{"doi":"10.4049/jimmunol.166.6.3846","title":"Human Vascular Endothelial Cells Stimulate a Lower Frequency of Alloreactive CD8+ Pre-CTL and Induce Less Clonal Expansion than Matching B Lymphoblastoid Cells: Development of a Novel Limiting Dilution Analysis Method Based on CFSE Labeling of Lymphocytes","abstract":"<jats:title>Abstract</jats:title>\n                  <jats:p>We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8+ T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8+ T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8+ T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8+ T cells by either BLC or HUVEC derived from the same donor. CD8+ T cells expanded by allostimulation were identified as CD8+, CFSElow cells and were categorized as CTL by the expression of intracellular perforin and IFN-γ. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p &amp;lt; 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8+ T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8+ T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.</jats:p>","journal":"The Journal of Immunology","year":2001,"id":606583,"datarank":0.519860385419959,"base_score":3.4657359027997265,"endowment":3.4657359027997265,"self_citation_contribution":0.519860385419959,"citation_network_contribution":0.0,"self_endowment_contribution":0.519860385419959,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":31,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1557276,"name":"David R Johnson","orcid":null,"position":1,"is_corresponding":false},{"id":1557277,"name":"Jordan S Pober","orcid":null,"position":2,"is_corresponding":false},{"id":1557273,"name":"Thomas J Dengler","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Human Vascular Endothelial Cells Stimulate a Lower Frequency of Alloreactive CD8+ Pre-CTL and Induce Less Clonal Expansion than Matching B Lymphoblastoid Cells: Development of a Novel Limiting Dilution Analysis Method Based on CFSE Labeling of Lymphocytes","abstract":"<jats:title>Abstract</jats:title>\n                  <jats:p>We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8+ T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8+ T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8+ T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8+ T cells by either BLC or HUVEC derived from the same donor. CD8+ T cells expanded by allostimulation were identified as CD8+, CFSElow cells and were categorized as CTL by the expression of intracellular perforin and IFN-γ. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p &amp;lt; 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8+ T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8+ T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.</jats:p>","is_dataset_classified":null,"base_score":0.0,"endowment":0.0,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"11238628","pmcid":null,"openalex_id":null,"authors":[],"funders":[{"funder_name":"NHLBI NIH HHS","grant_id":"HL51014","title":null}],"total_grants":1,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[],"oa_status":"bronze","license":"https://academic.oup.com/pages/standard-publication-reuse-rights","oa_locations":[{"url":"https://journals.aai.org/jimmunol/article-pdf/166/6/3846/1136419/3846.pdf","host_type":"publisher"},{"url":"https://academic.oup.com/jimmunol/article-pdf/166/6/3846/62536293/3846.pdf","host_type":"publisher"}],"fields_of_study":[],"mesh_terms":["Endothelium, Vascular","B-Lymphocyte Subsets","T-Lymphocyte Subsets","CD8-Positive T-Lymphocytes","Cells, Cultured","Cell Line, Transformed","Clone Cells","Stem Cells","Humans","Succinimides","Fluoresceins","Antigens, CD","Isoantigens","Fluorescent Dyes","Lymphocyte Count","Cytotoxicity Tests, Immunologic","Flow Cytometry","Coculture Techniques","Colony-Forming Units Assay","Lymphocyte Activation","Cell Division","Interphase","Immunologic Memory","T-Lymphocytes, Regulatory"],"keywords":[],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-30T04:50:39.519335Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}