John E. Coligan

Differential expression of two distinct T-cell receptors during thymocyte development

The product of the T-cell receptor (TCR) γ-gene1 has recently been found to be expressed on a subset of both peripheral cells2 and thymocytes3,4. As an initial approach to understanding the role of this γ-chain of TCR (TCRγ) in T-cell development, we have studied the ontogeny of TCR expression at the protein level in the developing murine thymus. We show here that the first T3-associated TCR to be expressed in the developing thymus is a disulphide-linked heterodimer composed of a γ-chain of relative molecular mass 35,000 (Mr 35K) and a 45K partner (termed TCR δ). This TCR γδ is first detected approximately two days before the appearance of cell-surface TCR αβ heterodimers. We report that N-glycosidase digestions reveal that all of the γ-protein expressed on fetal thymocytes, as in adult CD4−8− (L3T4−, Lyt2−) thymocytes4, bear N-linked carbohydrate side chains. The major γ-gene transcribed in mature, αβ-bearing T cells ( Vγ1.2 Cγ2) encodes no N-linked glycosylation site1 so these results suggest that the fetal γδ receptor defines a distinct T-cell lineage whose development in the thymus precedes classical αβ -bearing cells.

Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells.

Natural killer (NK) cells express receptors that are specific for MHC class I molecules. These receptors play a crucial role in regulating the lytic and cytokine expression capabilities of NK cells. In humans, three distinct families of genes have been defined that encode for receptors of HLA class I molecules. The first family identified consists of type I transmembrane molecules belonging to the immunoglobulin (Ig) superfamily and are called killer cell Ig-like receptors (KIR). A second group of receptors belonging to the Ig superfamily, named ILT (for immunoglobulin like transcripts), has more recently been described. ILTs are expressed mainly on B, T and myeloid cells, but some members of this group are also expressed on NK cells. They are also referred to as LIRs (for leukocyte Ig-like receptor) and MIRs (for macrophage Ig-like receptor). The ligands for the KIR and some of the ILT receptors include classical (class Ia) HLA class I molecules, as well as the nonclassical (class Ib) HLA-G molecule. The third family of HLA class I receptors are C-type lectin family members and are composed of heterodimers of CD94 covalently associated with a member of the NKG2 family of molecules. The ligand for most members is the nonclassical class I molecule HLA-E. NKG2D, a member of the NKG2 family, is expressed as a homodimer, along with the adaptor molecule DAP10. The ligands of NKG2D include the human class I like molecules MICA and MICB, and the recently described ULBPs. Each of these three families of receptors has individual members that can recognize identical or similar ligands yet signal for activation or inhibition of cellular functions. This dichotomy correlates with particular structural features present in the transmembrane and intracytoplasmic portions of these molecules. In this review we will discuss the molecular structure, specificity, cellular expression patterns, and function of these HLA class I receptors, as well as the chromosomal location and genetic organization.

Cutting Edge: NKG2D Is a Costimulatory Receptor for Human Naive CD8+ T Cells

In humans, all αβ CD8+ T cells express NKG2D, but in mouse, it is only expressed by activated and memory CD8+ T cells. We purified human naive CD8+ T cells to show that NKG2D serves as a costimulatory receptor for TCR induced Ca2+ mobilization and proliferation. The resulting effector cells are skewed toward a type 1 phenotype and produce high levels of IFN-γ and TNF-α. NKG2D ligands, MHC class I chain-related (MIC)A, MICB, and UL16-binding proteins are expressed on the proliferating cells and NKG2D is down-regulated. The addition of the homeostatic cytokines IL-7 and IL-15 to the culture medium not only enhances proliferation but also counteracts the down-regulation of NKG2D, more so than the addition of IL-2. These results indicate that NKG2D can regulate the priming of human naive CD8+ T cells, which may provide an alternative mechanism for potentiating and channeling the immune response.

Radioimmune assay of carcinoembryonic antigen

Abstract A double antibody technique for the detection and quantitation of carcino-embryonic antigen (CEA) in nanogram quantities is described. This antigen is characteristic of adenocarcinoma of digestive organs. The assay employs three γ-emitting radioisotopes. A computer program has been written to enable rapid calculation of results. The assay is of value in following the isolation and characterization of CEA and may also prove useful as a test for the detection of certain forms of cancer.

Inhibition of an allospecific T cell hybridoma by soluble class I proteins and peptides: Estimation of the affinity of a T cell receptor for MHC

To investigate the molecular basis of the interaction between the T cell receptor and the MHC class I antigen in an allogeneic response, a soluble counterpart of the murine class I molecule, H-2Kb, was genetically engineered. Cells secreting this soluble molecule, H-2Kb/Q10b, inhibited stimulation of an H-2Kb-reactive T cell hybridoma by cells transfected with H-2Kbm10, a weak stimulus, but not by H-2Kb- or H-2Kbm6-transfected cells. Soluble purified H-2Kb/Q10b protein also blocked T cell stimulation. In addition, a peptide from the wild-type H-2Kb molecule spanning the region of the bm10 mutation specifically inhibited activation of the T cell hybridoma by H-2Kbm10 cells, thus suggesting that amino acid residues 163-174 of H-2Kb define a region important for T cell receptor binding. An estimate for the Kd of the T cell receptor for soluble H-2Kb/Q10b was 10(-7) M, while the Kd for soluble peptide 163-174 was 10(-4) M.

A VASOACTIVE PEPTIDE, ENDOTHELIN-3, IS PRODUCED BY AND SPECIFICALLY BINDS TO PRIMARY ASTROCYTES

Primary rat astrocytes were found by immunohistochemistry to display positive staining for endothelin-3, located predominantly in the perinuclear area. The ability of these cells to produce and release endothelin-3 was confirmed by a combination of reverse-phase HPLC and radioimmunoassay, specific for endothelin-3, which demonstrated immunoreactive peptide in cellular extracts and astrocyte-conditioned medium. In addition, astrocytes were shown to possess a single class of binding sites for endothelin with comparable high affinity for endothelin-1, -2 and -3. These results suggest that astrocytes, by virtue of their ability to produce and secrete endothelin-3, serve as a potential extravascular source of intracerebral vasoregulation capable of influencing regional cerebral blood flow.

Structure of CD94 Reveals a Novel C-Type Lectin Fold: Implications for the NK Cell–Associated CD94/NKG2 Receptors

The crystal structure of the extracellular domain of CD94, a component of the CD94/NKG2 NK cell receptor, has been determined to 2.6 A resolution, revealing a unique variation of the C-type lectin fold. In this variation, the second alpha helix, corresponding to residues 102-112, is replaced by a loop, the putative carbohydrate-binding site is significantly altered, and the Ca2+-binding site appears nonfunctional. This structure may serve as a prototype for other NK cell receptors such as Ly-49, NKR-P1, and CD69. The CD94 dimer observed in the crystal has an extensive hydrophobic interface that stabilizes the loop conformation of residues 102-112. The formation of this dimer reveals a putative ligand-binding region for HLA-E and suggests how NKG2 interacts with CD94.

Isolation and characterization for carcinoembryonic antigen

Abstract Carcinoembryonic antigen (CEA) has been isolated from several tumors of the digestive tract. Comparisons of the products with one another and with CEA obtained from Dr. Gold have been made. CEA of two molecular sizes (6·8 S and 10·1 S) has been obtained, but the 6·8 S material is the one usually found. Considerable variation in yield has been observedp indicating that tumorsp may differ markedly in their content of CEA. Comparison of CEA isolated by Dr. Gold with material prepared by us has shown the two products to be immunologically indistinguishable.

IL-21 down-regulates NKG2D/DAP10 expression on human NK and CD8+ T cells

IL-21 is a recently described cytokine, produced by activated Th cells, that shares significant homology with members of the IL-2 family of cytokines. IL-21 mediates its biological effects via the IL-21R in conjunction with the common receptor γ-chain that is also shared by members of the IL-2 family. We report that culture of human primary NK and CD8+ T cells with IL-21 in combination with IL-2 results in significant reduction of the cell surface expression of NKG2D, compared with that in cells treated with IL-2 alone. The reduced expression of NKG2D after IL-21 culture had functional consequences for NK cell function, as assessed by NKG2D-mediated redirected lysis assays and degranulation assays, compared with NK cells treated with IL-2 alone. IL-21-mediated NKG2D down-regulation in human NK cells correlated with a marked reduction of DNAX-activating protein of 10 kDa (DAP10) transcription in cells treated with IL-2 in combination with IL-21 compared with cells stimulated with only IL-2. This was attributed to a dramatic reduction in DAP10 promoter activity, as assessed by a DAP10 luciferase reporter construct. In contrast to NKG2D expression, IL-21 was able to induce the expression of the NK activation receptors NKp30 and 2B4 as well as the costimulatory receptor CD28 on CD8+ T cells. These data indicate that IL-21 is able to channel NK and CD8+ T cell function by altering the expression pattern of activation/costimulatory receptors.

Human CD300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells.

CD300a is an immunoreceptor tyrosine-based inhibitory motif (ITIM) containing molecule that belongs to the CD300 family of paired activating/inhibitory receptors. It has been shown that its ligation inhibits activation signals on cells of both myeloid and lymphoid lineages. The ligands for CD300a have not been identified. Here, we show that a CD300a-Ig fusion protein specifically binds to apoptotic cells that are evolutionary apart, such as human and insect cells, suggesting that the ligand has to be conserved. Using surface plasmon resonance, ultracentrifugation, ELISA, and reporter cell assays, we identified phosphatidylethanolamine (PE) and phosphatidylserine (PS), 2 phospholipids that translocate to the outer leaflet of the plasma membrane of dead cells, as the ligands for CD300a. Mutational and structural modeling studies identified residues that are involved in the binding of CD300a to PE and PS and that form a cavity where the hydrophilic heads of PE and PS, can penetrate. CD300a down-regulates the uptake of apoptotic cells by macrophages and its ectopic expression in CD300a-negative cell lines also decreased the engulfment of dead cells. Collectively, our results indicate that PE and PS are ligands for CD300a, and that this interaction plays an important role in regulating the removal of dead cells.