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.

Questionable thymic nurse cell.

Since their discovery in 1980, thymic nurse cells (TNCs) have been controversial. Questions pertaining to the existence of the TNC as a "unit" cell with thymocytes completely enclosed within its cytoplasm were the focus of initial debates. Early skeptics proposed the multicellular complex to be an artifact of the procedures used to isolate TNCs from the thymus. Since that time, TNCs have been found in fish, frogs, tadpoles, chickens, sheep, pigs, rats, mice, and humans. Their evolutionary conservation throughout the animal kingdom relieved most speculations about the existence of TNCs and at the same time demonstrated their apparent importance to the thymus and T-cell development. In this review we will discuss and debate reports that describe (i) the organization or structure of TNCs, (ii) the thymocyte subset(s) found within the cytoplasm of TNCs and their uptake and release, and (iii) the function of this fascinating multicellular interaction that occurs during the process of T-cell development. Discussions about the future of the field and experimental approaches that will lead to answers to remaining questions are also presented.

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.

A single point mutation in HIV-1 V3 loop alters the immunogenic properties of rgp120.

The results of the study presented in this report show that clones of env derived from genetically divergent HIV-1 field isolates fall into two major subsets based on the predicted secondary structure of the V3 region in gp120. One subset exemplified by the clones A-UG06c, B-RT3.12 and C-UG045 is predicted to assume a beta-turn conformation in the V3 loop and comprises the GPGX residues. The other subset exemplified by the clones D-UG23c and D-UG042 (GXGX) are deficient in the expression of the beta-turn in the loop. Since secondary conformations are highly likely to confer antigenic properties in a protein backbone at least for B cells, we have used nucleic acid immunization to test the effect of the beta-turn deficiency on the immunogenic potential of rgp120 encoded in these field isolates. As hypothesized, inoculation of BALB/c mice with the env plasmid encoding the beta-turn expressing rgp120 molecules resulted in the development of a vigorous antibody response to the homologous V3 loop peptides. In contrast, immunization with an rgp120 clone deficient in the beta-turn in the V3 loop showed no evidence of antibody development to the V3 loop. Instead, the latter clones triggered T cell proliferative responses and markedly increased the level of IL-2 and IFN-gamma production by T cells. Significantly, reconstitution of the beta-turn conformation by site-directed mutagenesis of a single V3 loop residue yielded rgp120 molecules which restored antibody production while diminishing the cell-mediated immune (CMI) responses to the V3 residue. These observations demonstrate the marked impact of a single amino acid substitution on the immunogenic properties of V3 region in gp120 encoded by divergent HIV-1 field isolates.

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.

The husband's untold account of his wife's breast cancer: a chronologic analysis.

PURPOSE/OBJECTIVES: To identify husbands' perceived needs related to their wives' breast cancer and their view of how they can be helped to deal with the resultant challenges. DESIGN: Descriptive, qualitative. SETTING: Biobehavioral observational laboratory of a large urban research institute. SAMPLE: Nine husbands of women diagnosed with early-stage breast cancer, with a mean length of time since diagnosis of 2.4 years. METHODS: Two focus groups with five participants in the first and four in the second. Focus group questions were designed to facilitate the husbands' reports of their experiences. MAIN RESEARCH VARIABLES: The husbands' view of their lived experience with their wives' breast cancer and challenges across the illness trajectory; the content and process of how best to help them to deal with the challenges. FINDINGS: Husbands expressed their own personal and emotional concerns as well as a desire to support their wives. They asked for help to deal with their own personal concerns and to learn strategies to assist their wives with their experiences. CONCLUSIONS: Husbands' concerns related to their wives' breast cancer changed across the illness trajectory. Husbands' misunderstandings about their own personal emotions hindered their ability to provide support to their wives. Expectations stemming from the male gender role guided husbands' attempts to provide emotional support to their wives that was complicated by an awareness of their inability to meet their wives' needs. IMPLICATIONS FOR NURSING PRACTICE: An urgent need exists for nurses to adjust individual practices and develop programs that address husbands' concerns in the time frame in which they are experienced. Husbands must be explicitly included as participants in their wives' journey and allowed opportunities to express and deal with personal concerns apart from their wives.

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.

Immunogenic potential of rgp120 from African HIV-1 subtype A.

Previous studies have shown that the African strains of HIV-1 mostly cluster with the subtypes A, C or D based on phylogenetic analysis of the ENV nucleotide sequences. In the present investigation we have examined the immunogenic potential of full length gp120 derived from the Ugandan HIV-1 subtype A isolate, AUG06c, using computer-based prediction methods and a plasmid-mediated immunization technique. Computer-assisted analysis of the amino acid residues identified 15 potential B-cell epitopes in gp120 of AUG06c. Despite marked variation in the primary sequences, these epitopes were shown to correspond well to analogous sites in gp120 derived from the subtype B reference clones, MN and IIIBBH10. The relative positions of the epitopes indicated that E9[V3], E14[C3] and E15[V5] correspond to the previously defined principal neutralizing determinant (PND) located in the V3 loop, the CD4 binding site and gp120 "immunodominant" region, respectively. Intramuscular inoculation of BALB/c mice with the ENV clones from AUG06c or from the subtype C clone, CUG045 elicited antibodies which react with the homologous but not with the heterologous PND peptide in ELISA. However, cocktail inoculation with the ENV plasmids from AUG06c and CUG045 elicited antibodies which reacted with both peptides. Antibody response to the other predicted epitopes of AUG06c was not as strong as the response to the PND peptide. The response of the mice to DNA-mediated immunization was further tested in a proliferation assay. Spleen cells derived from the immunized mice exhibited a strong proliferative response to homologous and heterologous PND peptides in [3H]thymidine incorporation assay. DNA-mediated immunization with rgp120 of AUG06c appears to elicit cellular immune response of relatively broad specificity.