thymocyte development and in the activation and maintenance of peripheral T cells.

Specificity of T-cell alloreactivity.

T-cell alloreactivity is a well-established phenomenon, but its molecular basis has remained enigmatic. Although there are differences between T-cell recognition of conventional and allogeneic antigens, it has become increasingly clear that they share many similarities. Recent insights into the specificity of the T-cell receptor (TCR) for peptide and the seeming intrinsic affinity of the TCR for the surface of the MHC molecule have provided a better understanding of how the TCR and peptide-MHC complexes interact. Here, we highlight the similarities and differences between conventional and allogeneic recognition of TCR-peptide-MHC complexes, and discuss how our view of allorecognitionhas changed, as well as the implications for TCR specificity and T-cell development.

Alloreactive T cells respond specifically to multiple distinct peptide-MHC complexes.

The molecular basis underlying the specificity of alloreactive T cells for peptide-major histocompatibility complex ligands has been elusive. Here we describe a screen of 60 I-E(k)-alloreactive T cells and 83 naturally processed peptides that identified 9 reactive T cells. Three of the T cells responded to multiple, distinct peptides that shared no sequence homology. These T cells recognized each peptide-major histocompatibility complex ligand specifically and used a distinct constellation of I-E(k) contact residues for each interaction. Our studies show that alloreactive T cells have a 'germline-encoded' capacity to recognize multiple, distinct ligands and thus show 'polyspecificity', not degeneracy. Our findings help to explain the high frequency of alloreactive T cells and provide insight into the nature of T cell specificity.

I-Ep-bound self-peptides: identification, characterization, and role in alloreactivity.

T cell recognition of peptide/allogeneic MHC complexes is a major cause of transplant rejection. Both the presented self-peptides and the MHC molecules are involved; however, the molecular basis for alloreactivity and the contribution of self-peptides are still poorly defined. The murine 2.102 T cell is specific for hemoglobin(64-76)/I-Ek and is alloreactive to I-Ep. The natural self-peptide/I-Ep complex recognized by 2.102 remains unknown. In this study, we characterized the peptides that are naturally processed and presented by I-Ep and used this information to define the binding motif for the murine I-Ep class II molecule. Interestingly, we found that the P9 anchor residue preferred by I-Ep is quite distinct from the residues preferred by other I-E molecules, although the P1 anchor residue is conserved. A degree of specificity for the alloresponse was shown by the lack of stimulation of 2.102 T cells by 19 different identified self-peptides. The binding motif was used to search the mouse genome for candidate 2.102 reactive allopeptides that contain strong P1 and P9 anchor residues and possess previously identified allowable TCR contact residues. Two potential allopeptides were identified, but only one of these peptides, G protein-coupled receptor 128, was able to stimulate 2.102 T cells. Thus, the G protein-coupled receptor 128 peptide represents a candidate allopeptide that is specifically recognized by 2.102 T cells bound to I-Ep and was identified using bioinformatics. These studies highlight the specific involvement of self-peptides in alloreactivity.

Hierarchical contributions of allorecognition pathways in chronic lung rejection.

The role of allorecognition in initiating lung graft rejection is not clearly defined. Using the heterotopic tracheal transplantation model, we examined the contributions of the indirect and direct allorecognition pathways in chronic airway rejection. Fully mismatched, wild-type grafts were transplanted into major histocompatibility complex (MHC) II-/-, class II-like accessory molecule (H2-DMalpha)-/- using MHC I-/- and wild-type allorecipients as control subjects. Similarly, MHC I-/-, MHC II-/-, or MHC I/II-/- allografts were transplanted into wild-type mice with appropriate control subjects. Grafts from nonimmunosuppressed recipients were evaluated at Weeks 2, 4, and 6. Grafts transplanted into MHC II-/- and H2-DMalpha-/- allorecipients showed a more intact epithelium and reduced lumen obliteration compared with grafts transplanted into wild-type or MHC I-/- allorecipients (p < 0.05 for each). These grafts exhibited abundant CD4+ and CD8+ cell infiltrates similar to control allografts. MHC I-/- and MHC I/II-/- but not MHC II-/- allografts placed in wild-type animals demonstrated less severe rejection compared with allograft control subjects (p < 0.05 for each). Although the indirect allorecognition pathway has the strongest influence on rejection, the direct pathway is sufficient to ultimately cause chronic airway rejection. In addition, these results suggest that MHC class I molecules are the principal alloantigens in the mouse heterotopic tracheal model of obliterative bronchiolitis.

Prolonged survival of class II transactivator-deficient cardiac allografts.

BACKGROUND: Major histocompatibility complex (MHC) antigenic complexes trigger allogeneic T-cell responses and allograft rejection. MHC class II and related antigen processing genes, such as invariant chain (Ii) and H2-DM accessory molecules, are controlled by the master transcriptional regulator, class II transactivator (CIITA). CIITA also up-regulates MHC class I gene expression in vitro. Thus, disruption of a single factor, namely CIITA, represents an ideal strategy for reducing transplant rejection. METHODS: We studied the immunological advantages of transplanting CIITA deficient hearts into mismatched recipients in comparison to wild-type (B6) allografts or MHC class II-deficient (Abeta ) hearts. RESULTS: Elimination of CIITA greatly enhanced graft survival (median survival time [MST] 36 days) over the survival of wild-type (MST 9 days) and even over Abeta (MST 20 days) cardiac grafts. This was accompanied by greatly reduced mixed lymphocyte reactivity and in vivo antigen priming capacity. Analyses for CD4, CD8, and other inflammatory cells, plus cytotoxic T-cell activity and MHC class I specific alloantibody production, did not reveal significant differences in CIITA allograft tissues. Some cytokines that may support immunosuppression, such as transforming growth factor (TGF)-beta, were increased in mice receiving either Abeta or CIITA cardiac grafts. CONCLUSIONS: We conclude that disruption of CIITA function plays a beneficial role in preventing normal allogeneic T-cell responses. Even though inflammatory cells are present in CIITA allografts, the dramatic prolongation in allograft survival of CIITA hearts as compared with wild-type grafts provides a foundation for designing molecular therapies to interfere with MHC class II function and thereby reduce transplantation rejection.

Comparison of Abeta(b-/-), H2-DM(-), and CIITA(-/-) in second-set skin allograft rejection.

BACKGROUND: Responses against donor MHC antigens are the major contributor to allograft rejection. Currently, it is unclear whether both direct and indirect recognition pathways are necessary and/or sufficient for allograft rejection. Previously, we found donor MHC class II and H2-DM to have dramatic effects on cardiac allograft survival. METHODS: Here, we used H2-DM(-) mice, which express CLIP-MHC class II complexes, and CIITA(-/-) mice, which lack all class II proteins, to examine the role of direct and indirect recognition on skin allograft rejection. Recipients were primed with donor cultured keratinocytes and later tested for accelerated memory response by challenge with full-thickness tail skin grafts. RESULTS: As previously reported, Abeta(b-/-) grafts survived longer than wild-type grafts, while H2-DM(-) grafts were rejected as rapidly as wild-type grafts. Skin grafts deficient for both beta2m and H2-DM survived longer than grafts lacking only H2-DM, but not as long as Abeta(b-/-) grafts. Additionally, CIITA(-/-) grafts survived as long as Abeta(b-/-) grafts. CONCLUSIONS: The delayed rejection of Abeta(b-/-) compared to H2-DM(-) suggests that indirect recognition of surface-expressed donor MHC class II is sufficient to mediate rapid skin allograft rejection. The equivalent survival of CIITA(-/-) and Abeta(b-/-) grafts suggests that indirect presentation of donor class II molecules (Aalpha or Ebeta) present in Abeta(b-/-) but not CIITA(-/-) mice does not contribute to graft rejection. These results reveal a modest role for surface-expressed donor class II in primed keratinocyte rejection, but also reveal a dramatic contrast to the cardiac allograft system and indicate tissue/organ-specific mechanisms of rejection.