David N. Posnett

To determine whether T cells, like B cells, can become clonally expanded in normal individuals as a function of age, we compared the T cell V beta repertoire of cord blood to that of peripheral blood from normal donors over 65 yr of age. T cells from elderly subjects contained expanded subsets (greater than the mean+three standard deviations) of T cell receptor (TCR) V beta populations. These expanded subsets were observed primarily among CD8, but not CD4 cells, represented up to 37.5% of all CD8 cells, and were present in most elderly subjects. An expanded V beta 5.2/3 CD8 subset and a V beta 6.7a CD8 subset from separate donors were analyzed by reverse transcriptase-polymerase chain reaction, cloning and sequencing of the TCR beta chain VDJ junction. In both cases the expanded subsets were mono- or oligoclonal while control CD4 populations were polyclonal. Using two-color flow cytometry it was possible to identify the expanded V beta 6.7a subset as CD8+ CD28-CD11b+ cells. In three of five random old subjects similar expansions of V beta subsets were found specifically in the CD8+ CD28- subpopulation, an interesting subset of cytotoxic T lymphocytes, known to lack proliferative responses to TCR stimuli. It is common practice to use the demonstration of clonality as a diagnostic indicator for T cell lymphoma/leukemia. In view of the high frequency of expanded T clones of T cells in normal elderly subjects the diagnostic usefulness of this test should be reexamined.

The staphylococcal toxins are responsible for a number of diseases in man and other animals. Many of them have also long been known to be powerful T cell stimulants. They do not, however, stimulate all T cells. On the contrary, each toxin reacts with human T cells bearing particular V beta sequences as part of their receptors for major histocompatibility complex protein-associated antigen. The specificity of these toxins for V beta s puts them in the recently described class of superantigens and may account for the differential sensitivity of different individuals to the toxic effects of these proteins.

Peptide antigens used to generate site-specific antibodies to proteins are of interest in the development of vaccines. The need to conjugate them to a carrier protein for optimal immunogenicity results in a number of problems including a possible immune response to the carrier. Here we describe a new method of synthesizing an immunogenic peptide antigen, referred to as multiple antigenic peptide (MAP), which may render the need for a carrier protein obsolete. A 14-residue sequence derived from the human T cell antigen receptor beta-chain constant region was selected, and the peptide was synthesized directly onto a branching lysine core with 8 copies of the 14-residue peptide linked to the core by the COOH-terminal amino acid. The molecular weight of this structure was 13,422 of which only 7% represents the lysine residues of the core. The octameric MAP was highly immunogenic in mice and rabbits, allowing production of polyclonal and monoclonal antibodies. The majority of these antibodies reacted with the peptide in its monomeric form as well as its octameric form. Moreover, the antibodies reacted with the intact beta-chain protein. The antigenic determinants of the peptide that were recognized by the antibodies included continuous determinants and conformational determinants. The NH2-terminal residues of the octameric MAP appeared to be most immunogenic. There were no antibodies to the central lysine core. This method of direct synthesis of a polymeric peptide provides accurate knowledge of the conformation and quantity of the peptide prior to immunization, which is usually not the case when peptides are conjugated to carriers. The method is versatile because the possibility exists to synthesize MAP with 16 or 32 peptide arms or to synthesize polymers containing two different peptides.

We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

The diversity of the human TCR repertoire in aging has been studied by examining the profiles of complementarity-determining region 3 (CDR3) sizes expressed by the BV families. The TCRBV CDR3 profile, which shows size heterogeneity in young adult humans, is significantly restricted in aged humans. Clonal T cell expansions were identified using a PCR-based approach, in one or more BV families from all 14 healthy persons over the age of 65 that we studied. CD4+ T cell expansions were identified in 8 of 11 donors and CD8+ T cell expansions in 7 of 10 donors. These clonal expansions were stable during a 2-year period. Interestingly, more than half of the aged persons had clonal expansions within the BV3, -14, -16, and -23 families. Although there was no homology among the eight CDR3 sequences identified in clonal T cells from 8 aged persons, selective pressure on the expanded T cell clones was suggested by the fact that the BV families used by the T cell clones were not proportional to the number of genes in the different BV families.