Intrafamily fragment analysis of the T cell receptor beta chain CDR3 region.

The length distributions of the third complementarity determining region (CDR3) of the T cell receptor (TCR) beta chain were determined quantitatively from peripheral blood lymphocytes of healthy donors. RT-PCR products of 26 V beta families and subfamilies were analyzed by an A.L.F. DNA sequencer and Fragment Manager software. We established a normal reference of CDR3 lengths for most of the V beta families and subfamilies. The known range of CDR3 lengths for the V beta chain was expanded to 24 amino acids, and quantitative measurements were made for each fragment length allowing intrafamily CDR3 fragment length comparisons. Importantly, we were able to analyze intrafamily CDR3 fragment distribution without optimizing PCR conditions, thereby circumventing a major obstacle found in intrafamily TCR beta chain comparisons.

Immune modulation during treatment of systemic sclerosis with plasmapheresis and immunosuppressive drugs.

Immunologic effects of our previously reported treatment protocol were determined in eight systemic sclerosis (SSc) patients treated with plasmapheresis, cyclophosphamide, and prednisone as tolerated. Prior to treatment total serum IgG and circulating lymphocytes including the percentage of B, T, and NK cells were normal. Peripheral blood lymphocytes showed evidence of activation by increased spontaneous proliferation and expression of CD25 on T cells. In addition, CD4+ T cells showed increased expression of both activation (HLA-DR) and maturation (CD29) markers. The percentage of CD8+ T cells was low, resulting in a high CD4:CD8 T cell ratio. During treatment all patients showed clinical improvement. Laboratory testing showed that their NK cells had declined by 81%, B cells declined by 96%, and serum IgG declined by 49%; high titer ANA were abrogated. Total T cells declined by 52%, and the CD4:CD8 ratio fell to normal, due to an almost twofold increase in the percentage of CD8+ T cells. Spontaneous lymphocyte proliferation increased further and was accompanied by increased maturation and activation of both CD4+ and CD8+ T cells, as well as a reduction of immature CD4+ T cells. Among CD8+ T cells the percentage of cytotoxic cells increased as shown by an increase in the CD11b-, CD38+, and CD29+ phenotypes, a finding confirmed on follow-up three-color flow cytometry which showed an increase in activated CD8 cells bearing the cytotoxic CD28 marker. Three-color cytometry also showed that cells with the CD4+CD45RA+CD31+ suppressor inducer phenotype were low and those with the CD4+CD45RO+ CD31- helper inducer phenotype were high in treated SSc patients. Deficient CD4+CD45RA+CD31+ and CD8+ T cell populations suggest an immune regulatory imbalance in SSc which could have led to CD4+ T cell activation and autoimmunity. Depletion of B cells, in conjunction with augmentation of cytotoxic CD8+ T cells through combined therapy, may have diminished the autoimmune response.

Cotransductional mapping of the trp-his region of Bacillus megaterium.

Eight trp mutations (four trpE, two trpB, one trpC, and one trpD) have been mapped in Bacillus megaterium QM B1551 and were found to be linked to two hisH mutations and unlinked to several other his mutations.