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Although the CNS is immune privileged, continuous search for pathogens and tumours by immune cells within the CNS is indispensable. Thus, distinct immune-cell populations also cross the blood-brain barrier independently of inflammation/under homeostatic conditions. It was previously shown that effector memory T cells populate healthy CNS parenchyma in humans and, independently, that CCR5-expressing lymphocytes as well as CCR5 ligands are enriched in the CNS of patients with multiple sclerosis. Apart from the recently described CD8+ CNS tissue-resident memory T cells, we identified a population of CD4+CCR5high effector memory cells as brain parenchyma-surveilling cells. These cells used their high levels of VLA-4 to arrest on scattered VCAM1, their open-conformation LFA-1 to crawl preferentially against the flow in search for sites permissive for extravasation, and their stored granzyme K (GZMK) to induce local ICAM1 aggregation and perform trans-, rather than paracellular diapedesis through unstimulated primary brain microvascular endothelial cells. This study included peripheral blood mononuclear cell samples from 175 healthy donors, 29 patients infected with HIV, with neurological symptoms in terms of cognitive impairment, 73 patients with relapsing-remitting multiple sclerosis in remission, either 1-4 weeks before (n = 29), or 18-60 months after the initiation of natalizumab therapy (n = 44), as well as white matter brain tissue of three patients suffering from epilepsy. We here provide ex vivo evidence that CCR5highGZMK+CD4+ effector memory T cells are involved in CNS immune surveillance during homeostasis, but could also play a role in CNS pathology. Among CD4+ T cells, this subset was found to dominate the CNS of patients without neurological inflammation ex vivo. The reduction in peripheral blood of HIV-positive patients with neurological symptoms correlated to their CD4 count as a measure of disease progression. Their peripheral enrichment in multiple sclerosis patients and specific peripheral entrapment through the CNS infiltration inhibiting drug natalizumab additionally suggests a contribution to CNS autoimmune pathology. Our transcriptome analysis revealed a migratory phenotype sharing many features with tissue-resident memory and Th17.1 cells, most notably the transcription factor eomesodermin. Knowledge on this cell subset should enable future studies to find ways to strengthen the host defence against CNS-resident pathogens and brain tumours or to prevent CNS autoimmunity. [ABSTRACT FROM AUTHOR]

Cancer treatment with adoptively transferred tumor-associated antigen-specific CD4+ T-helper cells is a promising immunotherapeutic approach. In the pancreatic cancer model RIP-Tag2, the intraperitoneal (i.p.) application of Tag-specific TH1 cells exhibited a profound antitumoral efficiency. We investigated, whether an intravenous (i.v.) application of Tag-TH1 cells induces an equivalent therapeutic effect. Adoptively transferred fluorescent Tag-TH1 cells revealed a pronounced homing to the tumors after either i.p. or i.v. transfer, and both routes induced an almost equivalent therapeutic effect as demonstrated by magnetic resonance imaging, blood glucose level course and histology. The i.v. administration of Tag-TH1 cells induced p16INK4-positive/Ki67-negative tumor senescence more efficiently than i.p. administration. Both routes replenish host CD4+ T cells by transferred T cells and recruitment of B and dendritic cells to the tumors while reducing CD8+ T cells and depleting macrophages. Both administration routes efficiently induced a similar antitumoral efficiency despite the pronounced senescence induction after i.v. administration. Thus, a combinatory i.v./i.p. injection of therapeutic cells might overcome limitations of the individual routes and improve therapeutic efficacy in solid tumors. [ABSTRACT FROM AUTHOR]

The HLA-DR15 haplotype confers the largest part of the genetic risk to develop multiple sclerosis, a prototypic CD4+ T cell-mediated autoimmune disease. The mechanisms how certain HLA-class II molecules functionally contribute to autoimmune diseases are still poorly understood, but probably involve shaping an autoimmune-prone T cell repertoire during central tolerance in the thymus and subsequently maintaining or even expanding it in the peripheral immune system. Self-peptides that are presented by disease-associated HLA-class II molecules most likely play important roles during both processes. Here, we examined the functional involvement of the HLA-DR15 haplotype in autologous proliferation in multiple sclerosis and the contribution of HLA-DR15 haplotype-derived self-peptides in an in vitro system. We observe increased autologous T cell proliferation in patients with multiple sclerosis in relation to the multiple sclerosis risk-associated HLA-DR15 haplotype. Assuming that the spectrum of self-peptides that is presented by the two HLA-DR15 allelic products is important for sustaining autologous proliferation we performed peptide elution and identification experiments from the multiple sclerosis-associated DR15 molecules and a systematic analysis of a DR15 haplotype-derived self-peptide library. We identify HLA-derived self-peptides as potential mediators of altered autologous proliferation. Our data provide novel insights about perturbed T cell repertoire dynamics and the functional involvement of the major genetic risk factor, the HLA-DR15 haplotype, in multiple sclerosis. [ABSTRACT FROM AUTHOR]

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02+ glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients’ CD8+ T cells in vitro. In vivo, glioblastoma-specific CD8+ T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma. [ABSTRACT FROM AUTHOR]

MHC class II‐restricted autoreactive T cells play a major role in the development of autoimmune diabetes mellitus in both human and mouse. Two of our groups previously established panels of islet‐reactive CD4+ T cell clones from prediabetic non‐obese diabetic (NOD) mice. These clones express distinct sets of TCR Vα, Vβ, Jα and Jβ, and also differ in the structure of the junctional region of TCR. All of the T cell clones have been shown to cause insulitis and several induce diabetes when transferred to various recipients. The antigen specificities of these T cell clones have not been determined, but they do not react with defined islet cell antigens such as glutamic acid decarboxylase. To identify the peptide ligands recognized by these clones, we examined the reactivity of the T cell clones to peptide mixtures in which anchor residues for H2‐Ag7 were fixed. Most of the clones showed similar reactivity to the peptide mixtures. To further determine the peptide ligands of the T cell clones, we synthesized several peptides based on the favored amino acid motifs and examined clone reactivity to the synthetic peptides. Some of the peptides, e.g. HLAI‐RM and HIPI‐RM, could stimulate most of the T cell clones tested, even though the clones expressed different TCR. The results suggest that our islet‐reactive T cell clones recognize in islet β cells a natural ligand that is similar to these peptides. [ABSTRACT FROM PUBLISHER]

Alloreactive T cells recognize framework or peptide-dependent determinants on foreign MHC molecules. Among the peptide-dependent alloreactive T cells a significant proportion is specific for one particular peptide presented by the allo-MHC molecule as antigen-specific T cells would do. Such alloreactive, peptide-specific T cells are referred to as `allorestricted'. High-avidity HLA-A*02 allorestricted cytotoxic T lymphocyte (CTL) clones specific for peptide libraries can be generated from HLA-A*02– donors. We made use of this technique to study the role of closely related self-HLA molecules on shaping of the alloreactive T cell repertoire. Peripheral blood lymphocytes from HLA-A*0205 individuals were stimulated by HLA-A*0201 targets pulsed with an HLA-A*0201 peptide library. We did not observe a bias towards peptide-specific CTL in the HLA-A*0201-directed alloreactive repertoire of HLA-A*0205 donors as compared to HLA-A*02– donors. Comparison of the alloreactive T cell response between two donors having similar HLA haplotypes demonstrated that the allorestricted T cell repertoire is largely different between individuals. [ABSTRACT FROM PUBLISHER]

The specificity of peptide binding to MHC molecules is defined by binding motifs composed of several relatively conserved anchor positions. The peptide binding motifs of murine MHC class II I-A molecules are functionally important but poorly characterized. Here we use peptide binding studies and isolation of naturally presented peptides to characterize the peptide binding motif of the MHC class II I-A molecule, RT1.B1, a molecule that is involved in experimental autoimmunity in the Lewis rat. We now report that, similar to other class II motifs, the RT1.B1 motif consists of a nonamer sequence with four major anchor positions (P1, P4, P6 and P9). Residues at P4 and P9, rather than at P1, appeared to be particularly important for binding. Negatively charged residues were favored at P9, consistent with the presence of a serine at position 57 of the RT1.B1 β chain. This RT1.B1 motif could be observed in the dominant autoantigenic T cell epitopes mapped previously in the Lewis rat. These results highlight a general similarity and some important differences in the organization of MHC class II peptide binding motifs. The reported RT1.B1 motif should facilitate the prediction and design of T cell epitopes for the induction and control of experimental autoimmune diseases in Lewis rat models. [ABSTRACT FROM AUTHOR]

Motifs of peptldes naturally associated with H-2E and E molecules were determined by (i) pool sequencing of natural ligand mixtures and (ii) sequencing of individual natural ligands followed by their alignment to the basic motif suggested by pool sequencing. The data reveal nine amino acid motifs with interaction sites at relative positions P1, P4, P6 and P9, with specificities that are identical at some but different at other anchor positions between E and E motifs, illustrating the different requirements for peptides to be presented by these two MHC molecules. The anchors with the most restricted specificity are P1 and P9. P1 is aliphatic for E and predominantly aromatic for E. P9 is positively charged for both molecules. P4 and P6 show a totally different amino acid preference between E and E ligand motifs. An alignment of E and E protein sequences to the recently reported HLA-DR1 pocket residues is in agreement with observed anchor residues in E and E motifs, thus confirming the predicted similarity of mouse class II E molecules with human DR molecules. Furthermore, this alignment was extended to the putative pockets of class II E and E molecules, and allowed, together with sequence information of previously Identified natural ligands of E and E molecules, a prediction of their respective motifs. The information obtained by this study should be useful to identify putative class II E epltopes in proteins and to design peptides for blocking class II E molecules. [ABSTRACT FROM PUBLISHER]

Here we report peptide motifs of five HLA-B molecules, B5101, B5102, B5103, B5201 and B7801. Motifs were obtained by pool sequencing of natural ligands eluted from the respective molecules expressed in C1R cells upon transfection. A number of individual ligands that could be sequenced confirmed the motifs. All five HLA-B molecules belong to the HLA-B5, B35-crossreactive group and are closely related as indicated by similar sequences. B51 and B52 are associated with Bw4, whereas B78 Is associated with Bw6. One of the HLA-B molecules Investigated here, HLA-B5101, is associated with Behçet's disease, a multisystemic infiammatory disease affecting various organs. This disease is especially frequent among the Japanese population. The subtle differences between the peptide motifs of B5101 as compared with the motifs of the four other closely related but not disease-associated molecules could shed light on the nature of the HLA-associatlon of Behçet's disease [ABSTRACT FROM PUBLISHER]

The peptide motifs of two HLA molecules, B8 and DR3(17), which are associated with autoimmune diseases including myasthenia gravls, were determined from natural peptide pools using Edman degradation. The majority of HLA-B8 liganda are nonamere preferentially terminated by leucine. As a characteristic feature of the HLA-B8 motif, there is a high degree of conservation of positively charged amlno acids at position 3 and 5, exclusively lysine at position 3, and lysine or arglnine at position 5. Additional evidence for this allele-specific motif is the presence of these features in several viral peptldes recognized by HLA-B8 restricted T cells. The DR3(17) motif is characterized by four conserved anchor-like positions ordered in an almost symmetrical arrangement, as has been found for DR1 and DR5 motifs. A first hydrophobic/aromatic anchor three to four residues apart from the N-terminus (at relative position 1) appears to be a common feature of DR liganda. The second anchor is an aspartate at relative position 4, which is likely to be the DR3(17)-speclfIc contact site in the groove. Two additional conserved positions closer to the C-terminus are occupied by charged amino acids at relative position 6 and by hydrophobic/aromatic residues at positions 8, 9, or 10. Eight individual naturally processed DR17 ligands were sequenced and were found to be derived from exogenousiproteins and cytoplasmic membrane receptors. These natural peptldes conform well to the determined motif. A single exchange of the anchor-like positions in a model peptide abrogated binding to DR17 cells. On the basis of the DR17 motif, peptldes from the acetylcholine receptor(AChR), the autoimmune target in myasthenia gravls, were selected that should contain candidate epitopes for AChR-speclflc T helper cells. Several peptide sequences which were reported to activate T cells from DR3 individuals were among this collection. [ABSTRACT FROM PUBLISHER]

The target antigen recognized by H-2K-restricted cytotoxic T lymphocytes (CTLs) specific for a mutagen-induced antigen on DBA/2-derived tumor P815 was indentified as the product of a normal cellular gene encompassing a point mutation. Using synthetic peptides, the epitope recognized by these CTLs was narrowed down to be contained within the undecamer KYQAVTTTLEE, Incorporating the point mutation. The allele-specific peptide motif for H-2K molecules allowed us to predict the peptide naturally presented by the tumor cells to be the nonamer KYQAVTTTL. Isolation of the natural tum-specific peptide from P198.3 tumor cells and biochemical comparison with the synthetic nonamer confirmed the prediction. This natural nonapeptide is represented by ∼100 copies per tumor cell. [ABSTRACT FROM PUBLISHER]

Incompatibility of human minor histocompatibility (hmH) antigens induces rejection of grafts in organ transplantation and graft versus host disease In bone marrow transplantation if donor and recipient are matched for human leukocyte antigen (HLA) genes. These antigens are recognized only by T cells. We describe here the isolation of hmH peptides recognized by a hmH antigen specific, HLA-B35 restricted CTL clone which was derived from a patient who rejected the kidneys from two HLA-ldentlcal sisters. Naturally occurring hmH peptides were Isolated from a donor derived B cell line and an HLA-B35 transfected human B cell line by acid elution. Analysis of various HLA class I transfectant cells demonstrated that MHC class I molecules themselves determine the peptides which are naturally processed and presented to T cells. [ABSTRACT FROM PUBLISHER]

An alloreactive CTL clone, 27.B2, produced in the mouse strain combination B6.C-H-2 (bm1) against C57BL/6 (B6) and nominally directed against the K molecule has been shown to be peptlde dependent. The clone recognizes intact B6, but not intact bm1 or bm3 target cells. However, peptides recognized by 27.B2 CTL can be extracted from bm1 or B6 whole cell lysates as well as from purified K or K molecules. The target peptides recognized by 27.B2 appear to be Identical, whether isolated from K or K-expressing cells, as shown by identical behavior upon high-resolution reversed-phase HPLC separation. Thus, the same peptlde is presented by different MHC molecules including K and K, but is recognized by 27.B2 CTL only in the context of the foreign molecule, K, and not in the context of the self molecule, K. [ABSTRACT FROM PUBLISHER]