T cell affinity regulates asymmetric division, effector cell differentiation, and tissue pathology.

The strength of interactions between T cell receptors and the peptide-major histocompatibility complex (pMHC) directly modulates T cell fitness, clonal expansion, and acquisition of effector properties. Here we show that asymmetric T cell division is an important mechanistic link between increased signal strength, effector differentiation, and the ability to induce tissue pathology. Recognition of pMHC above a threshold affinity drove responding T cells into asymmetric cell division. The ensuing proximal daughters underwent extensive division and differentiated into short-lived effector cells expressing the integrin VLA-4, allowing the activated T cell to infiltrate and mediate destruction of peripheral target tissues. In contrast, T cells activated by below-threshold antigens underwent symmetric division, leading to abortive clonal expansion and failure to fully differentiate into tissue-infiltrating effector cells. Antigen affinity and asymmetric division are important factors that regulate fate specification in CD8(+) T cells and predict the potential of a self-reactive T cell to mediate tissue pathology.

A V beta 4-specific superantigen encoded by a new exogenous mouse mammary tumor virus.

The superantigen (SAg) expressed by mouse mammary tumor virus (MMTV) has been shown to play an essential role in the course of the viral life cycle. In the present study, we describe a V beta 4-specific SAg encoded by a new exogenous MMTV carried by the SIM mouse strain. This is the first report of a viral or bacterial SAg reacting with mouse V beta 4+ T cells. Injection of MMTV(SIM) into adult BALB/c mice leads to a rapid and strong stimulation of V beta 4+ CD4+ T cells, followed by a slow deletion of these cells. Neonatal exposure to the virus also leads to a progressive deletion of V beta 4+ T cells. In contrast to other strong MMTV SAg, this new SAg requires the presence of major histocompatibility complex class II I-E molecules to be presented efficiently to T cells. Sequence analysis revealed a new predicted amino acid sequence in the C-terminal polymorphic region of this SAg. Furthermore, sequence comparisons to the most closely related SAg with different V beta specificities hint at the specific residues involved in the interaction with the T cell receptor.

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

MHC class II expression is differentially regulated in plasmacytoid and conventional dendritic cells.

Major histocompatibility complex (MHC) class II-restricted antigen presentation is essential for the function of dendritic cells (DCs). We show here that plasmacytoid DCs (pDCs) differ from all other DC subsets with respect to expression of CIITA, the 'master regulator' of MHC class II genes. The gene encoding CIITA is controlled by three cell type-specific promoters: pI, pIII and pIV. With gene targeting in mice, we demonstrate that pDCs rely strictly on the B cell promoter pIII, whereas macrophages and all other DCs depend on pI. The molecular mechanisms driving MHC class II expression in pDCs are thus akin to those operating in lymphoid rather than myeloid cells.

Differential role of naïve and memory CD4 T-cell subsets in primary alloresponses.

The T cell response to major histocompatibility complex (MHC) alloantigens occurs via two main pathways. The direct pathway involves the recognition of intact allogeneic MHC:peptide complexes on donor cells and provokes uniquely high frequencies of responsive T cells. The indirect response results from alloantigens being processed like any other protein antigen and presented as peptide by autologous antigen-presenting cells. The frequencies of T cells with indirect allospecificity are orders of magnitude lower and comparable to other peptide-specific responses. In this study, we explored the contributions of naïve and memory CD4(+) T cells to these two pathways. Using an adoptive transfer and skin transplantation model we found that naive and memory CD4(+) T cells, both naturally occurring and induced by sensitization with multiple third-party alloantigens, contributed equally to graft rejection when only the direct pathway was operative. In contrast, the indirect response was predominantly mediated by the naïve subset. Elimination of regulatory CD4(+)CD25(+) T cells enabled memory cells to reject grafts through the indirect pathway, but at a much slower tempo than for naïve cells. These findings have implications for better targeting of immunosuppression to inhibit immediate and later forms of alloimmunity.

MHC-genotype of progeny influenced by parental infection.

In a previous series of in vitro fertilization experiments with mice we found non-random combination of major histocompatibility complex (MHC) haplotypes in the very early embryos. Our results suggested that two selection mechanisms were operating: (i) the eggs selected specific sperm; and (ii) the second meiotic division in the eggs was influenced by the type of sperm that entered the egg. Furthermore, the proportion of MHC-heterozygous embryos varied over time, suggesting that non-random fertilization was dependent on an external factor that changed over time. As a higher frequency of heterozygous individuals correlated with an uncontrolled epidemic by MHV (mouse hepatitis virus), we suggested that MHV-infection might have influenced the outcome of fertilization. Here, we present an experiment that tests this hypothesis. We infected randomly chosen mice with MHV and sham-infected control mice five days before pairing. We recovered the two-cell embryos from the oviduct, cultured them until the blastocyst stage, and determined the genotype of each resulting blastocyst by polymerase chain reaction. We found the pattern that we expected from our previous experiments: virus-infected mice produced more MHC-heterozygous embryos than sham-infected ones. This suggests that parents are able to promote specific combinations of MHC-haplotypes during fertilization according to the presence or absence of a viral infection.

MM-GBSA binding free energy decomposition and T cell receptor engineering.

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by class I major histocompatibility complexes (MHC) is the key event in the immune response against virus infected cells or tumor cells. The major determinant of T cell activation is the affinity of the TCR for the peptide-MHC complex, though kinetic parameters are also important. A study of the 2C TCR/SIYR/H-2Kb system using a binding free energy decomposition (BFED) based on the MM-GBSA approach had been performed to assess the performance of the approach on this system. The results showed that the TCR-p-MHC BFED including entropic terms provides a detailed and reliable description of the energetics of the interaction (Zoete and Michielin, 2007). Based on these results, we have developed a new approach to design sequence modifications for a TCR recognizing the human leukocyte antigen (HLA)-A2 restricted tumor epitope NY-ESO-1. NY-ESO-1 is a cancer testis antigen expressed not only in melanoma, but also on several other types of cancers. It has been observed at high frequencies in melanoma patients with unusually positive clinical outcome and, therefore, represents an interesting target for adoptive transfer with modified TCR. Sequence modifications of TCR potentially increasing the affinity for this epitope have been proposed and tested in vitro. T cells expressing some of the proposed TCR mutants showed better T cell functionality, with improved killing of peptide-loaded T2 cells and better proliferative capacity compared to the wild type TCR expressing cells. These results open the door of rational TCR design for adoptive transfer cancer therapy.

Cis interactions of immunoreceptors with MHC and non-MHC ligands

The conventional wisdom is that cell-surface receptors interact with ligands expressed on other cells to mediate cell-to-cell communication (trans interactions). Unexpectedly, it has recently been found that two classes of receptors specific for MHC class I molecules not only interact with MHC class I molecules expressed on opposing cells, but also with those on the same cell. These cis interactions are a feature of immunoreceptors that inhibit, rather than activate, cellular functions. Here, we review situations in which cis interactions have been observed, the characteristics of receptors that bind in trans and cis, and the biological roles of cis recognition.

Using transcription modules to identify expression clusters perturbed in Williams-Beuren syndrome.

The genetic dissection of the phenotypes associated with Williams-Beuren Syndrome (WBS) is advancing thanks to the study of individuals carrying typical or atypical structural rearrangements, as well as in vitro and animal studies. However, little is known about the global dysregulations caused by the WBS deletion. We profiled the transcriptomes of skin fibroblasts from WBS patients and compared them to matched controls. We identified 868 differentially expressed genes that were significantly enriched in extracellular matrix genes, major histocompatibility complex (MHC) genes, as well as genes in which the products localize to the postsynaptic membrane. We then used public expression datasets from human fibroblasts to establish transcription modules, sets of genes coexpressed in this cell type. We identified those sets in which the average gene expression was altered in WBS samples. Dysregulated modules are often interconnected and share multiple common genes, suggesting that intricate regulatory networks connected by a few central genes are disturbed in WBS. This modular approach increases the power to identify pathways dysregulated in WBS patients, thus providing a testable set of additional candidates for genes and their interactions that modulate the WBS phenotypes.

In vivo effects of a recombinant vaccinia virus expressing a mouse mammary tumor virus superantigen.

Early after infection, the mouse mammary tumor virus (MMTV) expresses a superantigen (SAg) at the surface of B lymphocytes. Interaction with the T-cell receptor Vbeta domain induces a polyclonal proliferative response of the SAg-reactive T cells. Stimulated T cells become anergic and are deleted from the T-cell repertoire. We have used a recombinant vaccinia virus encoding the MMTV(GR) SAg to dissect the effects of the retroviral SAg during an unrelated viral infection. Subcutaneous infection with this recombinant vaccinia virus induces a very rapid increase of Vbeta14 T cells in the draining lymph node. This stimulation does not require a large Plumber of infectious particles and is not strictly dependent on the expression of the major histocompatibility complex class II I-E molecule, as it is required after MMTV(GR) infection. In contrast to MMTV infection during which B cells are infected, we do not observe any clonal deletion of the reactive T cells following the initial stimulation phase. Our data show that contrary to the case with MMTV, macrophages but not B cells are the targets of infection by vaccinia virus in the lymph node, indicating the ability of these cells to present a retroviral SAg. The altered SAg expression in a different target cell observed during recombinant vaccinia virus infection therefore results in significant changes in the SAg response.

CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes.

Thymocytes and class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes express predominantly heterodimeric alpha/beta CD8. By interacting with non-polymorphic regions of MHC class I molecules CD8 can mediate adhesion or by binding the same MHC molecules that interact with the T-cell antigen receptor (TCR) function as coreceptor in TCR-ligand binding and T-cell activation. Using TCR photoaffinity labelling with a soluble, monomeric photoreactive H-2Kd-peptide derivative complex, we report here that the avidity of TCR-ligand interactions on cloned cytotoxic T cells is very greatly strengthened by CD8. This is primarily explained by coordinate binding of ligand molecules by CD8 and TCR, because substitution of Asp 227 of Kd with Lys severely impaired the TCR-ligand binding on CD8+, but not CD8- cells. Kinetic studies on CD8+ and CD8- cells further showed that CD8 imposes distinct dynamics and a remarkable temperature dependence on TCR-ligand interactions. We propose that the ability of CD8 to act as coreceptor can be modulated by CD8-TCR interactions.

MHC class II hierarchy of superantigen presentation predicts efficiency of infection with mouse mammary tumor virus.

Superantigens (SAgs) encoded by infectious mouse mammary tumor viruses (MMTVs) play a crucial role in the viral life cycle. Their expression by infected B cells induces a proliferative immune response by SAg-reactive T cells which amplifies MMTV infection. This response most likely ensures stable MMTV infection and transmission to the mammary gland. Since T cell reactivity to SAgs from endogenous Mtv loci depends on MHC class II molecules expressed by B cells, we have determined the ability of MMTV to infect various MHC congenic mice. We show that MHC class II I-E+ compared with I-E- mouse strains show higher levels of MMTV infection, most likely due to their ability to induce a vigorous SAg-dependent immune response following MMTV encounter. Inefficient infection is observed in MHC class II I-E- mice, which have been shown to present endogenous SAgs poorly. Therefore, during MMTV infection the differential ability of MHC class II molecules to form a functional complex with SAg determines the magnitude of the proliferative response of SAg-reactive T cells. This in turn influences the degree of T cell help provided to infected B cells and therefore the efficiency of amplification of MMTV infection.

NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment.

Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.