Plasticity within the αβ+CD4+ T-cell lineage: when,how and what for?

Following thymic output, αβ⁺CD4⁺ T cells become activated in the periphery when they encounter peptide–major histocompatibility complex. A combination of cytokine and co-stimulatory signals instructs the differentiation of T cells into various lineages and subsequent expansion and contraction during an appropriate and protective immune response. Our understanding of the events leading to T-cell lineage commitment has been dominated by a single fate model describing the commitment of T cells to one of several helper (T_H), follicular helper (T_FH) or regulatory (T_REG) phenotypes. Although a single lineage-committed and dedicated T cell may best execute a single function, the view of a single fate for T cells has recently been challenged. A relatively new paradigm in αβ⁺CD4⁺ T-cell biology indicates that T cells are much more flexible than previously appreciated, with the ability to change between helper phenotypes, between helper and follicular helper, or, most extremely, between helper and regulatory functions. In this review, we comprehensively summarize the recent literature identifying when T_H or T_REG cell plasticity occurs, provide potential mechanisms of plasticity and ask if T-cell plasticity is beneficial or detrimental to immunity.     

In Vivo Dynamical Interactions between CD4 Tregs,CD8 Tregs and CD4+CD25? Cells in Mice

Background

Regulatory T cells (Tregs) were shown to be central in maintaining immunological homeostasis and preventing the development of autoimmune diseases. Several subsets of Tregs have been identified to date; however, the dynamics of the interactions between these subsets, and their implications on their regulatory functions are yet to be elucidated.

Methodology/Principal Findings

We employed a combination of mathematical modeling and frequent in vivo measurements of several T cell subsets. Healthy BALB/c mice received a single injection of either hCDR1 - a tolerogenic peptide previously shown to induce Tregs, a control peptide or vehicle alone, and were monitored for 16 days. During this period, splenocytes from the treated mice were analyzed for the levels of CD4, CD25, CD8, CD28 and Foxp3. The collected data were then fitted to mathematical models, in order to test competing hypotheses regarding the interactions between the followed T cell subsets. In all 3 treatment groups, a significant, lasting, non-random perturbation of the immune system could be observed. Our analysis predicted the emergence of functional CD4 Tregs based on inverse oscillations of the latter and CD4⁺CD25⁻ cells. Furthermore, CD4 Tregs seemed to require a sufficiently high level of CD8 Tregs in order to become functional, while conversion was unlikely to be their major source. Our results indicated in addition that Foxp3 is not a sufficient marker for regulatory activity.

Conclusions/Significance

In this work, we unraveled the dynamics of the interplay between CD4, CD8 Tregs and effector T cells, using, for the first time, a mathematical-mechanistic perspective in the analysis of Treg kinetics. Furthermore, the results obtained from this interdisciplinary approach supported the notion that CD4 Tregs need to interact with CD8 Tregs in order to become functional. Finally, we generated predictions regarding the time-dependent function of Tregs, which can be further tested empirically in future work.     

Bcl6 is required for the development of mouse CD4+ and CD8α+ dendritic cells

Th2-type inflammation spontaneously shown in Bcl6-knockout (KO) mice is mainly caused by bone marrow (BM)-derived nonlymphoid cells. However, the function of dendritic cells (DCs) in Bcl6-KO mice has not been reported. We show in this article that the numbers of CD4(+) conventional DCs (cDCs) and CD8α(+) cDCs, but not of plasmacytoid DCs, were markedly reduced in the spleen of Bcl6-KO mice. Generation of cDCs from DC progenitors in BM cells was perturbed in the spleen of irradiated wild-type (WT) mice transferred with Bcl6-KO BM cells, indicating an intrinsic effect of Bcl6 in cDC precursors. Although cDC precursors were developed in a Bcl6-KO BM culture with Fms-like tyrosine kinase 3 ligand, the cDC precursors were more apoptotic than WT ones. Also p53, one of the molecular targets of Bcl6, was overexpressed in the precursors. The addition of a p53 inhibitor to Bcl6-KO BM culture protected apoptosis, suggesting that Bcl6 is required by cDC precursors for survival by controlling p53 expression. Furthermore, large numbers of T1/ST2(+) Th2 cells were naturally developed in the spleen of Bcl6-KO mice. Th2 skewing was accelerated in the culture of WT CD4 T cells stimulated with Ags and LPS-activated Bcl6-KO BM-derived DCs, which produced more IL-6 and less IL-12 than did WT DCs; the addition of anti-IL-6 Abs to the culture partially abrogated the Th2 skewing. These results suggest that Bcl6 is required in cDC precursors for survival and in activated DCs for modulating the cytokine profile.     

Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells,an Extrathymic αβT-cell Progenitor

The thymus, the primary organ for the generation of αβ T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of αβT cells. Yet, thymic involution begins early in life leading to a drastically reduced output of naïve αβT cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic αβT cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. γδ T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a γδ T cell subset by showing that the scarce entity of CD4⁺ Vδ1⁺γδ T cells can transdifferentiate into αβT cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. Vδ1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4⁺ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4⁺ Vδ1⁺γδ T cells. This knowledge is prerequisite for studies that relate to this αβT cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.     

Age-associated changes in interferon-γ and interleukin-4 secretion by purified human CD4+ and CD8+ T cells

Aging is associated with a decline in immune function. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), two important immune deviation-related cytokines, are mainly produced by type 1 and type 2 T cells, respectively. To investigate the age-associated changes in the secretion of these two cytokines, 20 elderly and 20 young subjects fulfilling the SENIEUR protocol were enrolled. The ratios of CD4+ to CD8+ T cells were not different between the two age groups. The CD4+ and CD8+ T cells were purified by a magnetic cell sorting system, and then activated by concurrent anti-CD3 and anti-CD28 stimulation. The released cytokines were determined by ELISA. Both the CD4+ and the CD8+ T cells of the elderly individuals secreted a significantly larger amount of IFN-gamma after activation. Profound IL-4 production by CD8+ T cells was observed in the older subjects compared with that of the young subjects. These data suggested that age-associated decrease in immunity may be related to an imbalance in the secretion of immune deviation cytokines. The number of IL-4-secreting CD8+ T cells (T cytotoxic 2) rose significantly in the older individuals. Our design also provided a useful way to differentiate the T cell subsets secreting the same cytokine, such as IFN-gamma-producing T helper 1 and T cytotoxic 1 cells.     

The quest for an AIDS vaccine: is the CD8+ T-cell approach feasible?

The rationale for developing anti-HIV vaccines that stimulate cytotoxic T-lymphocyte responses is given. We argue that such vaccines will work, provided that attention is paid to the development of memory T-cell responses that are strong and preferably activated. Furthermore, the vaccine should match the prevailing virus clade as closely as possible. Vaccines will have to stimulate a wide range of responses, but it is not clear how this can be achieved.     

Decreases in percentages of naïve CD4 and CD8 T cells and increases in percentages of memory CD8 T-cell subsets in the peripheral blood lymphocyte populations of A-bomb survivors

Our previous studies have revealed a clear dose-dependent decrease in the percentage of na?ve CD4 T cells that are phenotypically CD45RA+ in PBL among A-bomb survivors. However, whether there is a similar radiation effect on CD8 T cells has remained undetermined because of the unreliability of CD45 isoforms as markers of na?ve and memory subsets among the CD8 T-cell population. In the present study, we used double labeling with CD45RO and CD62L for reliable identification of na?ve and memory cell subsets in both CD4 and CD8 T-cell populations among 533 Hiroshima A-bomb survivors. Statistically significant dose-dependent decreases in the percentages of CD45RO-/CD62L+ na?ve cells were found in the CD8 T-cell population as well as in the CD4 T-cell population. Furthermore, the percentages of CD45RO+/CD62L+ and CD45RO+/CD62L- memory T cells were found to increase significantly with increasing radiation dose in the CD8 T-cell population but not in the CD4 T-cell population. These results suggest that the prior A-bomb exposure has induced long-lasting deficits in both na?ve CD4 and CD8 T- cell populations along with increased proportions of these particular subsets of the memory CD8 T-cell population.     

CD4~+T、CD8~+T、CD4~+/CD8~+与慢性乙肝不同中医体质存在相关性

探讨外周血CD4~+、CD8~+T水平与慢性乙型肝炎不同中医体质的关系。选取2016年2月至2017年3月在我院治疗的慢性乙型感染患者143例,其中平和质35例、气虚质40例、阴虚质21例、湿热质20例,气郁质16例、其他11例,采用流式细胞仪检测外周血T淋巴细胞亚群。不同中医体质患者性别、年龄、病程及HBV-DNA含量比较差异无统计学意义(p0.05);平和质患者AST为342.21(142.42,513.46)U/L,明显低于其他患者(p0.05);气虚质患者AST为512.40(322.81,726.50)U/L,明显高于其他患者(p0.05);气郁质及其他体质患者ALT分别为381.64(210.41,501.72)U/L和370.43(200.41,470.63)U/L,明显高于平和质、气虚质、阴虚质和湿热质患者(p0.05);平和质和气虚质患者外周血CD4~+T细胞分别为(39.10±2.01)%和(39.10±2.01)%,明显低于阴虚质、湿热质、气郁质及其他体质患者(p0.05);气郁质及其他体质患者CD8~+T细胞分别为(25.43±1.33)%和(25.24±1.31)%,明显低于平和质、气虚质、阴虚质和湿热质患者(p0.05);阴虚质、气郁质及其他体质患者分别为(1.71±0.09)、(1.75±0.08)和(1.78±0.09),明显高于平和质、气虚质和湿热质患者(p0.05)。慢性乙肝不同中医体质与CD4~+T、CD8~+T和以及肝功能有密切关系。关键词     

Distinct and overlapping effector functions of expanded human CD4+, CD8α+ and CD4-CD8α- invariant natural killer T cells

CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.     

Differences in the Large Extracellular Loop between the K+-Cl? Cotransporters KCC2 and KCC4

K⁺Cl⁻ cotransporters (KCCs) play fundamental physiological roles in processes such as inhibitory neurotransmission and cell volume regulation. Mammalian genomes encode four distinct KCC paralogs, which share basic transport characteristics but differ significantly in ion affinity, pharmacology, and relative sensitivity to cell volume. Studies to identify divergence in functional characteristics have thus far focused on the cytoplasmic termini. Here, we investigated sequence requirements of the large extracellular loop (LEL) for function in KCC2 and KCC4. Mutation of all four evolutionarily conserved cysteines abolished KCC2 transport activity. This behavior differs from that of its closest relative, KCC4, which is insensitive to this mutation. Chimeras supported the differences in the LEL of the two cotransporters, because swapping wild-type LEL resulted in functional KCC2 but rendered KCC4 inactive. Insertion of the quadruple cysteine substitution mutant of the KCC4 loop, which was functional in the parental isoform, abolished transport activity in KCC2. Dose-response curves of wild-type and chimeric KCCs revealed that the LEL contributes to the different sensitivity to loop diuretics; a KCC2 chimera containing the KCC4 LEL displayed an IC₅₀ of 396.5 μm for furosemide, which was closer to KCC4 (548.8 μm) than to KCC2 (184.4 μm). Cell surface labeling and immunocytochemistry indicated that mutations do not affect trafficking to the plasma membrane. Taken together, our results show a dramatic and unexpected difference in the sequence requirements of the LEL between the closely related KCC2 and KCC4. Furthermore, they demonstrate that evolutionarily highly conserved amino acids can have different functions within KCC members.     

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.     

CD8+ T-cell priming and boosting: more antigen-presenting DC, or more antigen per DC?

RNA transfection is a standard method to load dendritic cells (DC) with antigen for therapeutic cancer vaccination. While electroporation yields high transfection efficiency and satisfying expression levels, lipofection results in only few cells expressing high amounts of antigen. We compared antigen loading of human monocyte-derived DC by MelanA RNA electroporation and lipofection. No differences in phenotype or migrational capacity were detected, but lipofected DC induced stronger cytokine secretion by antigen-specific T cells and were superior in priming and boosting of MelanA-specific CD8⁺ T cells. Interestingly, T cells stimulated with the differently transfected DC did not differ in their functional avidity. To determine whether the amount of antigen per cell is indeed responsible for the superiority of the lipofected DC, we increased the amount of MelanA RNA fivefold and mixed those DC with mock-electroporated ones to mimic the antigen distribution of lipofected cells. This significantly improved the stimulatory capacity, indicating that indeed the amount of antigen per cell seems to be the responsible feature for the observed superiority of lipofected DCs. These data suggest that a few DC that express high amounts of antigen are more immunogenic than many DC expressing lower amounts, although this needs to be tested in a two-armed immunogenicity trial.     

Uneven distributions of naïve and memory T cells in the CD4 and CD8 T-cell populations derived from a single stem cell in an atomic bomb survivor: implications for the origins of the memory T-cell pools in adulthood

The processes that lead to the establishment and maintenance of memory T-cell pools in humans are not well understood. In this study, we examined the emergence of na?ve and memory T cells in an adult male who was exposed to an atomic bomb radiation dose of approximately 2 Gy in 1945 at the age of 17. The analysis presented here was made possible by our earlier observation that this particular individual carries a hematopoietic stem cell mutation at the hypoxanthine phosphoribosyltransferase (HPRT) locus that is almost certainly a result of his exposure to A-bomb radiation. Our key finding is that we detected a very much higher HPRT mutant frequency in the naive (CD45RA(+)) cell component of this individual's CD4 and CD8 T-cell populations than in the memory (CD45RA(-)) cell component of his CD4 and CD8 T-cell populations. This stands in marked contrast to our finding that HPRT mutant frequencies are fairly similar in the na?ve CD45RA(+) and memory CD45RA(-) components of the CD4 and CD8 T-cell populations of three unexposed individuals examined concurrently. In addition we found that the HPRT mutant frequencies were about 30-fold higher in the na?ve (CD45RA(+)) CD4 T cells of the exposed individual than in his memory (CD45RA(-)) cell populations, but that the effect was a little less striking in his CD8 cell populations, where the HPRT mutant frequencies were only about 15-fold higher in his na?ve T-cell pools than in his memory T-cell pools. We further found that 100% of the HPRT mutant cells in both his CD4 and CD8 na?ve cell subsets appeared to have originated from repeated divisions of the initial HPRT mutant stem cell, whereas only 4 of 24 and 5 of 6 mutant cells in his CD4 and CD8 memory cell subsets appeared to have originated from that same stem cell. The most straightforward conclusion may be that the great majority of the T cells produced by this individual since he was 17 years old have remained as na?ve-type T cells, rather than having become memory-type T cells. Thus the T cells that have been produced from the hematopoietic stem cells of this particular A-bomb-exposed individual seldom seem to enter and/or to remain in the memory T-cell pool for long periods. We speculate that this constraint on entry into memory T-cell pools may also apply to unirradiated individuals, but in the absence of genetic markers to assist us in obtaining evidential support, we must await clarifying information from radically different experimental approaches.