We therefore hypothesize that senescent\like CD8+ T? cells may not be able to reach T?cell survival niches in the BM, most likely because of their impaired manifestation of chemokine receptors. Interestingly, DP CD8+ T?cells showed features of activated cells, with increased manifestation of co\stimulatory molecules CD160 and 4\1BB. were found in rheumatoid arthritis patients. More DP and DN CD8+ T?cells were present in the bone marrow, in comparison with PBMCs. In summary, our results show that DP and DN cells are unique CD8+ T?cell subsets displaying defined properties. = 91, ***= 91) of na?ve, CM, EM, and TEMRA (defined using the markers CCR7 and CD45RA) within CD8+, CD28+CD57?CD8+, DP CD8+, DN CD8+, and CD28?CD57+CD8+ T?cells. We next investigated the distribution of CCR7+CD45RA+ na?ve, CCR7+CD45RA? CM, CCR7?CD45RA? EM, and CCR7?CD45RA+ TEMRA subpopulations within DP CD8+ T?cells, in comparison to the DN, CD28+CD57?, and CD28?CD57+ CD8+ T?cell subsets and the whole CD8+ T?cell human population (Fig.?1E; Assisting Info Fig. 1). CD28+CD57? and DP cells, similarly to CD8+ T?cells, were distributed in all, na?ve, CM, EM, and TEMRA subpopulations, with increased frequencies within the na?ve, CM, and EM subsets (Fig.?1E; Assisting Info Fig. 1ACC). DN and CD28?CD57+ CD8+ T?cells showed a similar distribution and were mainly included within the EM and TEMRA subsets, but not in the na?ve and CM subpopulations (Fig.?1E; Assisting Info Fig. 1D and E). As expected, the highest rate of recurrence of cells within TEMRA and the lowest levels of CM and na?ve were found out within CD28?CD57+ CD8+ T?cells. To further characterize the phenotype of the four cell subsets, gene manifestation profiles of FACS sorted CD28+CD57?, DP, DN, and CD28?CD57+CD8+ T?cells were studied using Affymetrix arrays. Gene Collection Enrichment Analysis (GSEA) was performed in order to determine pathways that are differentially controlled between the four subpopulations. A summary of the pathways significantly enriched after GSEA in the comparisons between the populations are reported in Assisting Information Number?2. Manifestation of genes coding for proteins involved in IFN\ reactions and G2/M checkpoint control improved both from CD28+CD57? to DP and from CD28+CD57? to DN cells. Mitotic spindle genes CRAC intermediate 2 were overexpressed in DN but not in DP cells when compared to CD28+CD57? cells. Furthermore, manifestation of MYC focuses on was reduced both from CD28+CD57? to DP and from DN to CD28?CD57+ cells, while DNA repair genes were downregulated from CD28+CD57? to DP cells and IL\2 STAT\5 signaling genes from DP to CD28?CD57+ cells, respectively. These results suggest that CD28+CD57?, DP, DN, and CD28?CD57+ CD8+ T?cells display distinct phenotypes. DP and DN CD8+ T?cells display differential manifestation of cytotoxic and effector molecules CD8+ T?cells are known to overexpress pro\inflammatory molecules when they become terminally differentiated and/or senescent 11, 12. Additionally, reduced levels of CD28 and improved manifestation of CD57 have been indicated as markers for highly differentiated T?cells. In order to assess the CRAC intermediate 2 stage of T?cell differentiation in DP CRAC intermediate 2 and DN cells in comparison to CD28+CD57? and CD28?CD57+ cells, we investigated the expression of cytotoxic molecules and markers for highly differentiated T?cells in CD28+CD57?, DP, DN, and CD28?CD57+ CD8+ T?cell subsets, first analyzing the data collected using microarrays. The gene manifestation of selected molecules involved in cytotoxic and terminal differentiation features in the four subsets is definitely reported in Table?1. Overall, genes encoding for pro\inflammatory and cytotoxic molecules, NK markers as well as transcription factors supporting CD8+ T?cell terminal differentiation progressively increased from CD28+CD57? to CD28?CD57+ subsets. Indeed, the manifestation of most of these genes was least expensive in CD28+CD57? cells, intermediate in DP, slightly higher in DN, and the highest was in CD28?CD57+ cells (Table?1). The chemokine CRAC intermediate 2 receptor CX3CR1 offers been shown CRAC intermediate 2 to reflect the degree of CD8+ T?cell differentiation 19. While the manifestation of CX3CR1 was low in the CD28+CD57? subset, the levels of this transcript gradually improved in DP, DN, and CD28?CD57+ Rabbit Polyclonal to FAKD2 cells (Table?1). These results were confirmed in the mRNA level using qPCR (data not demonstrated). The transcription element Hobit (ZNF 683) offers been shown to support the manifestation of granzyme B in highly differentiated T?cells 20. Again, ZNF 683 manifestation was least expensive in CD28+CD57?, intermediate in DP, significantly higher in DN compared to DP, and the highest in the CD28?CD57+ subset (Table?1). Similar results were observed for the manifestation of granzyme B, granzyme H, perforin 1, and the cytotoxic molecule FGFBP2 (Table?1). Manifestation of NK markers increase in highly differentiated T?cells 21. Indeed, most of NK receptors were low in CD28+CD57? cells, intermediate in DP, and higher in DN CD8+ T?cells (Table?1). No variations were found between the DN and CD28?CD57+ subsets. Table 1 Genes coding for proteins involved in cytotoxic and effector functions in CD28+CD57?, DP, DN, and CD28?CD57+CD8+ T?cells = 4 for each subset. Data are combined from one representative experiment. In order to reduce.