Expression of genes encoding cytotoxic cell-associated serine proteases in thymocytes

A family of homologous serine esterases designated granzyme A-H and the pore-forming protein perforin are present in cytoplasmic granules of mature peripheral cytolytic T lymphocytes and natural killer cells. In vivo, the majority of cytotoxic T cells containing these granule-associated proteins are of the CD4−CD8+ phenotype. It is generally assumed that these cells are derived from immature CD4−CD8− thymocytes. However, the precise intrathymic differentiation steps leading to functionally mature cytotoxic T cells are unclear. Thus we decided to analyze the expression of genes in the thymus which are preferentially expressed in mature cytotoxic cells, i.e. granzyme A, granzyme B, and perforin. In situ hybridization on tissue sections revealed the expression of genes coding for granzyme A and granzyme B in the thymus. No evidence was found, however, for thymocytes expressing the perforin gene. Granzyme A and granzyme B mRNA positive cells in the thymus are almost exclusively CD4−CD8− thymocytes, particularly of the CD3− IL2R− phenotyp

Expression of genes encoding the pre-TCR and CD3 complex during thymus development

The mature TCR is composed of a clonotypic heterodimer (αβor γδ) associated with the invariant CD3 components (γ, δ, ε and ζ). There is now considerable evidence that more immature forms of the TCR-CD3 complex (consisting of either CD3 alone or CD3 associated with a heterodimer of TCR β and pre-Tα) can be expressed at the cell surface on early thymocytes. These pre-TCR complexes are believed to be necessary for the ordered progression of early T cell development. We have analyzed in detail the expression of both the pre-TCR and CD3 complex at various stages of adult thymus development. Our data indicate that all CD3 components are already expressed at the mRNA level by the earliest identifiable (CD410) thymic precursor. In contrast, genes encoding the pre-TCR complex (pre-Tα and fully rearranged TCR β) are first expressed at the CD4410CD25+CD4−CD8− stage. Detectable surface expression of both CD3 and TCR β are delayed relative to expression of the corresponding genes, suggesting the existence of other (as yet unidentified) components of the pre-TCR comple

GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO : III. Velocity Sedimentation Studies of the Differentiation and Fate of Effector Cells in Long-Term Mixed Leukocyte Cultures

Separation of cells by velocity sedimentation at unit gravity was utilized to investigate the physical properties of cytotoxic thymus-derived lymphocytes (CTL) generated in long-term mixed leukocyte cultures (MLC). In kinetic studies, CTL were found almost exclusively in the large cell fractions at the peak of the response on day 4, whereas the majority of CTL in day 14 MLC had the sedimentation properties of small lymphocytes. Reculture until day 14 of cells fractionated on the basis of size on day 4 indicated that the small CTL were derived exclusively from cells which had been large on day 4. Re-exposure of day 14 MLC cells to the original stimulating alloantigens resulted in significant cell proliferation and rapid regeneration of CTL activity. Cell fractionation experiments demonstrated that the cells in the day 14 MLC population which responded to the secondary allogeneic stimulus were small T lymphocytes, and that these cells rapidly developed into large, highly cytotoxic CTL following stimulation. Moreover, by restimulating on day 14 fractions which were selected on the basis of size on day 4, it was found that the responding small lymphocytes were themselves the progeny of cells which were large at the peak of the response. Since CTL and CTL progenitors showed concomitant changes in physical properties with time, the possibility exists that they belong to the same cell lineage, and hence that CTL can differentiate into cells which are no longer cytotoxic, but capable of mounting an anamnestic response

An Adult Thymic Stromal-Cell Suspension Model for in Vitro Positive Selection

Presented here is a cell-suspension model for positive selection using thymocytes from αβ-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 –/–), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4-CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states

A cortisone sensitive CD3low subset of CD4+CD8− thymocytes represents an intermediate stage in intrathymic repertoire selection

Two populations of CD4 single positive (SP) thymocytes were found In transgenic mice bearing class l-restricted Mls-1a reactive (Vβ8.1) TCR genes in the absence of the restriction element. CD3high CD4 sp cells were deleted In the presence of Mls-15 and were cortisone resistant, whereas CD3low CD4 SP cells were not deleted In the presence of Mls-1* and were cortisone sensitive. Intravenous transfer of CD3low CD4 SP cells into nude mice resulted in significant peripheral expansion of these cells with apparent upregulation of CD3. These data Indicate that CD3low CD4 SP thymocytes represent an Intermediate stage In the transition from CDSlow double positive (DP) to CD3high SP thymocytes and raise the possibility that these cells may have undergone positive but not negative selection events (at least to Mls-1a). Furthermore the fact that CD3high DP thymocytes were also deleted by Mls-1a in these mice suggests strongly that sensitivity to Mls-1a deletion is dependent upon stage of thymic maturation (as revealed by TCR density) rather than CD4/CD8 phenotyp

Expression of genes encoding cytotoxic cell-associated serine proteases in thymocytes

A family of homologous serine esterases designated granzyme A-H and the pore-forming protein perforin are present in cytoplasmic granules of mature peripheral cytolytic T lymphocytes and natural killer cells. In vivo, the majority of cytotoxic T cells containing these granule-associated proteins are of the CD4-CD8+ phenotype. It is generally assumed that these cells are derived from immature CD4-CD8- thymocytes. However, the precise intrathymic differentiation steps leading to functionally mature cytotoxic T cells are unclear. Thus we decided to analyze the expression of genes in the thymus which are preferentially expressed in mature cytotoxic cells, i.e. granzyme A, granzyme B, and perforin. In situ hybridization on tissue sections revealed the expression of genes coding for granzyme A and granzyme B in the thymus. No evidence was found, however, for thymocytes expressing the perforin gene. Granzyme A and granzyme B mRNA positive cells in the thymus are almost exclusively CD4-CD8- thymocytes, particularly of the CD3- IL2R- phenotype

Successful ageing in an area of deprivation: Part 1—A qualitative exploration of the role of life experiences in good health in old age

Objectives: To determine the life histories and current circumstances of healthy and unhealthy older people who share an ecology marked by relative deprivation and generally poor health. Study design: In-depth interview study with a qualitative analysis. Methods: Matched pairs of healthy and unhealthy ‘agers’ were interviewed face-to-face. Healthy ageing was assessed in terms of hospital morbidity and self-reported health. Study participants consisted of 22 pairs (44 individuals), aged 72–89 years, matched for sex, age and deprivation category, and currently resident in the West of Scotland. All study participants were survivors of the Paisley/Renfrew (MIDSPAN) survey, a longitudinal study commenced in 1972 with continuous recording of morbidity and mortality since. Detailed life histories were obtained which focused on family, residence, employment, leisure and health. This information was supplemented by more focused data on ‘critical incidents’, financial situation and position in social hierarchies. Results: Data provided rich insights into life histories and current circumstances but no differences were found between healthy and unhealthy agers. Conclusions: It is important to understand what differentiates individuals who have lived in circumstances characterized by relative deprivation and poor health, yet have aged healthily. This study collected rich and detailed qualitative data. Yet, no important differences were detected between healthy and unhealthy agers. This is an important negative result as it suggests that the phenomenon of healthy ageing and the factors that promote healthy ageing over a lifetime are so complex that they will require even more detailed studies to disentangle