BAFF production by antigen-presenting cells provides T cell co-stimulation.

The B cell-activating factor from the tumor necrosis factor family (BAFF) is an important regulator of B cell immunity. Recently, we demonstrated that recombinant BAFF also provides a co-stimulatory signal to T cells. Here, we studied expression of BAFF in peripheral blood leukocytes and correlated this expression with BAFF T cell co-stimulatory function. BAFF is produced by antigen-presenting cells (APC). Blood dendritic cells (DC) as well as DC differentiated in vitro from monocytes or CD34+ stem cells express BAFF mRNA. Exposure to bacterial products further up-regulates BAFF production in these cells. A low level of BAFF transcription, up-regulated upon TCR stimulation, was also detected in T cells. Functionally, blockade of endogenous BAFF produced by APC and, to a lesser extent, by T cells inhibits T cell activation. Altogether, this indicates that BAFF may regulate T cell immunity during APC-T cell interactions and as an autocrine factor once T cells have detached from the APC.

T-Cell Receptors Binding Orientation over Peptide/MHC Class I Is Driven by Long-Range Interactions.

Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

Coat color dilution in mice because of inactivation of the melanoma antigen MART-1.

Melanoma antigen recognized by T cells 1 (MART-1) is a melanoma-specific antigen, which has been thoroughly studied in the context of immunotherapy against malignant melanoma and which is found only in the pigment cell lineage. However, its exact function and involvement in pigmentation is not clearly understood. Melanoma antigen recognized by T cells 1 has been shown to interact with the melanosomal proteins Pmel17 and OA1. To understand the function of MART-1 in pigmentation, we developed a new knockout mouse model. Mice deficient in MART-1 are viable, but loss of MART-1 leads to a coat color phenotype, with a reduction in total melanin content of the skin and hair. Lack of MART-1 did not affect localization of melanocyte-specific proteins nor maturation of Pmel17. Melanosomes of hair follicle melanocytes in MART-1 knockout mice displayed morphological abnormalities, which were exclusive to stage III and IV melanosomes. In conclusion, our results suggest that MART-1 is a pigmentation gene that is required for melanosome biogenesis and/or maintenance.

Specific binding of antigenic peptides to cell-associated MHC class I molecules.

T lymphocytes recognize antigen in the form of peptides that associate with specific alleles of class I or class II major histocompatibility (MHC) molecules. By contrast with the clear MHC allele-specific binding of peptides to purified class II molecules purified solubilized class I molecules either bind relatively poorly or show degenerate specificity. Using photo-affinity labelling, we demonstrate here the specific interaction of peptides with cell-associated MHC class I molecules and show that this involves metabolically active processes.

Distinct phenotypes of antigen-selected CD8 T cells emerge at different stages of an in vivo immune response.

We have previously described a unique system for identifying Ag-selected CD8 T cells during an in vivo response in normal mice. In this system, lymphocytes isolated from DBA/2 mice injected i.p. with HLA-CW3 transfected syngeneic (H-2d) P815 cells show a remarkable expansion of CD8 cells that utilize TCR expressing the V beta 10 gene segment and additional structural features characteristic of Kd-restricted CW3-specific CTL clones. We have now taken advantage of this system to characterize the surface phenotype of CD8 cells selected by Ag in vivo. We observed several distinct phenotypes at different stages of the response. At the peak of the response, Ag-selected cells were low in CD62L and CD45RB expression but displayed high levels of CD44. In addition, there was a partial down-regulation of CD8 and TCR. Cells of this phenotype were present in lymphoid tissues for several mo after immunization. Much later in the response, Ag-selected cells expressed higher levels of CD8 and TCR. Moreover, a distinct subset of these long-term immune cells emerged that now expressed CD62L and CD45RB. Analysis of CD8 cells from different tissues also revealed certain differences, particularly in TCR and co-receptor levels from liver-derived cells compared with circulating cells at the peak of the response. Our findings suggest that the function of Ag-selected CD8 cells may be regulated over time and according to location by subtle changes in cell-surface phenotype.

Combining MHC tetramer and intracellular cytokine staining for CD8(+) T cells to reveal antigenic epitopes naturally presented on tumor cells.

As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNgamma producing cells with unknown specificities and should be widely applicable.

Immunoscintigraphic localization of inflammatory lesions: concept, radiolabelling and in vitro testing of a granulocyte specific antibody.

Current nuclear medicine techniques for the localization of inflammatory processes are based on injection of 111In labelled autologous granulocytes which need to be isolated and radiolabelled in vitro before reinjection. A new technique is presented here that obviates the need for cell isolation by the direct intravenous injection of a granulocyte specific 123I labelled monoclonal antibody. In this publication the basic parameters of the antibody granulocyte interaction are described. Antibody binding does not inhibit vital functions of the granulocytes, such as chemotaxis and superoxide generation. Scatchard analysis of binding data reveals an apparent affinity of the antibody for granulocytes of 6.8 X 10(9) l/mol and approximately 7.1 X 10(4) binding sites per cell. Due to the high specificity of the antibody, the only expected interference is from CEA producing tumors.

A comparative study of T-cell receptor V beta usage in non-obese diabetic (NOD) and I-E transgenic NOD mice.

The non-obese diabetic (NOD) mouse is a model for the study of insulin-dependent diabetes mellitus (IDDM). Recently transgenic NOD mice have been derived (NOD-E) that express the major histocompatibility complex (MHC) class II I-E molecule. NOD-E do not become diabetic and show negligible pancreatic insulitis. The possibility pertained that NOD-E mice are protected from disease by a process of T-cell deletion or anergy. This paper describes our attempts to discover whether this was so, by comparing NOD and NOD-E mouse T-cell receptor V beta usage. Splenocytes and lymph node cells were therefore tested for their ability to proliferate in response to monoclonal anti-V beta antibodies. We were unable to show any consistent differences between NOD and NOD-E responses to the panel of antibodies used. Previously proposed V beta were shown to be unlikely candidates for deletion or anergy. T cells present at low frequency (V beta 5+) in both NOD and NOD-E mice were shown to be as capable of expansion in response to antigenic stimulation as were more frequently expressed V beta. Our data therefore do not support deletion or anergy as mechanisms which could account for the observed disease protection in NOD-E mice.

TCR-MHC class II interaction is required for peripheral expansion of CD4 cells in a T cell-deficient host.

It is well established that T cell-deficient nude and SCID mice can be reconstituted by i.v. injection of small numbers of purified peripheral CD4+ T cells; however, the requirements for expansion of the transferred T cells in such systems are not clear. We show here that blood and lymphoid organs of MHC class II-deficient mice (which selectively lack mature CD4+ T cells) cannot be reconstituted by transfer of purified splenic CD4+ T cells, whereas TCRalpha-deficient mice (which lack both CD4+ and CD8+ mature T cells) are readily reconstituted. The failure of CD4+ T cell reconstitution in MHC class II-deficient mice was not due to the presence of CD8+ T cells, since similar results were obtained in TCRalpha-MHC class II double-deficient mice. Consistent with most previous studies CD4+ T cells in reconstituted TCRalpha-deficient mice had a diverse TCR Vbeta repertoire and were predominantly of an activated/memory (CD44high) phenotype. Collectively our data demonstrate that the expansion of peripheral CD4+ T cells in a T cell-deficient host is dependent upon interactions of the TCR with MHC class II.

Thymic lineage commitment rather than selection causes genetic variations in size of CD4 and CD8 compartments.

During their development, immature CD4+ CD8+ thymocytes become committed to either the CD4 or CD8 lineage. Subsequent complete maturation of CD4+ and CD8+ cells requires a molecular match of the expressed coreceptor and the MHC specificity of the TCR. The final size of the mature CD4+ and CD8+ thymic compartments is therefore determined by a combination of lineage commitment and TCR-mediated selection. In humans and mice, the relative size of CD4+ and CD8+ peripheral T cell compartments shows marked genetic variability. We show here that genetic variations in thymic lineage commitment, rather than TCR-mediated selection processes, are responsible for the distinct CD4/CD8 ratios observed in common inbred mouse strains. Genetic variations in the regulation of lineage commitment open new ways to analyze this process and to identify the molecules involved.

Processing of tumor-associated antigen by the proteasomes of dendritic cells controls in vivo T-cell responses.

Dendritic cells are unique in their capacity to process antigens and prime naive CD8(+) T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-A(MART1) contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo.

Superantigens of mouse mammary tumor virus.

Superantigens (SAgs) are proteins of microbial origin that bind to major histocompatibility complex (MHC) class II molecules and stimulate T cells via interaction with the V beta domain of the T cell receptor (TCR). Mouse mammary tumor virus (MMTV) is a milk-transmitted type B retrovirus that encodes a SAg in its 3' long terminal repeat. Upon MMTV infection, B cells present SAg to the appropriate T cell subset, which leads to a strong "cognate" T-B interaction. This immune reaction results in preferential clonal expansion of infected B cells and differentiation of some of these cells into long-lived memory cells. In this way a stable MMTV infection is achieved that ultimately results in infection of the mammary gland and virus transmission via milk. Thus, in contrast to many microorganisms that attempt to evade the host immune system (reviewed in 1), MMTV depends upon a strong SAg-induced immune response for its survival. Because of their ability to stimulate very strong T cell responses in MHC-identical mice, minor lymphocyte stimulatory (Mls) antigens, discovered more than 20 years ago, are now known to be SAgs encoded by endogenous MMTV proviruses that have randomly integrated into germ cells. The aim of this review is to combine the extensive biology of Mls SAgs with our current understanding of the life cycle of MMTV.

Phenotypic heterogeneity of antigen-specific CD4 T cells under different conditions of antigen persistence and antigen load.

The factors responsible for the phenotypic heterogeneity of memory CD4 T cells are unclear. In the present study, we have identified a third population of memory CD4 T cells characterized as CD45RA(+)CCR7(-) that, based on its replication history and the homeostatic proliferative capacity, was at an advanced stage of differentiation. Three different phenotypic patterns of memory CD4 T cell responses were delineated under different conditions of antigen (Ag) persistence and load using CD45RA and CCR7 as markers of memory T cells. Mono-phenotypic CD45RA(-)CCR7(+) or CD45RA(-)CCR7(-) CD4 T cell responses were associated with conditions of Ag clearance (tetanus toxoid-specific CD4 T cell response) or Ag persistence and high load (chronic HIV-1 and primary CMV infections), respectively. Multi-phenotypic CD45RA(-)CCR7(+), CD45RA(-)CCR7(-) and CD45RA(+)CCR7(-) CD4 T cell responses were associated with protracted Ag exposure and low load (chronic CMV, EBV and HSV infections and HIV-1 infection in long-term nonprogressors). The mono-phenotypic CD45RA(-)CCR7(+) response was typical of central memory (T(CM)) IL-2-secreting CD4 T cells, the mono-phenotypic CD45RA(-)CCR7(-) response of effector memory (T(EM)) IFN-gamma-secreting CD4 T cells and the multi-phenotypic response of both IL-2- and IFN-gamma-secreting cells. The present results indicate that the heterogeneity of different Ag-specific CD4 T cell responses is regulated by Ag exposure and Ag load.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.