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...

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Previous work in this laboratory has led to the development of the hypothesis that the increased susceptibility of old mice to tuberculosis infection reflects a limited ability by immune CD4 mediator cells to accumulate at sites of bacterial implantation. To test this hypothesis with very low dose infections, the present study documented the course of a low-dose aerogenic infection with virulent Mycobacterium tuberculosis Erdman against time in the target organs of young (3-month-old) and old (24-month-old) B6D2F 1 hybrid mice. The results of the study indicated that the infection was controlled by the two groups of mice at similar rates, although the bacterial load in the old mice was eventually somewhat higher. Despite these similarities, some subtle differences between the young and old mice were also evident and included evidence of increased hematogenous spread of the infection from the lungs to other organs in the old mice. Interestingly, very poor expression of the cytokine interleukin-12 was observed in the lungs of infected old mice, leading to the hypothesis that the poor CD4 response in such animals could be partially attributed to the lack of this Th1-type, CD4 T-cell-enhancing cytokine. In this regard, treatment of old mice with exogenous interleukin-12 increased resistance and promoted gamma interferon secretion by CD4 T cells from these mice, although the effects were generally modest. These data suggest that old mice possess CD4-independent compensatory mechanisms by which to deal with low-dose pulmonary tuberculosis infections, although such mechanisms are less efficient than those seen in young animals.

SUMMARY Streptococcus sanguinis is a major component of the oral flora and an important cause of infective endocarditis. Although S. sanguinis is naturally competent, genome sequencing has suggested significant differences in the S. sanguinis competence system relative to those of other streptococci. An S. sanguinis mutant possessing an in-frame deletion in the comC gene, which encodes competence-stimulating peptide (CSP), was created. Addition of synthetic CSP induced competence in this strain. Gene expression in this strain was monitored by microarray analysis at multiple time points from 2.5 to 30 min after CSP addition, and verified by quantitative RT-PCR. Over 200 genes were identified whose expression was altered at least two-fold in at least one time point, with the majority upregulated. The “late” response was typical of that seen in previous studies. However, comparison of the “early” response in S. sanguinis with that of other oral streptococci revealed unexpected differences with regard to the number of genes induced, the nature of these genes, and their putative upstream regulatory sequences. S. sanguinis possesses a comparatively limited early response, which may define a minimal streptococcal competence regulatory circuit.

As mice age, spontaneous changes occur in the receptor repertoire of their T cells. The receptor repertoire of CD4+ T cells does not change with age. By contrast, however, the percentage of alpha beta+, CD8+ T cells bearing particular V elements varies considerably between individual aged mice, although it is remarkably consistent among individual young animals within a given strain. Changes of receptor V element use among CD8+ T cells in individual mice are unpredictable. However, when a large number of mice of the same strain is analyzed, strain-specific trends in V element skewing are found. Old C3H.SW and B10.BR mice have mono- or oligoclonal expansions of CD8+ T cells. These expansions of peripheral CD8+ T cells with age are probably due to deregulation of proliferation of individual CD8+ T cells after recognition of viral or environmental Ag, accompanied, perhaps, by partial transformation of particular T cell clones. Another phenomenon documented herein is the fact that the CD4/CD8 ratio drops steadily as a function of age. Shifts in CD4/CD8 ratio were not due to increased numbers of CD8+ T cells in spleen and lymph nodes, rather the CD4+ T cells disappeared from aging mice faster than CD8+ T cells.