Leonard Chess

The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4: a new member of the immunoglobulin gene family

The surface glycoproteins T4 and T8 define different functional subsets of T lymphocytes and may act as recognition molecules mediating appropriate interactions between the T cell and its target. Previously we employed gene transfer and subtractive hybridization to isolate a T8 cDNA; now we have isolated and sequenced a cDNA clone encoding the T4 molecule. The deduced protein sequence reveals that T4 is an integral membrane protein that shares significant amino acid and structural homologies with members of the immunoglobulin supergene family. The overall structure of T4 consists of an N-terminal variable (V)-like domain, a joining (J)-like region, a third extracellular domain, a membrane-spanning region homologous to class II MHC beta-chains, and a highly charged cytoplasmic domain. Comparison of the protein sequences deduced from the T4 and T8 cDNAs reveals structural similarities consistent with their postulated role as recognition molecules, as well as differences suggesting that the two proteins recognize different structures on the target cell.

The isolation and sequence of the gene encoding T8: A molecule defining functional classes of T lymphocytes

The T cell surface glycoproteins T4 and T8 are thought to mediate efficient cell-cell interactions in the immune system and in this way may be responsible for the appropriate targeting of subpopulations of T cells. We have used gene transfer combined with subtractive hybridization to isolate both cDNA and functional genomic clones encoding the T8 protein. The sequence of the cDNA reveals that T8 is a transmembrane protein with an N-terminal domain which shares significant homology to immunoglobulin variable region light chains. This immunoglobulin-like structure is likely to be important in the function of T8 during differentiation and in the course of the immune response.

A single amino acid substitution in a common African allele of the CD4 molecule ablates binding of the monoclonal antibody, OKT4

The CD4 molecule is a relatively non-polymorphic 55 kDa glycoprotein expressed on a subset of T lymphocytes. A common African allele of CD4 has been identified by non-reactivity with the monoclonal antibody, OKT4. The genetic basis for the OKT4- polymorphism of CD4 is unknown. In the present paper, the structure of the CD4 molecule from an homozygous CD4OKT4- individual was characterized at the molecular level. The size of the CD4OKT4- protein and mRNA were indistinguishable from those of the OKT4+ allele. The polymerase chain reaction (PCR) was used to map the structure of CD4OKT4- cDNAs by amplifying overlapping DNA segments and to obtain partial nucleotide sequence after asymmetric amplification. PCR was then used to clone CD4OKT4- cDNAs spanning the coding region of the entire, mature CD4 protein by amplification of two overlapping segments followed by PCR recombination. The nucleotide sequence of CD4OKT4- cDNA clones revealed a G----A transition at bp 867 encoding an arginine----tryptophan substitution at amino acid 240 relative to CD4OKT4+. Expression of a CD4OKT4- cDNA containing only this transition, confirmed that the arginine----tryptophan substitution at amino acid 240 ablates the binding of the mAb OKT4. A positively charged amino acid residue at this position is found in chimpanzee, rhesus macaque, mouse and rat CD4 suggesting that this mutation may confer unique functional properties to the CD4OKT4- protein.

LIGATION OF CD40 ON FIBROBLASTS INDUCES CD54 (ICAM-1) AND CD106 (VCAM-1) UP-REGULATION AND IL-6 PRODUCTION AND PROLIFERATION

CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon‐γ (rINF‐γ) induces fibroblast CD40 up‐regulation. This effect of rINF‐γ is augmented by recombinant interleukinlα or recombinant tumor necrosis factor‐α. CD40 expression on fibroblasts is functionally significant because CD40L‐CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule‐1) and CD106 (vascular cell adhesion molecule‐1) up‐regulation. Moreover, ligation of CD40 augments IL‐6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF‐γ enhances the effect of CD40L‐CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation. J. Leukoc. Biol. 58: 209–216; 1995.

2 å crystal structure of an extracellular fragment of human CD40 ligand

BACKGROUND The CD40 ligand (CD40L) is a member of the tumor necrosis factor (TNF) family of proteins and is transiently expressed on the surface of activated T cells. The binding of CD40L to CD40, which is expressed on the surface of B cells, provides a critical and unique pathway of cellular activation resulting in antibody isotype switching, regulation of apoptosis, and B cell proliferation and differentiation. Naturally occurring mutations of CD40L result in the clinical hyper-IgM syndrome, characterized by an inability to produce immunoglobulins of the IgG, IgA and IgE isotypes. RESULTS We have determined the crystal structure of a soluble extracellular fragment of human CD40L to 2 A resolution and with an R factor of 21.8%. Although the molecule forms a trimer similar to that found for other members of the TNF family, such as TNF alpha and lymphotoxin-alpha, and exhibits a similar overall fold, there are considerable differences in several loops including those predicted to be involved in CD40 binding. CONCLUSIONS The structure suggests that most of the hyper-IgM syndrome mutations affect the folding and stability of the molecule rather than the CD40-binding site directly. Despite the fact that the hyper-IgM syndrome mutations are dispersed in the primary sequence, a large fraction of them are clustered in space in the vicinity of a surface loop, close to the predicted CD40-binding site.

Murine CD8+ T cells that specifically delete autologous CD4+ T cells expressing Vβ8 TCR: a role of the Qa-1 molecule

Interactions mediated by TCRs expressed on different T cell subsets may play a role in immunoregulation. To investigate this idea, we studied the regulation of superantigen-induced TCR V beta-restricted responses. We asked whether the in vivo regulation of CD4+ V beta 8+ T cells following SEB injection is controlled by CD8+ T cells. We found that in mice deficient in CD8+ T cells, the down-regulation of CD4+ V beta 8+ T cells below baseline is not observed. Moreover, following SEB administration, CD8+ T cells emerge that preferentially kill subpopulations of activated CD4+ V beta 8+ but not CD4+ V beta 8- T cells in vitro. This TCR V beta-specific cytotoxicity is dependent on beta 2-microglobulin and is inhibited by antisera specific for Qa-1 but not by antibody to MHC class Ia. These data suggest the idea that the specificity of immune regulation may involve CD8+ T cell recognition of TCR V beta determinants and Qa-1 molecules expressed on CD4+ T cells.

Suppression of experimental autoimmune encephalomyelitis by selective blockade of encephalitogenic T-cell infiltration of the central nervous system

Multiple sclerosis (MS) is a devastating neuroinflammatory disorder of the central nervous system (CNS) in which T cells that are reactive with major components of myelin sheaths have a central role. The receptor for advanced glycation end products (RAGE) is present on T cells, mononuclear phagocytes and endothelium. Its pro-inflammatory ligands, S100-calgranulins, are upregulated in MS and in the related rodent model, experimental autoimmune encephalomyelitis (EAE). Blockade of RAGE suppressed EAE when disease was induced by myelin basic protein (MBP) peptide or encephalitogenic T cells, or when EAE occurred spontaneously in T-cell receptor (TCR)-transgenic mice devoid of endogenous TCR-α and TCR-β chains. Inhibition of RAGE markedly decreased infiltration of the CNS by immune and inflammatory cells. Transgenic mice with targeted overexpression of dominant-negative RAGE in CD4+ T cells were resistant to MBP-induced EAE. These data reinforce the importance of RAGE-ligand interactions in modulating properties of CD4+ T cells that infiltrate the CNS.

Human lymphocyte subpopulations.

Publisher Summary This chapter focuses the attention on some approaches to overcoming difficulties inherent in the investigation of the human system with particular attention to the three areas. The precise dissection of the cellular mechanisms and interactions involved in the generation of the human immune response has been facilitated by recent advances in three interrelated areas: (1) the development of in vitro methods for the characterization and identification of human lymphocyte classes by cell surface markers, (2) the development of new techniques for the isolation of highly purified subclasses of human lymphocytes and monocytes, and (3) the development of in vitro techniques to discriminate the functional properties and interactions of the isolated subsets of lymphocytes. Finally, the chapter concludes that one limitation has been the lack of genetically defined strains that have been so important in both the generation of alloantisera to unique subclasses of cells and in the genetic analysis of T, B, and macrophage cell interactions.

CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice

Trimolecular interactions between the T cell antigen receptor and MHC/peptide complexes, together with costimulatory molecules and cytokines, control the initial activation of naïve T cells and determine whether the helper precursor cell differentiates into either T helper (TH)1 or TH2 effector cells. We now present evidence that regulatory CD8+ T cells provide another level of control of TH phenotype during further evolution of immune responses. These regulatory CD8+ T cells are induced by antigen-triggered CD4+ TH1 cells during T cell vaccination and, in vitro, distinguish mature TH1 from TH2 cells in a T cell antigen receptor Vβ-specific and Qa-1-restricted manner. In vivo, protection from experimental autoimmune encephalomyelitis (EAE) induced by T cell vaccination depends on CD8+ T cells, and myelin basic protein-reactive TH1 Vβ8+ clones, but not TH2 Vβ8+ clones, used as vaccine T cells, protect animals from subsequent induction of EAE. Moreover, in vivo depletion of CD8+ T cells during the first episode of EAE results in skewing of the TH phenotype toward TH1 upon secondary myelin basic protein stimulation. These data provide evidence that CD8+ T cells control autoimmune responses, in part, by regulating the TH phenotype of self-reactive CD4+ T cells.

Induction of TCR Vβ-Specific CD8+ CTLs by TCR Vβ-Derived Peptides Bound to HLA-E

Previous studies have identified murine and human regulatory CD8+ T cells specific for TCR-Vβ families expressed on autologous activated CD4+ T cells. In the mouse, these regulatory CD8+ T cells were shown to be restricted by the MHC class Ib molecule, Qa-1. In the present study, we asked whether HLA-E, the human functional equivalent of Qa-1, binds Vβ peptides and whether the HLA-E/Vβ-peptide complex induces and restricts human CD8+ CTLs. We first created stable HLA-E gene transfectants of the C1R cell line (C1R-E). Two putative HLA-E binding nonapeptides identified in human TCR Vβ1 and Vβ2 chains (SLELGDSAL and LLLGPGSGL, respectively) were shown to bind to HLA-E. CD8+ T cells could be primed in vitro by C1R-E cells loaded with the Vβ1 (C1R-E/V1) or Vβ2 (C1R-E/V2) peptide to preferentially kill C1R-E cells loaded with the respective inducing Vβ peptide, compared with targets loaded with the other peptides. Priming CD8+ T cells with untreated C1R-E cells did not induce Vβ-specific CTLs. Of perhaps more physiological relevance was the finding that the CD8+ CTLs primed by C1R-E/V1 also preferentially killed activated autologous TCR Vβ1+. Similar results were observed in reciprocal experiments using C1R-E/V2 for priming. Furthermore, anti-CD8 and anti-MHC class I mAbs inhibited this Vβ-specific killing of C1R-E and CD4+ T cell targets. Taken together, the data provide evidence that certain TCR-Vβ peptides can be presented by HLA-E to further induce Vβ-specific CD8+ CTLs.