Peptide Centric Vß Specific Germline Contacts Shape a Specialist T Cell Response.

Certain CD8 T cell responses are particularly effective at controlling infection, as exemplified by elite control of HIV in individuals harboring HLA-B57. To understand the structural features that contribute to CD8 T cell elite control, we focused on a strongly protective CD8 T cell response directed against a parasite-derived peptide (HF10) presented by an atypical MHC-I molecule, H-2Ld. This response exhibits a focused TCR repertoire dominated by Vß2, and a representative TCR (TG6) in complex with Ld-HF10 reveals an unusual structure in which both MHC and TCR contribute extensively to peptide specificity, along with a parallel footprint of TCR on its pMHC ligand. The parallel footprint is a common feature of Vß2-containing TCRs and correlates with an unusual Vα-Vß interface, CDR loop conformations, and Vß2-specific germline contacts with peptides. Vß2 and Ld may represent "specialist" components for antigen recognition that allows for particularly strong and focused T cell responses.

Canonical T cell receptor docking on peptide-MHC is essential for T cell signaling.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.

The plasmacytoid dendritic cells evoke Salmonella-specific CTL effector response / Las células dendríticas plasmacitoides evocan la respuesta efectora de los linfocitos T citotóxicos específicos para Salmonella

Introduction: The immunological role of plasmacytoid dendritic cells (pDC) in bacterial infections such as Salmonella has been poorly documented. Therefore, we analyzed the effector function of these cells by presenting cytotoxic T lymphocytes (CTL) with Salmonella Typhimurium antigens. Objective: To analyze the Salmonella-specific CTL response evoked by pDCs. Materials and methods: We used plasmacytoid dendritic cells stained with carboxyfluorescein succinimidyl ester (CFSE) and pulsed with OmpC73, Salmonella Kb restricted epitopes or S. Typhimurium as targets for cytotoxicity assays. Results: Specific lysis was shown to be statistically significant in pDC + OmpC73 for all effector:target ratios (p≤0.05). For pDC + S. Typhimurium, statistical significance was only observed at a 1:100 ratio (p≤0.05) using OmpC73. Conclusion: Plasmacytoid dendritic cells evoke CTL response during S. Typhimurium infection.

Introducción. La función inmunológica de las células dendríticas plasmacitoides durante las infecciones bacterianas, como la de Salmonella spp., es poco conocida. En ese contexto, se analizó su función efectora para presentar antígenos de Salmonella Typhimurium ante linfocitos T citotóxicos. Objetivo. Analizar la respuesta de los linfocitos T citotóxicos específicos para Salmonella evocada por las células dendríticas plasmacitoides. Materiales y métodos. Se usaron células dendríticas plasmacitoides marcadas con éster de succinimidil-carboxifluoresceína, pulsadas con el epítopo de Salmonella OmpC73 Kb restringido o infectadas con S. Typhimurium como blanco en ensayos de citotoxicidad. Resultados. La lisis específica tuvo significación estadística usando células dendríticas plasmacitoides positivas pulsadas con OmpC73 en todas las relaciones de células efectoras y blanco (E:B) (p≤0,05); en cuanto a las células dendríticas plasmacitoides positivas para S. Typhimurium, solo se observó significación estadística en la relación de 1:100 (p≤0,05) usando las células efectoras OmpC73. Conclusión. Las células dendríticas plasmacitoides pueden evocar la respuesta de los linfocitos T citotóxicos durante la infección con S. Typhimurium.

Las células dendríticas plasmacitoides evocan la respuesta efectora de los linfocitos T citotóxicos específicos para Salmonella / The plasmacytoid dendritic cells evoke Salmonella-specific CTL effector response

Resumen Introducción. La función inmunológica de las células dendríticas plasmacitoides durante las infecciones bacterianas, como la de Salmonella spp., es poco conocida. En ese contexto, se analizó su función efectora para presentar antígenos de Salmonella Typhimurium ante linfocitos T citotóxicos. Objetivo. Analizar la respuesta de los linfocitos T citotóxicos específicos para Salmonella evocada por las células dendríticas plasmacitoides. Materiales y métodos. Se usaron células dendríticas plasmacitoides marcadas con éster de succinimidil-carboxifluoresceína, pulsadas con el epítopo de Salmonella OmpC73 Kb- restringido o infectadas con S. Typhimurium como blanco en ensayos de citotoxicidad. Resultados. La lisis específica tuvo significación estadística usando células dendríticas plasmacitoides positivas pulsadas con OmpC73 en todas las relaciones de células efectoras y blanco (E:B) (p≤0,05); en cuanto a las células dendríticas plasmacitoides positivas para S. Typhimurium, solo se observó significación estadística en la relación de 1:100 (p≤0,05) usando las células efectoras OmpC73. Conclusión. Las células dendríticas plasmacitoides pueden evocar la respuesta de los linfocitos T citotóxicos durante la infección con S. Typhimurium.

Abstract Introduction: The immunological role of plasmacytoid dendritic cells (pDC) in bacterial infections such as Salmonella has been poorly documented. Therefore, we analyzed the effector function of these cells by presenting cytotoxic T lymphocytes (CTL) with Salmonella Typhimurium antigens. Objective: To analyze the Salmonella-specific CTL response evoked by pDCs. Materials and methods: We used plasmacytoid dendritic cells stained with carboxyfluorescein succinimidyl ester (CFSE) and pulsed with OmpC73, Salmonella Kb- restricted epitopes or S. Typhimurium as targets for cytotoxicity assays. Results: Specific lysis was shown to be statistically significant in pDC + OmpC73 for all effector:target ratios (p≤0.05). For pDC + S. Typhimurium, statistical significance was only observed at a 1:100 ratio (p≤0.05) using OmpC73. Conclusion: Plasmacytoid dendritic cells evoke CTL response during S. Typhimurium infection.

A Tolerogenic Artificial APC Durably Ameliorates Experimental Autoimmune Encephalomyelitis by Directly and Selectively Modulating Myelin Peptide-Autoreactive CD4+ and CD8+ T Cells.

In this study, a tolerogenic artificial APC (TaAPC) was developed to directly and selectively modulate myelin-autoreactive CD4+ and CD8+ T cells in the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide-induced experimental autoimmune encephalomyelitis in C57BL/6J mice. Cell-sized polylactic-coglycolic acid microparticles were generated to cocouple target Ags (MOG40-54/H-2Db-Ig dimer, MOG35-55/I-Ab multimer), regulatory molecules (anti-Fas and PD-L1-Fc), and "self-marker" CD47-Fc and encapsulate inhibitory cytokine (TGF-ß1). Four infusions of the TaAPCs markedly and durably inhibited the experimental autoimmune encephalomyelitis progression and reduced the local inflammation in CNS tissue. They circulated throughout vasculature into peripheral lymphoid tissues and various organs, but not into brain, with retention of 36 h and exerted direct effects on T cells in vivo and in vitro. Two infusions of the TaAPCs depleted 65-79% of MOG35-55-specific CD4+ and 46-62% of MOG40-54-specific CD8+ T cells in peripheral blood, spleen, and CNS tissues in an Ag-specific manner and regulatory molecule-dependent fashion; induced robust T cell apoptosis; inhibited the activation and proliferation of MOG peptide-reactive T cells; reduced MOG peptide-reactive Th1, Th17, and Tc17 cells; and expanded regulatory T cells. They also inhibited IFN-γ/IL-17A secretion and elevated IL-10/TGF-ß1 production in splenocytes but not in CNS tissue. More importantly, the TaAPCs treatment did not obviously suppress the overall immune function of host. To our knowledge, this study provides the first experimental evidence for the capability of TaAPCs to directly modulate autoreactive T cells by surface presentation of multiple ligands and paracrine release of cytokine, thus suggesting a novel Ag-specific immunotherapy for the T cell-mediated autoimmune diseases.

The Immunogenicity of a Proline-Substituted Altered Peptide Ligand toward the Cancer-Associated TEIPP Neoepitope Trh4 Is Unrelated to Complex Stability.

Human cancers frequently display defects in Ag processing and presentation allowing for immune evasion, and they therefore constitute a significant challenge for T cell-based immunotherapy. We have previously demonstrated that the antigenicity of tumor-associated Ags can be significantly enhanced through unconventional residue modifications as a novel tool for MHC class I (MHC-I)-based immunotherapy approaches. We have also previously identified a novel category of cancer neo-epitopes, that is, T cell epitopes associated with impaired peptide processing (TEIPP), that are selectively presented by MHC-I on cells lacking the peptide transporter TAP. In this study, we demonstrate that substitution of the nonanchoring position 3 into a proline residue of the first identified TEIPP peptide, the murine Trh4, results in significantly enhanced recognition by antitumor CTLs toward the wild-type epitope. Although higher immunogenicity has in most cases been associated with increased MHC/peptide complex stability, our results demonstrate that the overall stability of H-2Db in complex with the highly immunogenic altered peptide ligand Trh4-p3P is significantly reduced compared with wild-type H-2Db/Trh4. Comparison of the crystal structures of the H-2Db/Trh4-p3P and H-2Db/Trh4 complexes revealed that the conformation of the nonconventional methionine anchor residue p5M is altered, deleting its capacity to form adequate sulfur-π interactions with H-2Db residues, thus reducing the overall longevity of the complex. Collectively, our results indicate that vaccination with Thr4-p3P significantly enhances T cell recognition of targets presenting the wild-type TEIPP epitope and that higher immunogenicity is not necessarily directly related to MHC/peptide complex stability, opening for the possibility to design novel peptide vaccines with reduced MHC/peptide complex stability.

Modification of host dendritic cells by microchimerism-derived extracellular vesicles generates split tolerance.

Maternal microchimerism (MMc) has been associated with development of allospecific transplant tolerance, antitumor immunity, and cross-generational reproductive fitness, but its mode of action is unknown. We found in a murine model that MMc caused exposure to the noninherited maternal antigens in all offspring, but in some, MMc magnitude was enough to cause membrane alloantigen acquisition (mAAQ; "cross-dressing") of host dendritic cells (DCs). Extracellular vesicle (EV)-enriched serum fractions from mAAQ+, but not from non-mAAQ, mice reproduced the DC cross-dressing phenomenon in vitro. In vivo, mAAQ was associated with increased expression of immune modulators PD-L1 (programmed death-ligand 1) and CD86 by myeloid DCs (mDCs) and decreased presentation of allopeptide+self-MHC complexes, along with increased PD-L1, on plasmacytoid DCs (pDCs). Remarkably, both serum EV-enriched fractions and membrane microdomains containing the acquired MHC alloantigens included CD86, but completely excluded PD-L1. In contrast, EV-enriched fractions and microdomains containing allopeptide+self-MHC did not exclude PD-L1. Adoptive transfer of allospecific transgenic CD4 T cells revealed a "split tolerance" status in mAAQ+ mice: T cells recognizing intact acquired MHC alloantigens proliferated, whereas those responding to allopeptide+self-MHC did not. Using isolated pDCs and mDCs for in vitro culture with allopeptide+self-MHC-specific CD4 T cells, we could replicate their normal activation in non-mAAQ mice, and PD-L1-dependent anergy in mAAQ+ hosts. We propose that EVs provide a physiologic link between microchimerism and split tolerance, with implications for tumor immunity, transplantation, autoimmunity, and reproductive success.

Kinetic and thermodynamic studies of the interaction between activating and inhibitory Ly49 natural killer receptors and MHC class I molecules.

Natural killer (NK) cells are lymphocytes of the innate immune system that eliminate virally infected or malignantly transformed cells. NK cell function is regulated by diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 receptors control NK cell cytotoxicity by sensing major histocompatibility complex class I molecules (MHC-I) on target cells. Although crystal structures have been reported for Ly49/MHC-I complexes, the underlying binding mechanism has not been elucidated. Accordingly, we carried out thermodynamic and kinetic experiments on the interaction of four NK Ly49 receptors (Ly49G, Ly49H, Ly49I and Ly49P) with two MHC-I ligands (H-2Dd and H-2Dk). These Ly49s embrace the structural and functional diversity of the highly polymorphic Ly49 family. Combining surface plasmon resonance, fluorescence anisotropy and far-UV circular dichroism (CD), we determined that the best model to describe both inhibitory and activating Ly49/MHC-I interactions is one in which the two MHC-I binding sites of the Ly49 homodimer present similar binding constants for the two sites (∼106 M-1) with a slightly positive co-operativity in some cases, and without far-UV CD observable conformational changes. Furthermore, Ly49/MHC-I interactions are diffusion-controlled and enthalpy-driven. These features stand in marked contrast with the activation-controlled and entropy-driven interaction of Ly49s with the viral immunoevasin m157, which is characterized by strong positive co-operativity and conformational selection. These differences are explained by the distinct structures of Ly49/MHC-I and Ly49/m157 complexes. Moreover, they reflect the opposing roles of NK cells to rapidly scan for virally infected cells and of viruses to escape detection using immunoevasins such as m157.

Protective T Cell Responses Featured by Concordant Recognition of Middle East Respiratory Syndrome Coronavirus-Derived CD8+ T Cell Epitopes and Host MHC.

The coordinated recognition of virus-derived T cell epitopes and MHC molecules by T cells plays a pivotal role in cellular immunity-mediated virus clearance. It has been demonstrated that the conformation of MHC class I (MHC I) molecules can be adjusted by the presented peptide, which impacts T cell activation. However, it is still largely unknown whether the conformational shift of MHC I influences the protective effect of virus-specific T cells. In this study, utilizing the Middle East respiratory syndrome coronavirus-infected mouse model, we observed that through the unusual secondary anchor Ile5, a CD8+ T cell epitope drove the conformational fit of Trp73 on the α1 helix of murine MHC I H-2Kd In vitro renaturation and circular dichroism assays indicated that this shift of the structure did not influence the peptide/MHC I binding affinity. Nevertheless, the T cell recognition and the protective effect of the peptide diminished when we made an Ile to Ala mutation at position 5 of the original peptide. The molecular bases of the concordant recognition of T cell epitopes and host MHC-dependent protection were demonstrated through both crystal structure determination and tetramer staining using the peptide-MHC complex. Our results indicate a coordinated MHC I/peptide interaction mechanism and provide a beneficial reference for T cell-oriented vaccine development against emerging viruses such as Middle East respiratory syndrome coronavirus.

Recognition of the Major Histocompatibility Complex (MHC) Class Ib Molecule H2-Q10 by the Natural Killer Cell Receptor Ly49C.

Murine natural killer (NK) cells are regulated by the interaction of Ly49 receptors with major histocompatibility complex class I molecules (MHC-I). Although the ligands for inhibitory Ly49 were considered to be restricted to classical MHC (MHC-Ia), we have shown that the non-classical MHC molecule (MHC-Ib) H2-M3 was a ligand for the inhibitory Ly49A. Here we establish that another MHC-Ib, H2-Q10, is a bona fide ligand for the inhibitory Ly49C receptor. H2-Q10 bound to Ly49C with a marginally lower affinity (∼5 µm) than that observed between Ly49C and MHC-Ia (H-2K(b)/H-2D(d), both ∼1 µm), and this recognition could be prevented by cis interactions with H-2K in situ To understand the molecular details underpinning Ly49·MHC-Ib recognition, we determined the crystal structures of H2-Q10 and Ly49C bound H2-Q10. Unliganded H2-Q10 adopted a classical MHC-I fold and possessed a peptide-binding groove that exhibited features similar to those found in MHC-Ia, explaining the diverse peptide binding repertoire of H2-Q10. Ly49C bound to H2-Q10 underneath the peptide binding platform to a region that encompassed residues from the α1, α2, and α3 domains, as well as the associated ß2-microglobulin subunit. This docking mode was conserved with that previously observed for Ly49C·H-2K(b) Indeed, structure-guided mutation of Ly49C indicated that Ly49C·H2-Q10 and Ly49C·H-2K(b) possess similar energetic footprints focused around residues located within the Ly49C ß4-stand and L5 loop, which contact the underside of the peptide-binding platform floor. Our data provide a structural basis for Ly49·MHC-Ib recognition and demonstrate that MHC-Ib represent an extended family of ligands for Ly49 molecules.

Identification of the murine H-2D(b) and human HLA-A*0201 MHC class I-restricted HPV6 E7-specific cytotoxic T lymphocyte epitopes.

Recurrent respiratory papillomatosis is caused by human papillomavirus (HPV) infection, most commonly types 6 (HPV-6) and 11 (HPV-11). Due to failed host immune responses, HPV is unable to be cleared from the host, resulting in recurrent growth of HPV-related lesions that can obstruct the lumen of the airway within the upper aerodigestive tract. In our murine model, the HPV-6b and HPV-11 E7 antigens are not innately immunogenic. In order to enhance the host immune responses against the HPV E7 antigen, we linked calreticulin (CRT) to HPV-6b E7 and found that vaccinating C57BL/6 mice with the HPV-6b CRT/E7 DNA vaccine is able to induce a CD8+ T cell response that recognizes an H-2D(b)-restricted E7aa21-29 epitope. Additionally, vaccination of HLA-A*0201 transgenic mice with HPV-6b CRT/E7 DNA generated a CD8+ T cell response against the E7aa82-90 epitope that was not observed in the wild-type C57BL/6 mice, indicating this T cell response is restricted to HLA-A*0201. In vivo cytotoxic T cell killing assays demonstrated that the vaccine-induced CD8+ T cells are able to efficiently kill target cells. Interestingly, the H-2D(b)-restricted E7aa21-29 sequence and the HLA-A*0201-restricted E7aa82-90 sequence are conserved between HPV-6b and HPV-11 and may represent shared immunogenic epitopes. The identification of the HPV-6b/HPV-11 CD8+ T cell epitopes facilitates the evaluation of various immunomodulatory strategies in preclinical models. More importantly, the identified HLA-A*0201-restricted T cell epitope may serve as a peptide vaccination strategy, as well as facilitate the monitoring of vaccine-induced HPV-specific immunologic responses in future human clinical trials.

The MHC Class I Cancer-Associated Neoepitope Trh4 Linked with Impaired Peptide Processing Induces a Unique Noncanonical TCR Conformer.

MHC class I downregulation represents a significant challenge for successful T cell-based immunotherapy. T cell epitopes associated with impaired peptide processing (TEIPP) constitute a novel category of immunogenic Ags that are selectively presented on transporter associated with Ag processing-deficient cells. The TEIPP neoepitopes are CD8 T cell targets, derived from nonmutated self-proteins that might be exploited to prevent immune escape. In this study, the crystal structure of H-2D(b) in complex with the first identified TEIPP Ag (MCLRMTAVM) derived from the Trh4 protein has been determined to 2.25 Å resolution. In contrast to prototypic H-2D(b) peptides, Trh4 takes a noncanonical peptide-binding pattern with extensive sulfur-π interactions that contribute to the overall complex stability. Importantly, the noncanonical methionine at peptide position 5 acts as a main anchor, altering only the conformation of the H-2D(b) residues Y156 and H155 and thereby forming a unique MHC/peptide conformer that is essential for recognition by TEIPP-specific T cells. Substitution of peptide residues p2C and p5M to the conservative α-aminobutyric acid and norleucine, respectively, significantly reduced complex stability, without altering peptide conformation or T cell recognition. In contrast, substitution of p5M to a conventional asparagine abolished recognition by the H-2D(b)/Trh4-specific T cell clone LnB5. We anticipate that the H-2D(b)/Trh4 complex represents the first example, to our knowledge, of a broader repertoire of alternative MHC class I binders.

Anti-CD45 radioimmunotherapy without TBI before transplantation facilitates persistent haploidentical donor engraftment.

Many patients with hematologic malignancies cannot tolerate hematopoietic cell transplantation (HCT), whereas others may not have a compatible human leukocyte antigen-matched donor. To overcome these limitations, we optimized a conditioning regimen employing anti-CD45 radioimmunotherapy (RIT) replacing total body irradiation (TBI) before haploidentical HCT in a murine model. Mice received 200 to 400 µCi (90)Y-anti-CD45 antibody (30F11), with or without fludarabine (5 days starting day -8), with cyclophosphamide (CY; days -2 and +2) for graft-versus-host disease prophylaxis, and 1.5 × 10(7) haploidentical donor bone marrow cells (day 0). Haploidentical bone marrow transplantation (BMT) with 300 µCi (90)Y-anti-CD45 RIT and CY, without TBI or fludarabine, led to mixed chimeras with 81.3 ± 10.6% mean donor origin CD8(+) cells detected 1 month after BMT, and remained stable (85.5 ± 11% mean donor origin CD8(+) cells) 6 months after haploidentical BMT. High chimerism levels were induced across multiple hematopoietic lineages 28 days after haploidentical BMT with 69.3 ± 14.1%, 75.6 ± 20.2%, and 88.5 ± 11.8% CD3(+) T cells, B220(+) B cells, and CD11b(+) myeloid cells, respectively. Fifty percent of SJL leukemia-bearing mice treated with 400 µCi (90)Y-DOTA-30F11, CY, and haploidentical BMT were cured and lived >200 days. Mice treated with 200 µCi (90)Y-DOTA-30F11 had a median overall survival of 73 days, while untreated leukemic mice had a median overall survival of 34 days (P < .001, Mantel-Cox test). RIT-mediated haploidentical BMT without TBI may increase treatment options for aggressive hematologic malignancies.

An endocytic YXXΦ (YRRF) cargo sorting motif in the cytoplasmic tail of murine cytomegalovirus AP2 'adapter adapter' protein m04/gp34 antagonizes virus evasion of natural killer cells.

Viruses have evolved proteins that bind immunologically relevant cargo molecules at the cell surface for their downmodulation by internalization. Via a tyrosine-based sorting motif YXXΦ in their cytoplasmic tails, they link the bound cargo to the cellular adapter protein-2 (AP2), thereby sorting it into clathrin-triskelion-coated pits for accelerated endocytosis. Downmodulation of CD4 molecules by lentiviral protein NEF represents the most prominent example. Based on connecting cargo to cellular adapter molecules, such specialized viral proteins have been referred to as 'connectors' or 'adapter adapters.' Murine cytomegalovirus glycoprotein m04/gp34 binds stably to MHC class-I (MHC-I) molecules and suspiciously carries a canonical YXXΦ endocytosis motif YRRF in its cytoplasmic tail. Disconnection from AP2 by motif mutation ARRF should retain m04-MHC-I complexes at the cell surface and result in an enhanced silencing of natural killer (NK) cells, which recognize them via inhibitory receptors. We have tested this prediction with a recombinant virus in which the AP2 motif is selectively destroyed by point mutation Y248A, and compared this with the deletion of the complete protein in a Δm04 mutant. Phenotypes were antithetical in that loss of AP2-binding enhanced NK cell silencing, whereas absence of m04-MHC-I released them from silencing. We thus conclude that AP2-binding antagonizes NK cell silencing by enhancing endocytosis of the inhibitory ligand m04-MHC-I. Based on a screen for tyrosine-based endocytic motifs in cytoplasmic tail sequences, we propose here the new hypothesis that most proteins of the m02-m16 gene family serve as 'adapter adapters,' each selecting its specific cell surface cargo for clathrin-assisted internalization.

Identification of an atypical CD8 T cell epitope encoded by murine cytomegalovirus ORF-M54 gaining dominance after deletion of the immunodominant antiviral CD8 T cell specificities.

Control of murine cytomegalovirus (mCMV) infection is mediated primarily by CD8 T cells, with four specificities dominating in BALB/c mice. Functional deletion of the respective immunodominant epitopes (IDEs) in mutant virus Δ4IDE revealed a still efficient control of infection. In a murine model of hematopoietic cell transplantation and infection with Δ4IDE, an mCMV-specific open reading frame (ORF) library screening assay indicated a strong CD8 T cell reactivity against the ORF-M54 product, the highly conserved and essential mCMV homolog of human CMV DNA polymerase UL54, which is a known inducer of in vivo protection against mCMV by DNA immunization. Applying bioinformatic algorithms for CD8 T cell epitope prediction, the top-scoring peptides were used to stimulate ex vivo-isolated CD8 T cells and to generate cytolytic T cell lines; yet, this approach failed to identify M54 epitope(s). As an alternative, a peptide library consisting of 549 10-mers with an offset of two amino acids (aa), covering the complete aa-sequence of the M54 protein, was synthesized and used for the stimulation. A region of 12 aa proved to encompass an epitope. An 'alanine walk' over this antigenic 12-mer and all possible 11-, 10- and 9-mers derived thereof revealed aa-residues critical for antigenicity, and terminal truncations identified the H-2D(d) presented 8-mer M5483-90 as the optimal epitope. An increased frequency of the corresponding CD8 T cells in the absence of the 4 IDEs indicated immunodomination by the IDE-specific CD8 T cells as a mechanism by which the generation of M54-specific CD8 T cells is inhibited after infection with wild-type mCMV.

Genetic investigation of MHC-independent missing-self recognition by mouse NK cells using an in vivo bone marrow transplantation model.

MHC-I-specific receptors play a vital role in NK cell-mediated "missing-self" recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b(-/-) bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2D(b-/-) MHC-I-deficient BM cells. Selective rejection of Clr-b(-/-) BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b(-/-) BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1B(hi) NK cells, leaving the remaining NKR-P1B(lo) NK subset and MHC-I-dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b(-/-) hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I-deficient NK cells, Clr-b(-/-) NK cells were hyporesponsive to both NK1.1 (NKR-P1C)-stimulated and IL-12/18 cytokine-primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.

Ocular inflammation in HLA-B27 transgenic mice reveals a potential role for MHC class I in corneal immune privilege.

PURPOSE: HLA-B27 is a major histocompatibility complex class I (MHCI) allele that has been closely associated with the development of ankylosing spondylitis and acute anterior uveitis (AAU), the most common form of uveitis worldwide. We have been characterizing the phenotypes of transgenic mice carrying a human HLA-B27 allele, but that are deficient in endogenous mouse MHCI genes (H-2K(-/-) and H-2D(-/-) double knockout, or DKO) to create the HLA-B27/DKO line. In maintaining and expanding this colony, we observed a rare sporadic severe central keratitis that developed in transgenic animals, but that was not present in wild-type (WT) animals. METHODS: The corneas of affected HLA-B27/DKO and DKO mice were compared to their WT counterparts by staining with standard histological methods for markers of inflammation and neovascularization. A model of experimental corneal inflammation was subsequently used to test the responses of each genotype to insult. RESULTS: We identified a previously unreported corneal pathology in naïve HLA-B27/DKO mice, and we describe significantly prolonged CD4(+)- and CD8(+)-associated inflammation in these animals following an experimentally induced corneal injury. CONCLUSIONS: These results demonstrate an increased T-cell response in B27/DKO corneas due to the expression of the HLA-B27 allele, suggesting that low MHCI expression in WT corneas is an important contributor to immune privilege.

CD8+ T cells control Ross River virus infection in musculoskeletal tissues of infected mice.

Ross River virus (RRV), chikungunya virus, and related alphaviruses cause debilitating polyarthralgia and myalgia. Mouse models of RRV and chikungunya virus have demonstrated a role for the adaptive immune response in the control of these infections. However, questions remain regarding the role for T cells in viral control, including the magnitude, location, and dynamics of CD8(+) T cell responses. To address these questions, we generated a recombinant RRV expressing the H-2(b)-restricted glycoprotein 33 (gp33) determinant derived from the glycoprotein of lymphocytic choriomeningitis virus. Using tetramers, we tracked gp33-specific CD8(+) T cells during RRV-lymphocytic choriomeningitis virus infection. We found that acute RRV infection induces activation of CD8(+) T cell responses in lymphoid and musculoskeletal tissues that peak from 10-14 d postinoculation, suggesting that CD8(+) T cells contribute to control of acute RRV infection. Mice genetically deficient for CD8(+) T cells or wild-type mice depleted of CD8(+) T cells had elevated RRV loads in skeletal muscle tissue, but not joint-associated tissues, at 14 d postinoculation, suggesting that the ability of CD8(+) T cells to control RRV infection is tissue dependent. Finally, adoptively transferred T cells were capable of reducing RRV loads in skeletal muscle tissue of Rag1(-/-) mice, indicating that T cells can contribute to the control of RRV infection in the absence of B cells and Ab. Collectively, these data demonstrate a role for T cells in the control of RRV infection and suggest that the antiviral capacity of T cells is controlled in a tissue-specific manner.

Antigen-specific expansion and differentiation of natural killer cells by alloantigen stimulation.

Natural killer (NK) cells provide important host defense against microbial pathogens and can generate a population of long-lived memory NK cells after infection or immunization. Here, we addressed whether NK cells can expand and differentiate after alloantigen stimulation, which may be important in hematopoietic stem cell and solid tissue transplantation. A subset of NK cell in C57BL/6 mice expresses the activating Ly49D receptor that is specific for H-2D(d). These Ly49D(+) NK cells can preferentially expand and differentiate when challenged with allogeneic H-2D(d) cells in the context of an inflammatory environment. H-2D(d) is also recognized by the inhibitory Ly49A receptor, which, when coexpressed on Ly49D(+) NK cells, suppresses the expansion of Ly49D(+) NK cells. Specificity of the secondary response of alloantigen-primed NK cells was defined by the expression of activating Ly49 receptors and regulated by the inhibitory receptors for MHC class I. Thus, the summation of signals through a repertoire of Ly49 receptors controls the adaptive immune features of NK cells responding to allogeneic cells.

A novel murine cytomegalovirus vaccine vector protects against Mycobacterium tuberculosis.

Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.