Interaction of Staphylococcus aureus toxin "superantigens" with human T cells.

Abstract

A modification of the polymerase chain reaction has been used to establish the fact that a collection of Staphylococcus aureus toxins are "superantigens," each of which interacts with the T-cell a4i receptor of human T cells by means of a specific set of V,8 elements.The antigen receptor [T-cell receptor (TCR)] on most peripheral T cells is a heterodimer made up of a and P chains. Five germ-line-encoded variable elements (Va, Ja, Vp, Dp, and JP) as well as non-germ-line-encoded amino acids contribute to the receptor combining site (1-4). The ligands for aB TCRs are combinations of antigen-derived peptides, bound to major histocompatibility complex proteins (MHC) (5)(6)(7)(8). Usually the specificity of TCRs for antigen plus MHC is determined by all of the variable elements of both a and p chains (1-4). Exceptions to this rule have, however, recently been documented by us and others (9-18). In the examples studied so far, the exceptions involve an antigen/MHC combination that can stimulate T cells bearing a particular VP, almost regardless of the composition of the rest of the receptor on these cells. We have suggested the term "superantigen" to describe receptor ligands of this type. These superantigens and the VP elements that engage them have been well documented in mice with the help of an increasing number of VP-specific antibodies and DNA probes.Some superantigens are endogenously synthesized-for example, a mouse B-cell-derived self-superantigen bound to IE reacts with nearly all T cells bearing Vl17a and mice that express IE delete nearly all V,817a' T cells (9, 10). Exogenous superantigens have also been described. We and others have recently shown that some bacterial proteins are also able to stimulate T cells in a Vp-specific fashion (15-19). For example, Staphylococcus aureus enterotoxin B (SEB) stimulates mouse T cells bearing Vp83, and has no effect on most other cells. Since the S. aureus toxins are important contributors to morbidity and mortality in man (20), we wished to extend our studies on bacterial superantigens to human T cells. In an initial study, we used monoclonal antibodies (mAbs) to four human VP elements to show that T cells expressing these VP elements were differentially stimulated by a panel of S. aureus toxins (18). However, since there are at least 20 V, families in human (2, 21), we were limited by the lack of a complete set of anti-VP mAbs. We have, therefore, modified the methodology of the polymerase chain reaction (PCR) (22,23) MATERIALS AND METHODSPreparation of RNA and cDNA Synthesis. Total RNA was prepared from anti-CD3-stimulated peripheral T cells as described (18,24). Two micrograms of total RNA was used for the synthesis of first strand cDNA using reverse transcriptase (Amersham) and random hexanucleotides. The reaction was stopped by heating for 5 min at 950C before PCR.Amplification of cDNA by PCR and Quantitation of Amplified Products. One-twentieth of each cDNA sample was coamplified using a Vp-specific primer with a C,3 primer and two Ca primers (Table 1...

T‐Cell Receptor BJ Gene Segment Expression in Human Umbilical Cord Blood CD4+ and CD8+ T‐Cell Subsets

“…Additionally, TCR BV genes and their products have been reported to be expressed in a highly disproportional manner, varying in frequencies during ontogeny [21,39,40,42,45]. Although usage of individual BVs in unfractionated thymocytes from different individuals is statistically similar [42], they appear to be expressed at different frequencies within the peripheral Tcell pool whereas the BJ usage patterns in PBL T cells and thymocytes are similar [15,16,42].…”

“…The cycle profile included 94ЊC for denaturation, annealing at 55ЊC and extension at 72ЊC (1 min each) followed by additional 9 min at 72ЊC at the end of the final cycle. Oligonucleotides specific for BV 3, 5S1, 6S1-3, 8, 9, 12 and 18 as well as an oligonucleotide specific for both CB 1 and CB 2 (for primer sequences, see [21]) were used as 5 0 sense and 3 0 anti-sense primers, respectively.…”

Structural Analysis of TCRα and β Chains from Human T‐Cell Clones Specific for Small Nuclear Ribonucleoprotein Polypeptides Sm‐D, Sm‐B and U1–70 kDa: TCR Complementarity Determining Region 3 Usage Appears Highly Conserved

“…Briefly, T cells were stimulated for 48 h in the presence of 20 U/ml IL-2 and immobilized anti-CD3 for 48 h mRNA was extracted from the cells using an oligo (dT)-cellulose microaffinity column adsorption method (Micro-Fast Tract, Invitrogen, San Diego, CA, USA). First strand cDNA was synthesized using Reverse Transcriptase and RNase H (Superscript Preamplification System, Life Technologies), then amplified using Taq Polymerase (Fisher Scientific, St. Louis, MO, USA) in the presence of primers specific for one of 29 V alpha [18] or one of 24 V beta [19,20] regions of the TCR. Kinetics studies were performed with serial sampling during each PCR to ensure that the PCR was analyzed during the linear phase of the amplification.…”

Emergence of oligoclonal t cell populations following therapeutic t cell depletion in rheumatoid arthritis

“…Total RNA was purified from established T cell clones by guanidinium thiocyanate and phenol-chloroform extraction (RNAzol; Tel Test, Friendswood, TX). Complementary DNA (cDNA) was synthesized and amplified by polymerase chain reaction (PCR) using Vp-Cpspecific primer sets (23). The Vp primer was attached to a T7 promotor.…”