The relationship between high ratios of CD4/FOXP3 and CD8/CD163 and the improved survivability of metastatic triple-negative breast cancer patients: a multicenter cohort study.

BACKGROUND: Triple-negative breast cancer (TNBC) has been documented as the most aggressive subtype of breast cancer. This study aimed to analyze antitumor and protumor immune activities, and their ratios as significant prognostic biomarkers in metastatic TNBC (mTNBC). METHODS: A multicenter cohort study was conducted among 103 de novo mTNBC patients. The expression of CD8 and CD163 was evaluated using immunohistochemistry staining, CD4 and FOXP3 using double-staining immunohistochemistry, and PD-L1 using immunohistochemistry and RT-PCR. RESULTS: Multivariate analysis revealed that high CD4/FOXP3 (HR 1.857; 95% CI 1.049-3.288; p = 0.034) and the CD8/CD163 ratio (HR 2.089; 95% CI 1.174-3.717; p = 0.012) yield significantly improved 1 year overall survival (OS). Kaplan-Meier analysis showed that high levels of CD4 (p = 0.023), CD8 (p = 0.043), CD4/FOXP3 (p = 0.016), CD8/FOXP3 (p = 0.005), CD8/CD163 (p = 0.005) ratios were significantly associated with higher rate of 1 year OS. Furthermore, 1 year OS was directly correlated with antitumor CD4 (R = 0.233; p = 0.018) and CD8 (R = 0.219; p = 0.026) and was indirectly correlated with protumor CD163 and FOXP3 through CD4/FOXP3 (R = 0.282; p = 0.006), CD4/CD163 (R = 0.239; p = 0.015), CD8/FOXP3 (R = 0.260; p = 0.008), and CD8/CD163 (R = 0.258; p = 0.009). CONCLUSION: This is the first study to demonstrate that high levels of CD4/FOXP3 and CD8/CD163 significantly improved the 1 year OS in de novo mTNBC patients. Thus, we recommend the application of these markers as prognosis determination and individual treatment decision.

BCL6 is required for the thymic development of TCRαß+CD8αα+ intraepithelial lymphocyte lineage.

TCRαß+CD8αα+ intraepithelial lymphocytes (CD8αα+ αß IELs) are a specialized subset of T cells in the gut epithelium that develop from thymic agonist selected IEL precursors (IELps). The molecular mechanisms underlying the selection and differentiation of this T cell type in the thymus are largely unknown. Here, we found that Bcl6 deficiency in αß T cells resulted in the near absence of CD8αα+ αß IELs. BCL6 was expressed by approximately 50% of CD8αα+ αß IELs and by the majority of thymic PD1+ IELps after agonist selection. Bcl6 deficiency blocked early IELp generation in the thymus, and its expression in IELps was induced by thymic TCR signaling in an ERK-dependent manner. As a result of Bcl6 deficiency, the precursors of IELps among CD4+CD8+ double-positive thymocytes exhibited increased apoptosis during agonist selection and impaired IELp differentiation and maturation. Together, our results elucidate BCL6 as a crucial transcription factor during the thymic development of CD8αα+ αß IELs.

Unraveling the potential of CD8, CD68, and VISTA as diagnostic and prognostic markers in patients with pancreatic ductal adenocarcinoma.

Introduction: Pancreatic cancer is a truculent disease with limited treatment options and a grim prognosis. Immunotherapy has shown promise in treating various types of cancer, but its effectiveness in pancreatic cancer has been lacking. As a result, it is crucial to identify markers associated with immunological pathways in order to improve the treatment outcomes for this deadly cancer. The purpose of this study was to investigate the diagnostic and prognostic significance of three markers, CD8, CD68, and VISTA, in pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer. Methods: We analyzed gene expression data from Gene Expression Omnibus (GEO) database using bioinformatics tools. We also utilized the STRING online tool and Funrich software to study the protein-protein interactions and transcription factors associated with CD8, CD68, and VISTA. In addition, tissue microarray (TMA) and immunohistochemistry (IHC) staining were performed on 228 samples of PDAC tissue and 10 samples of normal pancreatic tissue to assess the expression levels of the markers. We then correlated these expression levels with the clinicopathological characteristics of the patients and evaluated their survival rates. Results: The analysis of the GEO data revealed slightly elevated levels of VISTA in PDAC samples compared to normal tissues. However, there was a significant increase in CD68 expression and a notable reduction in CD8A expression in pancreatic cancer. Further investigation identified potential protein-protein interactions and transcription factors associated with these markers. The IHC staining of PDAC tissue samples showed an increased expression of VISTA, CD68, and CD8A in pancreatic cancer tissues. Moreover, we found correlations between the expression levels of these markers and certain clinicopathological features of the patients. Additionally, the survival analysis revealed that high expression of CD8 was associated with better disease-specific survival and progression-free survival in PDAC patients. Conclusion: These findings highlight the potential of CD8, CD68, and VISTA as diagnostic and prognostic indicators in PDAC.

The transcription factor Aiolos restrains the activation of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3-/- CD8αα+ IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3-/- and Ikzf3+/+ IELs showed an amplified effector machinery in Ikzf3-/- CD8αα+ IELs compared to Ikzf3+/+ counterparts. Ikzf3-/- CD8αα+ IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs.

CD3 downregulation identifies high-avidity human CD8 T cells.

CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.

Single cell and spatial transcriptomics analysis of kidney double negative T lymphocytes in normal and ischemic mouse kidneys.

T cells are important in the pathogenesis of acute kidney injury (AKI), and TCR+CD4-CD8- (double negative-DN) are T cells that have regulatory properties. However, there is limited information on DN T cells compared to traditional CD4+ and CD8+ cells. To elucidate the molecular signature and spatial dynamics of DN T cells during AKI, we performed single-cell RNA sequencing (scRNA-seq) on sorted murine DN, CD4+, and CD8+ cells combined with spatial transcriptomic profiling of normal and post AKI mouse kidneys. scRNA-seq revealed distinct transcriptional profiles for DN, CD4+, and CD8+ T cells of mouse kidneys with enrichment of Kcnq5, Klrb1c, Fcer1g, and Klre1 expression in DN T cells compared to CD4+ and CD8+ T cells in normal kidney tissue. We validated the expression of these four genes in mouse kidney DN, CD4+ and CD8+ T cells using RT-PCR and Kcnq5, Klrb1, and Fcer1g genes with the NIH human kidney precision medicine project (KPMP). Spatial transcriptomics in normal and ischemic mouse kidney tissue showed a localized cluster of T cells in the outer medulla expressing DN T cell genes including Fcer1g. These results provide a template for future studies in DN T as well as CD4+ and CD8+ cells in normal and diseased kidneys.

A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4+, CD8+, Caspase-3 and NF-κB in mice.

BACKGROUND: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. METHODS: Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. RESULTS: The IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. CONCLUSIONS: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.

Cross-Activation of Regulatory T Cells by Self Antigens Limits Self-Reactive and Activated CD8+ T Cell Responses.

The interaction between regulatory T (Treg) cells and self-reactive T cells is a crucial mechanism for maintaining immune tolerance. In this study, we investigated the cross-activation of Treg cells by self-antigens and its impact on self-reactive CD8+ T cell responses, with a focus on the P53 signaling pathway. We discovered that major histocompatibility complex (MHC) I-restricted self-peptides not only activated CD8+ T cells but also induced the delayed proliferation of Treg cells. Following HLA-A*0201-restricted Melan-A-specific (pMelan) CD8+ T cells, we observed the direct expansion of Treg cells and concurrent suppression of pMelan+CD8+ T cell proliferation upon stimulation with Melan-A peptide. Transcriptome analysis revealed no significant alterations in specific signaling pathways in pMelan+CD8+ T cells that were co-cultured with activated Treg cells. However, there was a noticeable upregulation of genes involved in P53 accumulation, a critical regulator of cell survival and apoptosis. Consistent with such observation, the blockade of P53 induced a continuous proliferation of pMelan+CD8+ T cells. The concurrent stimulation of Treg cells through self-reactive TCRs by self-antigens provides insights into the immune system's ability to control activated self-reactive CD8+ T cells as part of peripheral tolerance, highlighting the intricate interplay between Treg cells and CD8+ T cells and implicating therapeutic interventions in autoimmune diseases and cancer immunotherapy.

Factors contributing to the potency of CD8+ T cells.

CD8+ cytotoxic T lymphocytes (CTLs) play a crucial role in targeting virus-infected and cancer cells. Although other cytotoxic lymphocytes such as CD4+ T and natural killer (NK) cells, as well as chimeric antigen receptor (CAR)-T cells, can also identify and destroy aberrant cells, they seem to be significantly less potent based on available experimental data. Here, I contemplate the molecular mechanisms controlling the sensitivity and kinetics of granule-mediated CD8+ T cell cytolytic responses. I posit that the clustering of MHC-I molecules and T cell receptors (TCRs) on the cell surface, as well as the contribution of the CD8 co-receptor, are major factors driving exceptionally potent cytolytic responses. I also contend that CD8+ T cells with known specificity and engineered TCR-T cells might be among the most efficient cytolytic effectors for treating patients suffering from viral infections or cancer.

Drug hypersensitivity and eosinophilia: The decisive role of p-i stimulation.

Eosinophilia is a common finding in drug hypersensitivity reactions (DHR). Its cause is unclear, as neither antigen/allergen-driven inflammation nor clonal expansion is involved. Most delayed-DHRs are due to p-i (pharmacologic interaction of drugs with immune receptors). These are off-target activities of drugs with immune receptors that result in various types of T-cell stimulation, some of which involve excessive IL-5 production. Functional and phenotypic studies of T-cell clones and their TCR-transfected hybridoma cell lines revealed that some p-i-induced drug stimulations occur without CD4/ CD8 co-receptor engagement. The CD4/CD8 co-receptors link Lck (lymphocyte-specific protein tyrosine kinase) and LAT (linker for activation of T cells) to the TCR. Alteration of Lck or LAT can result in a TCR signalosome with enhanced IL-5 production. Thus, if a more affine TCR-[drug/peptide/HLA] interaction allows bypassing the CD4 co-receptor, a modified Lck/LAT activation may lead to a TCR signalosome with elevated IL-5 production. This "IL-5-TCR-signalosome" hypothesis could also explain eosinophilia in superantigen or allo-stimulation (graft-versus-host disease), in which evasion of CD4/CD8 co-receptors has also been described. It may open new therapeutic possibilities in certain eosinophilic diseases by directly targeting the IL-5-TCR signalosome.

Effects of Cefixime on Immune Functions and Inflammatory Factors in Children with Urinary Tract Infection and Targeted Nursing Strategies

Objective: This study aims to explore the effects of cefixime on immune functions and inflammatory factors in children with urinary tract infection and to investigate its nursing strategies. Methods: A total of 161 children with urinary tract infection who were diagnosed in our hospital from November 2019 to November 2021 were selected. All children were treated with cefixime and received targeted nursing strategies. The indices of immune functions and the levels of inflammatory factors were compared before and after the treatment. The satisfaction degree of children’s family members, recurrence rate and incidence of adverse reactions were measured. Results: The levels of CD3+, CD4+ and CD4+/CD8+ in children after the treatment were significantly higher but the CD8+ level was significantly lower than those before the treatment (p < 0.001). The levels of C-reactive protein, tumour necrosis factor-α and interleukin-6 after the treatment were lower than those before the treatment (p < 0.001). The average score of nursing satisfaction of children’s family members was (84.53 ± 13.65) points, with the total satisfaction degree of 90.68% (146/161). Within 6 months after the treatment, only six children had urinary tract infection again and the recurrence rate was 3.73% (6/161). During the treatment, seven children had adverse reactions to the drug, with an incidence rate of 4.35% (7/161). Conclusions: Cefixime can improve the immune function of children with urinary tract infection and reduce the levels of inflammatory factors. The implementation of targeted nursing strategies can improve clinical satisfaction and reduce the recurrence rate of the disease and thus can be helpful to establish a comprehensive and efficient clinical program for children with urinary tract infection (AU)

High-affinity CD8 variants enhance the sensitivity of pMHCI antigen recognition via low-affinity TCRs.

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.

The first crystal structure of CD8αα from a cartilaginous fish.

Introduction: Cartilaginous fishes are the most evolutionary-distant vertebrates from mammals and possess an immunoglobulin (Ig)- and T cell-mediated adaptive immunity. CD8 is the hallmark receptor of cytotoxic T cells and is required for the formation of T cell receptor-major histocompatibility complex (TCR-MHC) class I complexes. Methods: RACE PCR was used to obtain gene sequences. Direct dilution was applied for the refolding of denatured recombinant CD8 protein. Hanging-drop vapor diffusion method was performed for protein crystallization. Results: In this study, CD8α and CD8ß orthologues (termed ScCD8α and ScCD8ß) were identified in small-spotted catshark (Scyliorhinus canicula). Both ScCD8α and ScCD8ß possess an extracellular immunoglobulin superfamily (IgSF) V domain as in previously identified CD8 proteins. The genes encoding CD8α and CD8ß are tandemly linked in the genomes of all jawed vertebrates studied, suggesting that they were duplicated from a common ancestral gene before the divergence of cartilaginous fishes and other vertebrates. We determined the crystal structure of the ScCD8α ectodomain homodimer at a resolution of 1.35 Å and show that it exhibits the typical topological structure of CD8α from endotherms. As in mammals, the homodimer formation of ScCD8αα relies upon interactions within a hydrophobic core although this differs in position and amino acid composition. Importantly, ScCD8αα shares the canonical cavity required for interaction with peptide-loaded MHC I in mammals. Furthermore, it was found that ScCD8α can co-immunoprecipitate with ScCD8ß, indicating that it can form both homodimeric and heterodimeric complexes. Conclusion: Our results expand the current knowledge of vertebrate CD8 dimerization and the interaction between CD8α with p/MHC I from an evolutionary perspective.

Destined for the intestine: thymic selection of TCRαß CD8αα intestinal intraepithelial lymphocytes.

The immune system is composed of a variety of different T-cell lineages distributed through both secondary lymphoid tissue and non-lymphoid tissue. The intestinal epithelium is a critical barrier surface that contains numerous intraepithelial lymphocytes that aid in maintaining homeostasis at that barrier. This review focuses on T-cell receptor αß (TCRαß) CD8αα intraepithelial lymphocytes, and how recent advances in the field clarify how this unique T-cell subset is selected, matures, and functions in the intestines. We consider how the available evidence reveals a story of ontogeny starting from agonist selection of T cells in the thymus and finishing through the specific signaling environment of the intestinal epithelium. We conclude with how this story raises further key questions about the development of different ontogenic waves of TCRαß CD8αα IEL and their importance for intestinal epithelial homeostasis.

Aberrant expression of T cell receptors in monocyte/macrophage RAW 264.7 cells: FCγRII/III compensates the need for CD3.

Conventionally T-cell receptors (TCRs) have so far been considered as a T-lymphocyte privilege. However, recent findings also place TCR expression in non-lymphoid cells, namely neutrophils, eosinophils and macrophages. In order to examine the ectopic expression of TCR, this study focused on RAW 264.7 cells, which have been broadly used for their macrophage properties. Immunofluorescence staining detected 70% and 40% of the cells to express TCRαß and TCRγδ respectively, which was also verified by RT-PCR experiments and confocal microscopy analysis. Interestingly, except from the predicted 292 and 288 bp gene products for the α- and γ-chain, additional products at 220 and 550 bp could be detected, respectively. RAW 264.7 cells also expressed the co-stimulatory CD4 and CD8 markers at a percentage of 61% and 14% respectively, which supported the expression of TCRs. However, only low numbers of cells expressed CD3ε and CD3ζ (9% and 7% respectively). Such observations contradicted the existing knowledge, and indicated that TCRs would be supported by other molecules for reaching the membrane and transducing their signal. Such candidate molecules could be the Fcγ receptors (FcγRs). Indeed, the FcγRII/III receptor was found to be expressed in 75% of the cells, which also expressed at a percentage of 25% major histocompatibility complex (MHC) class II molecules. Engagement of the FcγRII/III receptor by a recombinant IgG2aCH2 fragment, except from stimulating the macrophage-dependent properties of the cells, was shown to reduce expression of TCRαß and γδ indicating that FcγRII/III was indeed used by TCRs for their transport to the cell membrane. In order to examine the ability of RAW 264.7 cells to simultaneously display antigen presenting- and T-cell properties, functional experiments as to antigen-specific antibody and IL-2 production were performed. In in vitro immunization assays in the presence of naïve B cells, RAW264.7 failed to promote antibody production. However, RAW 264.7 cells could compete with antigen-stimulated macrophages but not T cells when applied to a system of in vivo antigen-sensitized cells followed by an in vitro immunization protocol. Interestingly, simultaneous addition of antigen and the IgG2aCH2 fragment to RAW 264.7 cells could promote IL-2 production from the cells, indicating that FcγRII/III activation could also support TCR stimulation. Extrapolating these findings to cells of the myeloid origin, the above results dictate novel regulatory mechanisms towards the alteration of the immune response.

The emerging roles of leukocyte cell-derived chemotaxin-2 in immune diseases: From mechanisms to therapeutic potential.

Leukocyte cell-derived chemotaxin-2 (LECT2, also named ChM-II), initially identified as a chemokine mediating neutrophil migration, is a multifunctional secreted factor involved in diverse physiological and pathological processes. The high sequence similarity of LECT2 among different vertebrates makes it possible to explore its functions by using comparative biology. LECT2 is associated with many immune processes and immune-related diseases via its binding to cell surface receptors such as CD209a, Tie1, and Met in various cell types. In addition, the misfolding LECT2 leads to the amyloidosis of several crucial tissues (kidney, liver, and lung, etc.) by inducing the formation of insoluble fibrils. However, the mechanisms of LECT2-mediated diverse immune pathogenic conditions in various tissues remain to be fully elucidated due to the functional and signaling heterogeneity. Here, we provide a comprehensive summary of the structure, the "double-edged sword" function, and the extensive signaling pathways of LECT2 in immune diseases, as well as the potential applications of LECT2 in therapeutic interventions in preclinical or clinical trials. This review provides an integrated perspective on the current understanding of how LECT2 is associated with immune diseases, with the aim of facilitating the development of drugs or probes against LECT2 for the theranostics of immune-related diseases.

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans.

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. Here, we identify, confirm, and replicate the biomarker performance characteristics of candidate proteins in patients with DILI at onset (DO; n = 133) and follow-up (n = 120), acute non-DILI at onset (NDO; n = 63) and follow-up (n = 42), and healthy volunteers (HV; n = 104). Area under the receiver operating characteristic curve (AUC) for cytoplasmic aconitate hydratase, argininosuccinate synthase, carbamoylphosphate synthase, fumarylacetoacetase, fructose-1,6-bisphosphatase 1 (FBP1) across cohorts achieved near complete separation (range: 0.94-0.99) of DO and HV. In addition, we show that FBP1, alone or in combination with glutathione S-transferase A1 and leukocyte cell-derived chemotaxin 2, could potentially assist in clinical diagnosis by distinguishing NDO from DO (AUC range: 0.65-0.78), but further technical and clinical validation of these candidate biomarkers is needed.

Shaping immunity for life: Layered development of CD8+ T cells.

Historically, the immune system was believed to develop along a linear axis of maturity from fetal life to adulthood. Now, it is clear that distinct layers of immune cells are generated from unique waves of hematopoietic progenitors during different windows of development. This model, known as the layered immune model, has provided a useful framework for understanding why distinct lineages of B cells and γδ T cells arise in succession and display unique functions in adulthood. However, the layered immune model has not been applied to CD8+ T cells, which are still often viewed as a uniform population of cells belonging to the same lineage, with functional differences between cells arising from environmental factors encountered during infection. Recent studies have challenged this idea, demonstrating that not all CD8+ T cells are created equally and that the functions of individual CD8+ T cells in adults are linked to when they were created in the host. In this review, we discuss the accumulating evidence suggesting there are distinct ontogenetic subpopulations of CD8+ T cells and propose that the layered immune model be extended to the CD8+ T cell compartment.

Ca2+ Homeostasis by Plasma Membrane Ca2+ ATPase (PMCA) 1 Is Essential for the Development of DP Thymocytes.

The strength of Ca2+ signaling is a hallmark of T cell activation, yet the role of Ca2+ homeostasis in developing T cells before expressing a mature T cell receptor is poorly understood. We aimed to unveil specific functions of the two plasma membrane Ca2+ ATPases expressed in T cells, PMCA1 and PMCA4. On a transcriptional and protein level we found that PMCA4 was expressed at low levels in CD4-CD8- double negative (DN) thymocytes and was even downregulated in subsequent stages while PMCA1 was present throughout development and upregulated in CD4+CD8+ double positive (DP) thymocytes. Mice with a targeted deletion of Pmca1 in DN3 thymocytes had an almost complete block of DP thymocyte development with an accumulation of DN4 thymocytes but severely reduced numbers of CD8+ immature single positive (ISP) thymocytes. The DN4 thymocytes of these mice showed strongly elevated basal cytosolic Ca2+ levels and a pre-mature CD5 expression, but in contrast to the DP thymocytes they were only mildly prone to apoptosis. Surprisingly, mice with a germline deletion of Pmca4 did not show any signs of altered progression through the developmental thymocyte stages, nor altered Ca2+ homeostasis throughout this process. PMCA1 is, therefore, non-redundant in keeping cellular Ca2+ levels low in the early thymocyte development required for the DN to DP transition.