Melanosomal targeting sequences from gp100 are essential for MHC class II-restricted endogenous epitope presentation and mobilization to endosomal compartments

CD4+ T lymphocytes play an important role in CD8+ T cell-mediated responses against tumors. Considering that about 20% of melanomas express major histocompatibility complex (MHC) class II, it is plausible that concomitant antigenic presentation by MHC class I and class II complexes shapes positive (helper T cells) or negative (regulatory T cells) anti-tumor responses. Interestingly, gp100, a melanoma antigen, can be presented by both MHC class I and class II when expressed endogenously, suggesting that it can reach endosomal/MHC class II compartments (MIIC). Here, we demonstrated that the gp100 putative amino-terminal signal sequence and the last 70 residues in carboxy-terminus, are essential for MIIC localization and MHC class II presentation. Confocal microscopy analyses confirmed that gp100 was localized in LAMP-1+ endosomal/MIIC. Gp100-targeting sequences were characterized by deleting different sections in the carboxy-terminus (residues 590 to 661). Transfection in 293T cells, expressing MHC class I and class II molecules, revealed that specific deletions in carboxy-terminus resulted in decreased MHC class II presentation, without effects on MHC class I presentation, suggesting a role in MIIC trafficking for these deleted sections. Then, we used these gp100-targeting sequences to mobilize the green fluorescent protein (GFP) to endosomal compartments, and to allow MHC class II and class I presentation of minimal endogenous epitopes. Thus, we concluded that these specific sequences are MIIC targeting motifs. Consequently, these sequences could be included in expression cassettes for endogenously expressed tumor or viral antigens to promote MHC class II and class I presentation and optimize in vivo T cell responses, or as an in vitro tool for characterization of new MHC class II epitopes.

Specific T cell recognition of kinetic isomers in the binding of peptide to class II major histocompatibility complex

Helper T cells are triggered by molecular complexes of antigenic peptides and class II proteins of the major histocompatibility complex . The formation of stable complexes between class II major histocompatibility complex proteins and antigenic peptides is often accompanied by the formation of a short-lived complex. In this report, we describe T cell recognition of two distinct complexes, one short-lived and the other long-lived, formed during the binding of an altered myelin basic protein peptide to I-Ak. One myelin basic protein-specific T cell clone is triggered by only the short-lived complex, and another is triggered by only the stable complex. Thus, a single peptide bound to a particular class II molecule can activate different T cells depending on the conditions of the binding reaction.

Restricted and conserved T-cell repertoires involved in allorecognition of class II major histocompatibility complex.

The nature of the alloreactive T-cell response is not yet clearly understood. These strong cellular responses are thought to be the basis of allograft rejection and graft-vs.-host disease. The question of the extent of responding T-cell repertoires has so far been addressed by cellular cloning, often combined with molecular T-cell receptor (TCR) analysis. Here we present a broad repertoire analysis of primed responder cells from mixed lymphocyte cultures in which two different DR1/3 responders were stimulated with DR3/4 cells. Repertoire analysis was performed by TCR spectratyping, a method by which T cells are analyzed on the basis of the complementarity-determining region 3 length of different variable region (V) families. Strikingly, both responders showed very similar repertoires when the TCR V beta was used as a lineage marker. This was not seen when TCR V alpha was analyzed. A different pattern of TCR V beta was observed if the stimulating alloantigen was changed. This finding indicates that alloreactive T cells form a specific repertoire for each alloantigen. Since conservation appears to be linked to TCR V beta, the question of different roles of alpha and beta chains in allorecognition is raised.

A general model of invariant chain association with class II major histocompatibility complex proteins.

The binding of invariant chain to major histocompatibility complex (MHC) proteins is an important step in processing of MHC class II proteins and in antigen presentation. The question of how invariant chain can bind to all MHC class II proteins is central to understanding these processes. We have employed molecular modeling to predict the structure of class II-associated invariant chain peptide (CLIP)-MHC protein complexes and to ask whether the predicted mode of association could be general across all MHC class II proteins. CLIP fits identically into the MHC class II alleles HLA-DR3, I-Ak, I-Au, and I-Ad, with a consistent pattern of hydrogen bonds, contacts, and hydrophobic burial and without bad contacts. Our model predicts the burial of CLIP residues Met-91 and Met-99 in the deep P1 and P9 anchor pockets and other detailed interactions, which we have compared with available data. The predicted pattern of I-A allele-specific effects on CLIP binding is very similar to that observed experimentally by alanine-scanning mutations of CLIP. Together, these results indicate that CLIP may bind in a single, general way across products of MHC class II alleles.

Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120.

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.

Toward prediction of class II mouse major histocompatibility complex peptide binding affinity:in silico bioinformatic evaluation using partial least squares, a robust multivariate statistical technique

The accurate identification of T-cell epitopes remains a principal goal of bioinformatics within immunology. As the immunogenicity of peptide epitopes is dependent on their binding to major histocompatibility complex (MHC) molecules, the prediction of binding affinity is a prerequisite to the reliable prediction of epitopes. The iterative self-consistent (ISC) partial-least-squares (PLS)-based additive method is a recently developed bioinformatic approach for predicting class II peptide−MHC binding affinity. The ISC−PLS method overcomes many of the conceptual difficulties inherent in the prediction of class II peptide−MHC affinity, such as the binding of a mixed population of peptide lengths due to the open-ended class II binding site. The method has applications in both the accurate prediction of class II epitopes and the manipulation of affinity for heteroclitic and competitor peptides. The method is applied here to six class II mouse alleles (I-Ab, I-Ad, I-Ak, I-As, I-Ed, and I-Ek) and included peptides up to 25 amino acids in length. A series of regression equations highlighting the quantitative contributions of individual amino acids at each peptide position was established. The initial model for each allele exhibited only moderate predictivity. Once the set of selected peptide subsequences had converged, the final models exhibited a satisfactory predictive power. Convergence was reached between the 4th and 17th iterations, and the leave-one-out cross-validation statistical terms - q2, SEP, and NC - ranged between 0.732 and 0.925, 0.418 and 0.816, and 1 and 6, respectively. The non-cross-validated statistical terms r2 and SEE ranged between 0.98 and 0.995 and 0.089 and 0.180, respectively. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made freely available online (http://www.jenner.ac.uk/MHCPred).

MHC class II binding prediction by molecular docking

Proteins of the Major Histocompatibility Complex (MHC) bind self and nonself peptide antigens or epitopes within the cell and present them at the cell surface for recognition by T cells. All T-cell epitopes are MHC binders but not all MCH binders are T-cell epitopes. The MHC class II proteins are extremely polymorphic. Polymorphic residues cluster in the peptide-binding region and largely determine the MHC's peptide selectivity. The peptide binding site on MHC class II proteins consist of five binding pockets. Using molecular docking, we have modelled the interactions between peptide and MHC class II proteins from locus DRB1. A combinatorial peptide library was generated by mutation of residues at peptide positions which correspond to binding pockets (so called anchor positions). The binding affinities were assessed using different scoring functions. The normalized scoring functions for each amino acid at each anchor position were used to construct quantitative matrices (QM) for MHC class II binding prediction. Models were validated by external test sets comprising 4540 known binders. Eighty percent of the known binders are identified in the best predicted 15% of all overlapping peptides, originating from one protein. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Major Histocompatibility Complex (MHC) Class II sequence polymorphism in long-finned pilot whale (Globicephala melas) from the North Atlantic

Funding Silvia S. Monteiro and Marisa Ferreira were supported by a Ph.D. grant from Fundação para a Ciência e Tecnologia (ref SFRH/BD/38735/2007 and SFRH/BD/30240/2006, respectively). Alfredo López was supported by a postdoctoral grant from Fundação para a Ciência e Tecnologia (ref SFRH/BPD/82407/2011). Catarina Eira is supported by CESAM (UID/AMB/50017), from FCT/MEC through national funds and FEDER (PT2020, Compete 2020). The work related with strandings and tissue collection in Portugal was partially supported by the SafeSea Project EEAGrants PT 0039 (supported by Iceland, Liechtenstein and Norway through the EEA Financial Mechanism), by the Project MarPro–Life09 NAT/PT/000038 (funded by the European Union–Program Life+) and by the project CetSenti FCT RECI/AAG-GLO/0470/2012; FCOMP-01-0124-FEDER-027472 (Funded by the Program COMPETE and Fundação para a Ciência e Tecnologia).

Major histocompatibility complex class I involvement in the rejection of allogeneic erythrocytes in rainbow trout (Oncorhynchus mykiss)

Major histocompatibility complex genes are thought to be involved in allogeneic graft rejection but not many reports are available on their functional analysis in fish. Analysis of available sequences of MHC genes suggests functions in antigen presentation similar to those found in higher vertebrates. In mammals, the MHC class I and class II molecules are major determinants of allogeneic graft rejection due to their polymorphism in conjunction with their antigen presenting function. In fish, MHC class H molecules are found to be involved in rejection of allogeneic scale grafts. The present study was designed to investigate the involvement of MHC class I molecules in allograft rejection. Erythrocytes were collected from donors of rainbow trout expressed different class MHC class I alleles, stained with two dyes, mixed and grafted to the recipients that were of the same sibling group as the donors. The grafts were rejected by allogeneic recipients and the MHC class I linkage group was the major determinant for the rejection.

Analysis of the peripheral T-cell compartment in the MHC class II deficiency syndrome.

To analyse the impact of lack of MHC class II expression on the composition of the peripheral T-cell compartment in man, the expression characteristics of several membrane antigens were examined on peripheral blood lymphocytes (PBL) and cultured T cells derived from an MHC-class-II-deficient patient. No MHC class II expression could be detected on either PBL or activated T cells. Moreover, the expression of MHC class I was reduced both on PBL and in vitro activated T cells compared to the healthy control. However, the reduced expression of CD26 observed on the PBL of the patient was restored after in vitro expansion. Despite the presumably class-II-deficient thymic environment, a distinct but reduced single CD4+ T-cell population was observed in the PBL of the patient. After in vitro expansion, the percentage of CD4+ cells dropped even further, most likely due to a proliferative disadvantage, compared to the single CD8+ T-cell population. However, proliferation analysis showed that T-cell activation via the TcR/CD3 pathway is not affected by the MHC class II deficiency.

TCR V alpha- and V beta-gene segment use in T-cell subcultures derived from a type-III bare lymphocyte syndrome patient deficient in MHC class-II expression.

Previously, we and others have shown that MHC class-II deficient humans have greatly reduced numbers of CD4+CD8- peripheral T cells. These type-III Bare Lymphocyte Syndrome patients lack MHC class-II and have an impaired MHC class-I antigen expression. In this study, we analyzed the impact of the MHC class-II deficient environment on the TCR V-gene segment usage in this reduced CD4+CD8- T-cell subset. For these studies, we employed TcR V-region-specific monoclonal antibodies (mAbs) and a semiquantitative PCR technique with V alpha and V beta amplimers, specific for each of the most known V alpha- and V beta-gene region families. The results of our studies demonstrate that some of the V alpha-gene segments are used less frequent in the CD4+CD8- T-cell subset of the patient, whereas the majority of the TCR V alpha- and V beta-gene segments investigated were used with similar frequencies in both subsets in the type-III Bare Lymphocyte Syndrome patient compared to healthy control family members. Interestingly, the frequency of TcR V alpha 12 transcripts was greatly diminished in the patient, both in the CD4+CD8- as well as in the CD4-CD8+ compartment, whereas this gene segment could easily be detected in the healthy family controls. On the basis of the results obtained in this study, it is concluded that within the reduced CD4+CD8- T-cell subset of this patient, most of the TCR V-gene segments tested for are employed. However, a skewing in the usage frequency of some of the V alpha-gene segments toward the CD4-CD8+ T-cell subset was noticeable in the MHC class-II deficient patient that differed from those observed in the healthy family controls.

Melan-A/MART-1-specific CD4 T cells in melanoma patients: identification of new epitopes and ex vivo visualization of specific T cells by MHC class II tetramers.

Over the past decade, many efforts have been made to identify MHC class II-restricted epitopes from different tumor-associated Ags. Melan-A/MART-1(26-35) parental or Melan-A/MART-1(26-35(A27L)) analog epitopes have been widely used in melanoma immunotherapy to induce and boost CTL responses, but only one Th epitope is currently known (Melan-A51-73, DRB1*0401 restricted). In this study, we describe two novel Melan-A/MART-1-derived sequences recognized by CD4 T cells from melanoma patients. These epitopes can be mimicked by peptides Melan-A27-40 presented by HLA-DRB1*0101 and HLA-DRB1*0102 and Melan-A25-36 presented by HLA-DQB1*0602 and HLA-DRB1*0301. CD4 T cell clones specific for these epitopes recognize Melan-A/MART-1+ tumor cells and Melan-A/MART-1-transduced EBV-B cells and recognition is reduced by inhibitors of the MHC class II presentation pathway. This suggests that the epitopes are naturally processed and presented by EBV-B cells and melanoma cells. Moreover, Melan-A-specific Abs could be detected in the serum of patients with measurable CD4 T cell responses specific for Melan-A/MART-1. Interestingly, even the short Melan-A/MART-1(26-35(A27L)) peptide was recognized by CD4 T cells from HLA-DQ6+ and HLA-DR3+ melanoma patients. Using Melan-A/MART-1(25-36)/DQ6 tetramers, we could detect Ag-specific CD4 T cells directly ex vivo in circulating lymphocytes of a melanoma patient. Together, these results provide the basis for monitoring of naturally occurring and vaccine-induced Melan-A/MART-1-specific CD4 T cell responses, allowing precise and ex vivo characterization of responding T cells.

HLA class II mismatch alleles as targets of the alloreactive CD4 T-cell response

In allogeneic hematopoietic stem cell transplantation (allo-HSCT), alloreactive T lymphocytes of donor origin mediate the beneficial graft-versus-leukemia effect but also induce graft-versus-host disease (GvHD). Since human leukocyte antigens (HLA) mismatch alleles represent major targets of alloreactive T lymphocytes, patient and donor are usually matched for the class I molecules A, B, C, and for the class II molecules DRB1 and DQB1, in order do reduce the risk of GvHD. The HLA-DPB1 locus, however, is still ignored in donor selection. Interestingly, clinical studies have demonstrated that disparities at HLA-DQB1 alleles as well as distinct HLA DPB1 mismatch constellations do not adversely affect the outcome of allo-HSCT. It has also been shown that HLA class II is predominantly expressed on hematopoietic cells under non-inflammatory conditions. Therefore, this PhD thesis focused on the application of CD4 T cells in adoptive immunotherapy of leukemias.rnIn the first part of this thesis we developed a rapid screening approach to detect T-cell reactivity of donors to single HLA class II mismatch alleles. Allo-HLA reactivity was measured in naive, memory, and entire CD4 T cells isolated from PBMC of healthy donors by flow cytometric cell sorting according to expression of the differentiation markers CD45RA, CD45RO, CD62L, and CCR7. T-cell populations were defined by a single marker to facilitate translation into a clinical-grade allo-depletion procedure. Alloreactivity to single HLA-DR/-DQ mismatch alleles was analyzed in short-term mixed lymphocyte reactions (MLR) in vitro. As standard antigen-presenting cells, we used the HLA-deficient cell line K562 upon electroporation with single HLA-DR/-DQ allele mRNA. We observed in IFN-γ ELISpot assays that allo-HLA-reactivity preferentially derived from subsets enriched for naive compared to memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD62L (P=0.008) or CD45RA (P=0.011) were used as marker. Median numbers of allo-HLA-reactive effector cells were 3.5-fold and 16.6-fold lower in CD62Lneg and CD45RAneg memory CD4 T cells than in entire CD4 T cells, respectively. In allele-specific analysis, alloreactivity to single HLA-DR alleles clearly exceeded that to HLA-DQ alleles. In terms of alloproliferation no significant difference could be observed between individual CD4 T-cell subsets. rnThe second part of this thesis dealed with the generation of allo-HLA-DQ/-DP specific CD4 T cells. Naive CD45RApos CD4 T cells isolated from healthy donor PBMC by flow cytometric cell sorting were stimulated in MLR against single allo-HLA-DQ/-DP alleles transfected into autologous mature monocyte-derived dendritic cells by mRNA electroporation. Rapidly expanding HLA-DQ/-DP mismatch reactive T cells significantly recognized and cytolysed primary acute myeloid leukemia (AML) blasts, fibroblasts (FB) and keratinocytes (KC) in IFN-γ ELISpot and 51chromium release assays if the targets carried the HLA DQ/ DP allele used for T cell priming. While AML blasts were recognized independent of pre-incubating them with IFN-γ, recognition of FB and KC required IFN-γ pre treatment. We further investigated HLA class II expression on hematopoietic and non-hematopoietic cells by flow cytometry. HLA class II was not detected on primary FB, KC, and non-malignant kidney cells, but was expressed at significant levels on primary AML blasts and B-LCL. Up-regulation of HLA class II expression was observed on all cell types after pre-incubation with IFN-γ.rnIn summary, the novel K562-HLA based MLR approach revealed that naive-depleted CD4 T-cell subsets of healthy individuals contain decreased allo-HLA reactivity in vitro. We propose the application of CD45RAneg naive-depleted CD4 T cells as memory T cell therapy, which might be beneficial for HLA-mismatched patients at high-risk of GvHD and low-risk of leukemia relapse. Memory T cells might also provide important post-transplant immune functions against infectious agents. Additionally, the screening approach could be employed as test system to detect donors which have low risks for the emergence of GvHD after allo-HSCT. In the second part of this thesis we developed a protocol for the generation of allo-HLA-DQ/-DP specific CD4 T cell lines, which could be applied in situations in which patient and donor are matched in all HLA alleles but one HLA-DQ/-DP allele with low GvHD potential. These T cells showed lytic activity to leukemia cells while presumably sparing non-hematopoietic tissues under non-inflammatory conditions. Therefore, they might be advantageous for allo-HSCT patients with advanced stage AML after reduced-intensity conditioning and T-cell depletion for the replenishment of anti-leukemic reactivity if the risk for disease relapse is high. rn

Selection and recombination drive the evolution of MHC class II DRB diversity in ungulates

Major histocompatibility complex (MHC) antigen-presenting genes are the most variable loci in vertebrate genomes. Host-parasite co-evolution is assumed to maintain the excessive polymorphism in the MHC loci. However, the molecular mechanisms underlying the striking diversity in the MHC remain contentious. The extent to which recombination contributes to the diversity at MHC loci in natural populations is still controversial, and there have been only few comparative studies that make quantitative estimates of recombination rates. In this study, we performed a comparative analysis for 15 different ungulates species to estimate the population recombination rate, and to quantify levels of selection. As expected for all species, we observed signatures of strong positive selection, and identified individual residues experiencing selection that were congruent with those constituting the peptide-binding region of the human DRB gene. However, in addition for each species, we also observed recombination rates that were significantly different from zero on the basis of likelihood-permutation tests, and in other non-quantitative analyses. Patterns of synonymous and non-synonymous sequence diversity were consistent with differing demographic histories between species, but recent simulation studies by other authors suggest inference of selection and recombination is likely to be robust to such deviations from standard models. If high rates of recombination are common in MHC genes of other taxa, re-evaluation of many inference-based phylogenetic analyses of MHC loci, such as estimates of the divergence time of alleles and trans-specific polymorphism, may be required.

Transcriptional regulation of the invariant chain associated with MHC class II molecules

The invariant chain associated with the major histocompatibility complex (MHC) class II molecules is a non-polymorphic glycoprotein implicated in antigen processing and class II molecule intracellular transport. Class II molecules and invariant chain (In) are expressed primarily by B lymphocytes and antigen-presenting cells such as macrophages and can be induced by interferon gamma (IFN-$\gamma$) in a variety of cell types such as endothelial cells, fibroblasts, and astrocytes. In this study the cis-acting sequences involved in the constitutive, tissue-specific, and IFN-$\gamma$ induced expression of the human In gene were investigated and nuclear proteins which specifically bound these sequences were identified.^ To define promoter sequences involved in the regulation of the human In gene, 790 bp 5$\sp\prime$ to the initiation of transcription were subcloned upstream of the gene encoding chloramphenicol acetyl transferase (CAT). Transfection of this construct into In expressing and non-expressing cell lines demonstrated that this 790 bp In promoter sequence conferred tissue specificity to the CAT gene. Deletion mutants were created in the promoter to identify sequences important for transcription. Three regulatory regions were identified $-$396 to $-$241, $-$241 to $-$216, and $-$216 to $-$165 bp 5$\sp\prime$ to the cap site. Transfection into a human glioblastoma cell line, U-373 MG, and treatment with IFN-$\gamma$, demonstrated that this 5$\sp\prime$ region is responsive to IFN-$\gamma$. An IFN-$\gamma$ response element was sublocalized to the region $-$120 to $-$61 bp. This region contains homology to the interferon-stimulated response element (ISRE) identified in other IFN responsive genes. IFN-$\gamma$ induces a sequence-specific DNA binding factor which binds to an oligonucleotide corresponding to $-$107 to $-$79 bp of the In promoter. This factor also binds to an oligonucleotide corresponding to $-$91 to $-$62 of the interferon-$\beta$ gene promoter, suggesting this factor may be member of the IRF-1/ISGF2, IRF-2, ICSBP family of ISRE binding proteins. A transcriptional enhancer was identified in the first intron of the In gene. This element, located in a 2.6 kb BamHI/PstI fragment, enhances the IFN-$\gamma$ response of the promoter in U-373 MG. The majority of the In enhancer activity was sublocalized to a 550 bp region $\sim$1.6 kb downstream of the In transcriptional start site. ^