Ned S. Braunstein

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.

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.

An MHC interaction site maps to the amino-terminal half of the T cell receptor α chain variable domain

We have used cloned T cell receptor (TCR) genes from closely related CD4 T cell lines to probe the interaction of the TCR with several specific major histocompatibility complex (MHC) class II ligands. Complementarity determining region 3 (CDR3) equivalents of both alpha and beta TCR chains are required for antigen-MHC recognition. Our data provide novel information about the rotational orientation of TCR-MHC contacts in that exchange of the amino terminal portion of the TCR alpha chain containing the putative CDR1 and CDR2 regions results in both gain and loss of MHC class II specificity by the resulting receptor. These two TCRs differ primarily in recognition of polymorphisms in the second hypervariable region of the MHC class II alpha chain. These results document the involvement of CDR1 and/or CDR2 of the TCR alpha chain in MHC recognition and suggest a rotational orientation of this TCR to its MHC ligand.

Human CD8+ T lymphocyte clones specific for T cell receptor Vβ families expressed on autologous CD4+ T cells

CD8+ T cells control immune responses, and recent studies suggest that this regulation is, in part, specifically directed towards TCR structures expressed by CD4+ cells. To develop a system to study the role of the TCR in regulatory interactions, we isolated clones of CD4+ cells expressing identified TCR V beta chains. These CD4+ clones were used to stimulate and expand autologous CD8+ cells, which kill the inducing CD4+ clone as well as independently isolated autologous CD4+ clones sharing the same TCR V beta as the inducing cell but not CD4+ T cells expressing different V beta TCRs. This V beta-specific cytotoxicity is dependent on the state of activation of the target cells and is not inhibited by an anti-class I monoclonal antibody, W6/32. We envision that V beta-specific CD8+ T cells of this type may regulate immune responses by direct interaction with antigen-activated CD4+ cells.

Controlled Cell Deformation Produces Defined Areas of Contact between Cells and Ligand-Coated Surfaces

AbstractA method which allows precise control of the time of initiation and the area of contact of T cells with immobilized ligands has been developed. Cells are trapped in an asymmetric film that can be quantitatively thinned by reducing the films capillary pressure. Ligands adsorbed to the base of the apparatus are forced into close contact with the cells as the air–liquid interface is drawn down. Using interference microscopy and microbeads to indicate the film height, the amount of thinning can be controlled to within 1 μm. In this study, this system was used to produce contact areas of 182 and 356 μm2 between T cells and anti-CD3 coated surfaces. These contact areas were measured using fluorescent dye exclusion microscopy. This apparatus can be used for quantitative studies of T cell activation, as is reported in Patrick et al., J. Immunol. Method. 24:97–108, 2000.

Regulation of IFN-γ signaling is essential for the cytotoxic activity of CD8+ T cells

Previous studies have demonstrated that, as naive murine CD4+ cells differentiate into Th1 cells, they lose expression of the second chain of IFN-γR (IFN-γR2). Hence, the IFN-γ-producing subset of Th cells is unresponsive to IFN-γ. Analysis of IFN-γ-producing CD8+ T cells demonstrates that, like Th1 cells, these cells do not express IFN-γR2. To define the importance of IFN-γ signaling for the development of functional CD8+ T cells, mice either lacking IFN-γR2 or overexpressing this protein were examined. While CD8+ T cell development and function appear normal in IFN-γR2−/− mice, CD8+ T cell function in IFN-γR2 transgenic is altered. IFN-γR2 transgenic CD8+ T cells are unable to lyse target cells in vitro. However, these cells produce Fas ligand, perforin, and granzyme B, the effector molecules required for killing. Interestingly, TG CD8+ T cells proliferate normally and produce cytokines, such as IFN-γ in response to antigenic stimulation. Therefore, although IFN-γ signaling is not required for the generation of normal cytotoxic T cells, constitutive IFN-γ signaling can selectively impair the cytotoxic function of CD8+ T cells.

Dependence of T cell activation on area of contact and density of a ligand-coated surface

An apparatus which allows precise control of the time of initiation and the area of contact of cells with immobilized ligands has been developed. Cells are trapped in an asymmetric film that can be quantitatively thinned, forcing the cells into close contact with ligands adsorbed on the base of the apparatus. Using microbeads to indicate the film height, the amount of thinning can be controlled to within 1 microm, producing known contact areas between cells and the ligand-coated surface. This system was used with anti-CD3-coated surfaces of different densities to examine the effect of ligand density on T cell activation, while keeping the number of ligands presented to the cells constant. T cell activation was observed individually in each cell as intracellular calcium mobilization. In these experiments both the percent of T cell activation and the rate of calcium rise were found to depend only on the number of anti-CD3 molecules presented and not on their density. This implies that the spacing between molecules is not important in the range studied, and suggests that receptor clustering to levels higher than dimers may not be necessary for induction of calcium mobilization by anti-CD3.

Modeling of early events in T cell signal transduction after controlled T cell activation by peptide major histocompatibility complex.

AbstractCalcium signaling was observed in murine T cells over time, starting at a precise moment of contact with a layer of fibroblasts expressing a stimulatory major histocompatibility class II–peptide complex. The contact was controlled by a film-thinning apparatus. Intracellular calcium levels were followed with the ratiometric dye, Fura-2. The calcium response was highly synchronized and well fitted by a mathematical model. The model includes three components: a sequence of reactions occurring after T cell receptor (TCR) triggering; InsP3-mediated calcium release from intracellular stores (Meyer and Stryer, Proc. Natl. Acad. Sci. USA 85: 5051–5055, 1988); and slow changes in levels phospholipase C-γ1 (PLCγ1) reflecting a decrease in receptor triggering rate. Each component in the model controls a different part of the response—the initial delay, the sharp rise, and the slow decay, respectively. Kinetic parameters determined from curve fitting were the initial delay in calcium signaling defined as the time when [PLCγ1] reached its half of its maximum (76 s), the coefficient characterizing calcium efflux from endoplasmic reticulum (ER) (2.86 μM s--1, expressed per liter of cell volume), and a rate constant characterizing the diminishing yield of production of PLCγ1 (0.00046 s--1) by active TCR. Only the parameter representing PLCγ1 production varied much from cell to cell.

Controlling duration of contact between T cells and antigen-presenting cells.

A new method which allows precise control of the duration of contact between T cells and antigen-presenting cells (APCs) has been developed. A glass coverslip coated with poly-L-lysine, and then with T cells, was placed at the base of a cylindrical well, and the well was filled with liquid medium. A round coverslip, on which APCs were adhered, was supported on the surface of the medium by surface tension, cell-side down. By withdrawing medium from four capillary holes near the base of the well, the coverslip could be lowered to initiate contact between T cells and APCs at a defined time zero. The contact was broken at desired time points by re-introducing medium into the well in order to separate the two coverslips. Each cell type remained adherent to its original surface after separation for all contact times studied. The T cells were monitored for intracellular calcium mobilization using the fluorescent dye, Fura-2. Contact durations of less than 1 min did not trigger calcium signals. Contact durations of 3 and 5 min induced strong calcium signals. Breaking the contact caused a rapid decrease in intracellular calcium levels. This method of cell manipulation allows precise control of the duration of contact of T cells with APCs, while keeping the cells under continuous observation. The measurements so obtained provide a quantitative understanding of the dynamics of early T cell activation.