Thymic Nurse Cells Participate in Heterotypic Internalization and Repertoire Selection of Immature Thymocytes; Their Removal from the Thymus of Autoimmune Animals May be Important to Disease Etiology.

Thymic nurse cells (TNCs) are specialized epithelial cells that reside in the thymic cortex. The initial report of their discovery in 1980 showed TNCs to contain up to 200 thymocytes within specialized vacuoles in their cytoplasm. Much has been reported since that time to determine the function of this heterotypic internalization event that exists between TNCs and developing thymocytes. In this review, we discuss the literature reported that describes the internalization event and the role TNCs play during T cell development in the thymus as well as why these multicellular complexes may be important in inhibiting the development of autoimmune diseases.

Circulating macrophages as well as developing thymocytes are enclosed within thymic nurse cells.

Both thymic nurse cells (TNCs) and macrophages have been reported to function as antigen-presenting cells during the process of MHC restriction. Negative selection, which results in the apoptosis of potentially autoreactive thymocytes, is believed to be associated with both macrophages and TNCs in the cortex. Both cell types have also been reported to ingest thymocytes undergoing positive and negative selection. However, macrophages ingest apoptotic thymocytes, while TNCs have been shown to internalize viable cells. A subset of the TNC-engulfed population is allowed to mature and is released, while the remaining fraction becomes apoptotic and is absorbed within the TNC cytoplasm through lysosomal activity. A recent report described a subset of rat TNCs that contain macrophages as well as thymocytes within their cytoplasm. We examined freshly isolated TNCs from C57BL/6 mice and found that, of the TNC population recovered, 1.7% contained macrophages within its cytoplasm. There also were macrophages tightly bound but not internalized into the multicellular structure at a rate of 2.9%. The total association of macrophages with TNCs was approximately 4.6%. This unique association of macrophages with TNCs was also observed in vitro when freshly isolated thymocytes (containing macrophages) were added to cultures of cells from the TNC cell line tsTNC-1. The macrophage-TNC interaction was found to be dynamic, with macrophages moving rapidly into and out of TNCs containing cytoplasmic thymocytes. Macrophages within TNCs showed a close association with cytoplasmic thymocytes. We then labeled peritoneal macrophages with CFDA SE, a cell tracking dye, and returned them to the mouse peritoneum. Within 1 h, labeled macrophages were detectable in the thymus. This is the first investigation to show a direct interaction between peripheral macrophages and TNCs. These results suggest that TNCs and macrophages work together as antigen-presenting cells.

TNF and Fas-induced apoptosis during negative selection in thymic nurse cells.

Apoptosis of thymocytes associated with thymic nurse cells (TNCs) has been well-documented. TNCs selectively bind and internalize immature alphabeta TCRlo CD4+ CD8+ thymocytes in vitro. A subset of the internalized population matures to the alphabeta TCRhi CD69hi stage of development while the fraction that remains within the cytoplasm dies through the process of apoptosis. Negative selection by thymic cortical epithelial cells has been reported, but little is known about the apoptotic pathway(s) employed to facilitate the death signal. Using the TNC line tsTNC-1 that was reported earlier to maintain the ability to internalize alphabeta TCRlo CD4+ CD8+ cells in vitro, we investigated the role of Fas and TNFalpha in TNC-induced apoptosis. Our initial studies revealed that tsTNC-1 cells express both FasL and TNFalpha apoptosis of triple positive cells was shown to be reduced approximately 50% in co-cultures of tsTNC-1 cells and thymocytes in the presence of either anti-TNFalpha or Fas-Fc. When maximum effective concentrations of both TNFalpha, and Fas-Fc were added to these co-cultures, apoptotic death was further reduced to approximately 68%. These results suggest that both TNFalpha and Fas apoptotic pathways are active during thymocyte selection by TNCs.

Lysosomal-mediated degradation of apoptotic thymocytes within thymic nurse cells.

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature alphabetaTCR(lo)CD4(+)CD8(+) thymocytes in vitro was used in long-term coincubation experiments to determine the ultimate fate of thymocytes that remained within intracytoplasmic vacuoles of thymic nurse cells (TNCs). In an earlier report, a subset of the population released from the TNC interaction was shown to mature to the alphabetaTCR(hi)CD69(hi) stage of development, while thymocytes that bided within the TNC cytoplasm died through the process of apoptosis. Here, we show the presence of both apoptotic and nonapoptotic thymocytes within the cytoplasm of freshly isolated TNCs as well as in tsTNC-1 cells in culture. A microscopic analysis revealed total degradation of the cytoplasmic apoptotic thymocyte population that remained in tsTNC-1 cells after an 8- to 10-h incubation period. A quantitative analysis showed an increase of cytoplasmic thymocyte degradation over time to almost 80% after 9 h of incubation. However, in the presence of bafilomycin A1, which is used to inhibit acidification of lysosomal vesicles, degradation of apoptotic thymocytes never reached 10%. These data suggest that lysosomes within TNCs play a role in the degradation of apoptotic thymocytes. We examined tsTNC-1 cells before the addition of thymocytes to cultures and found lysosomes to be clustered around the nucleus in the cytoplasm of TNCs. Shortly after the internalization event, apoptotic thymocytes move to the area of the cytoplasm containing lysosomes. Using the confocal microscope, we obtained evidence that shows the degradation event to be facilitated through the fusion of lysosomes with the specialized vacuoles within TNCs containing apoptotic cells.

A thymic nurse cell-specific monoclonal antibody.

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature alpha beta TCRloCD4+CD8+ thymocytes in vitro was used in the development of a monoclonal antibody that is specific to the cell surface of thymic nurse cells (TNCs) in the thymus. The rat monoclonal antibody ph91 showed specificity to cells of the subcapsular region of the thymic cortex. Upon mechanical dispersion of the thymus in vitro, ph91 recognized cells displaying the multicellular morphology unique to TNCs. Ph91 staining was not detected on fresh thymocytes, stromal cells of the inner thymic cortex, thymic medullary cells, B cells or fibroblasts. Ph91 recognized a 43-kDa protein on the surface of TNCs. Exposure of tsTNC-1 cells to ph91 in tissue culture significantly reduced the percentage of binding of the alpha beta TCRloCD4+CD8+ thymocyte subset previously shown to target TNCs. In organ culture, ph91 reduced the viability of developing thymocytes by 70%. The largest reduction was found in the alpha beta TCR+CD4+CD8+ thymocyte subset. These results represent the first report of a TNC-specific monoclonal antibody. Further, the antigen to which ph91 binds may play a role in the process of thymocyte binding and their subsequent internalization which is unique to TNCs and important to the T cell developmental process.

Positive selection by thymic nurse cells requires IL-1 beta and is associated with an increased Bcl-2 expression.

A temperature-sensitive line of thymic nurse cells (tsTNC-1) that maintains the ability to selectively internalize immature alpha beta TCRloCD4+CD8+ thymocytes in vitro was used in long-term coincubation experiments to determine nurse cell function during the process of MHC restriction. The thymocyte subset released from its association with TNCs contained both viable and apoptotic cells. The cells that remained within intracytoplasmic vacuoles died through the process of programmed cell death. Surviving or rescued thymocytes in the released population displayed an increase in Bcl-2 protein expression. The rescue activity of TNCs was drastically reduced with the addition of antibodies against either class I or class II MHC antigens to cocultures. A subset of the TNC-rescued population matured from the alpha beta TCRloCD69- phenotype to alpha beta TCRhiCD(69+)-expressing cells only when IL-1 beta was added to cocultures. These results suggest that TNC rescue of early double-positive thymocytes from apoptosis is associated with an interaction between the TCR and the MHC and the onset of Bcl-2 expression. Maturation of thymocytes within the TNC-rescued population requires the costimulatory effects of IL-1 beta.

Thymic nurse cell rescue of early CD4+CD8+ thymocytes from apoptosis.

We have now developed temperature sensitive lines of thymic nurse cells (TNCs), using the SV40 viral mutant tsA58, that maintain the ability to selectively internalize a subpopulation of alpha beta TCR+CD4+CD8+ thymocytes in vitro. One line, tsTNC-1, was shown to be able to rescue a subset of CD4+CD8+ thymocytes from programmed cell death at 32 degrees C, the temperature at which binding and internalization were detected. Rescue was significantly diminished at 38 degrees C, the temperature at which thymocyte binding was not observed. The rescued population of thymocytes showed a reduced level of apoptosis as measured by the DNA fragmentation assay. TNC rescue resulted in a shift of CD4+CD8+ thymocytes from immature TCRlow PNArhigh cells to the more mature TCRint PNArlow phenotype but no changes in cell surface levels of HSA nor CD69 were detected. The rescue activity of tsTNC-1 cells at 32 degrees C was significantly reduced with the addition of antibodies to either class I or class II MHC antigens. These results suggest that we have established TNC lines, using the SV40 viral mutant tsA58, that have the ability to rescue a subset of the TNC interactive thymocyte population from programmed cell death. The thymocyte population rescued by TNCs matures to a phenotype within the double positive stage of development that is indicative of positive selection.