Default development of cloned human naive CD4 T cells into interleukin-4- and interleukin-5-producing effector cells

It was recently demonstrated that naive human and mouse CD4 T cells release low but sufficient levels of interleukin (IL)-4 at priming to support their development into IL-4 producers. To determine whether this IL-4 is produced by a minor subset of cells, freshly isolated human naive CD4 T cells were directly cloned by limiting dilution in the absence of exogenous IL-4. More than 95% of neonatal and 60% of adult naive T cells seeded in single-cell cultures could be expanded upon stimulation with anti-CD3 mAb immobilized on CD32-B7.1 L cell transfectants in the presence of IL-2. All 171 clones derived from four neonates and two adults produced IL-4 and IL-5 at generally high levels. Like the allergen-specific human Th2 clones described in the literature, these T cell clones produced little or no interferon γ upon stimulation via their T cell receptor/CD3 complex, whereas they released high levels of this cytokine when activated with phorbol 12-myristate 13-acetate + ionomycin. Cells cloned and expanded in the presence of anti-IL4 + anti-IL-4R mAb produced much lower levels of IL-4 and IL-5. It is concluded that almost every single naive human CD4 T cell primed and expanded in the absence of exogenous IL-4 releases sufficient autocrine IL-4 to support its clonal expansion into high IL-4/IL-5 producers.     

Antigen-independent responsiveness to interleukin-4 demonstrates differential regulation of newborn human T cells

The low incidence of graft-versus-host disease following clinical use of umbilical cord blood compared to adult bone marrow as a source of stem cells for bone marrow reconstitution, leads to questions concerning the level of immunocom-petence of newborn T cells. The maturation and functional status of newborn CD4⁺ T cells, which are almost exclusively CD45RA⁺ naive T cells, compared with their adult phenotypic counterparts, is poorly understood. We examined the proliferative response to mitogens and cytokines of CD4/CD45RA⁺ T cells from adults and newborns, with and without accessory cells. Newborn CD4/CD45RA⁺ T cells demonstrated a distinct proliferative response profile which was determined by the number of accessory cells present in co-cultures with various stimuli. Newborn CD4/CD45RA⁺ T cells were particularly responsive to interleukin (IL)-4, IL-4 plus anti-CD2 monoclonal antibodies (mAb) and IL-4 plus phytohemagglutinin (PHA), whereas adult CD4/CD45RA⁺ T cells were unresponsive under similar conditions. The mitogenic responses of newborn and adult CD4/CD45RA⁺ T cells to PHA and anti-CD2 mAb, which were equivalent, were directly proportional to the number of accessory cells present, whereas the responsiveness to cytokines was inversely proportional to the number of co-cultured accessory cells. Anti-CD2 responses were much more sensitive to low numbers of accessory cells than PHA. The particular sensitivity of newborn CD4/CD45RA⁺ T cells to IL-4 represents an antigen-independent T cell activation response which could help promote a Th2 immune response resulting in the newborn.     

Interleukin-13 is produced by activated human CD45RA+ and CD45R0+ T cells: modulation by interleukin-4 and interleukin-12

Interleukin (IL)-13 is a cytokine originally identified as a product of activated T cells. Little is known, however, about IL-13 production by human T cells and its modulation by other cytokines. Here, we show that IL-13 is produced by activated human CD4⁺ and CD8⁺ CD45R0⁺ memory T cells and CD4⁺ and CD8⁺ CD45RA⁺ naive T cells. In contrast, IL-4, which shares many biological activities with IL-13, is only produced by CD45R0⁺ T cells following activation. Analysis of intracellular cytokine production by single CD45RA⁺ and CD45R0⁺ T cells indicated that IL-13 continued to be produced for more than 24 h after stimulation, whereas IL-4 could not be detected after 24 h. These data were confirmed by measurement of specific mRNA and suggest that IL-13, unlike IL-4, but like interferon-γ (IFN-γ), is a cytokine with long-lasting kinetics. The majority of human CD45R0⁺ T cells produced IL-4 and IL-13 simultaneously. In contrast, IFN-γ protein was generally not co-expressed with IL-4 or IL-13. IL-4 added to primary cultures of highly purified peripheral blood T cells activated by the combination of anti-CD3+anti-CD28 mAb enhanced IL-13 production by CD45RA⁺ and to a lesser extent by CD45R0⁺ T cells. Under these conditions, however, IL-12 inhibited IL-13 production by CD45RA⁺ T cells and to a lesser extent by CD45R0⁺ T cells in a dose-dependent fashion. These inhibiting effects were not related to enhanced IFN-γ production induced by IL-12, since IFN-γ by itself did not affect IL-13 production. Collectively, our data indicate that IL-13 is produced by peripheral blood T cells which also produce IL-4, but not IFN-γ, and by naive CD45RA⁺ T cells which, in contrast, fail to produce IL-4. These observations, together with the long-lasting production of IL-13, suggest that IL-13 may have IL-4-like functions in situations where T cell-derived IL-4 is still absent or where its production has already been down-regulated.     

In vitro responses of human CD45R0brightRA− and CD45R0−RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0^brightRA^? or CD45R0^?-RA^bright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0^?RA^bright T cells, which converted to CD45R0^brightRA^? after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0^brightRA^?T cells compared to the CD45R0^?RA^bright subset. The poor responses of CD45R0^?RA^bright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0^?RA^bright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0⁺RA⁺). Alternatively, if primed CD45R0^?RA^bright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0^bright and CD45RA^bright expression identify memory and naive CD4⁺ T cells, respectively.     

Interleukin-4 and interleukin-5 are rarely co-expressed by human T cells

Interleukin (IL)-4 and IL-5 are two cytokines which synergize in the induction of several biological effector functions. They are produced by mouse and human T helper 2 (Th2) and T helper 0 (Th0) cells. Little is known about the regulation of the two cytokines at the single-cell level. Here we show, using a flow cytometric intracellular staining technique, that IL-4 and IL-5 are predominantly produced by different human peripheral CD4⁺ and CD8⁺ T cells, whereas interferon (IFN)-γ and IL-2 are produced by the same cells. In contrast, cloned human Th0 and Th2 cells were able to produce IL-4 and IL-5 simultaneously. The segregation of IL-4 and IL-5 in activated peripheral T cells was found within 72 h of activation upon anti-CD3 or phorbol ester + ionomycin stimulation. The kinetics of IL-4 and IL-5 production were different at the mRNA and the intra-and extracellular protein level, indicating that the cytokines are regulated differently. T cells from three patients with hyper-IgE syndrome did not display a substantial proportion of IL-4/IL-5 double-positive cells. However, simultaneous production could be induced in normal human T cells after prolonged stimulation with a minimum of two restimulation cycles. We conclude that the simultaneous production of IL-4 and IL-5 is a feature of repetitively activated human T cells.     

IL-15-independent antiviral function of primary and memory CD8+ T cells

Memory CD8+ T cells are comprised of CD122hi IL-15-dependent and CD122lo IL-15-independent subsets. Induction and retention of IL-15-independent memory CD8+ T cells was assessed in IL-15-/- and wild-type (wt) mice immunized with recombinant vaccinia virus (rVV) or Sindbis virus (rSIN) vectors expressing the identical foreign epitope. Both vectors induced epitope-specific CD8+ T cell expansion and function, independent of IL-15. Similar kinetics of rVV clearance confirmed effective CD8+ T cell function in IL-15-/- mice. CD44hi CD122hi CD8+ T cells, mainly of the CD62L-/lo phenotype, increased more dramatically and declined more rapidly in IL-15-/- mice, independent of the vector. Rapid IL-15-independent memory CD8+ T cell expansion following challenge of immune mice compensated for the limited memory CD8+ populations in IL-15-/- mice. However, despite expansion and expression of potent effector function, viral clearance was delayed in the absence of IL-15, coinciding with a rapid loss in cytolytic function.     

Early regulation of CD8 T cell alloreactivity by CD4+CD25- T cells in recipients of anti-CD154 antibody and allogeneic BMT is followed by rapid peripheral deletion of donor-reactive CD8+ T cells, precluding a role for sustained regulation

While acquisition of regulatory function by CD4+CD25- T cells has been reported following antigenic stimulation, "naturally occurring" regulatory CD4+ T cells (Treg) are believed to express CD25. We examined the mechanisms involved in peripheral CD8 T cell tolerance by induction of mixed chimerism using non-myeloablative conditioning with low-dose (3 Gy) total body irradiation and anti-CD154 antibody. Recipient CD4+ T cells were initially required for the induction of CD8 cell tolerance, but were not needed beyond 2 weeks. Depletion of CD25+ Treg prior to bone marrow transplantation and blockade of IL-2 with neutralizing antibody did not impede tolerance induction. Tolerance was dependent on CTLA4, but not on IFN-gamma. In C57BL/6 mice containing a fraction of 2C TCR transgenic CD8+ T cells, which recognize the MHC class I alloantigen Ld, induction of chimerism with L(d+), but not Ld-, bone marrow cells led to deletion of peripheral 2C+ CD8+ cells within 1 week in peripheral blood and spleen. Complete deletion required the presence of recipient CD4+ T cells. Thus, a novel, rapid form of regulation by CD4+CD25- T cells permits initial CD8 T cell tolerance in this model. Rapid peripheral deletion of donor-specific CD8 T cells precludes an ongoing requirement for CD4 T cell-mediated regulation.     

Hyaluronan‐binding by CD44 reduces the memory potential of activated murine CD8 T cells

Expansion and death of effector CD8 T cells are regulated to limit immunopathology and cells that escape contraction go on to generate immunological memory. CD44, a receptor for the extracellular matrix component hyaluronan, is a marker of activated and memory T cells. Here, we show with a murine model that the increase in CD44 expression and hyaluronan binding induced upon CD8 T cell activation was proportional to the strength of TCR engagement, thereby identifying the most strongly activated T cells. When CD44^?/? and CD44^+/+ OT‐I CD8 T cells were adoptively transferred into mice challenged with Listeria‐OVA, there was a slight increase in the percentage of CD44^+/+ cells at the effector site. However, CD44^+/+ cells were out‐competed by CD44^?/? cells after the contraction phase in the lymphoid tissues, and the CD44^?/? cells preferentially formed more memory cells. The hyaluronan‐binding CD44^+/+ CD8 effector T cells showed increased pAkt expression, higher glucose uptake, and were more susceptible to cell death during the contraction phase compared to non‐binding CD44^+/+ and CD44^?/? OT‐I CD8 T cells, suggesting that CD44 and its engagement with hyaluronan skews CD8 T cells toward a terminal effector differentiation state that reduces their ability to form memory cells.     

Human bone marrow contains a subset of quiescent early memory CD8+ T cells characterized by high CD127 expression and efflux capacity

Even today it is still not completely understood how CD8⁺ T‐cell memory is maintained long term. Since bone marrow (BM) is a niche for immunological memory, we sought to identify long‐lasting early memory CD8⁺ T cells in this compartment. To achieve this, we looked for CD8⁺ T cells that are able to efflux Rhodamine 123, a typical property of stem cells. Indeed, we identified a distinct subset of CD8⁺ T cells in BM, with the capacity to efflux and high CD127 expression. These CD127^hi effluxers are conventional CD8⁺ T cells exhibiting a broad TCR‐Vβ repertoire and are generated in response to viral peptides in vitro. CD127^hi effluxer CD8⁺ T cells have an early memory phenotype defined by preferential TNF‐α production and a Bcl‐2^hi, KLRG‐1^low profile. This population has long telomeres and shows constitutively low frequencies of Ki‐67 expression ex vivo, but has a high proliferative and differentiation capacity in vitro. However, IL‐15 downmodulates CD127 in CD127^hi effluxer CD8⁺ T cells in vitro. Consequently, the CD127^low effluxer subset may comprise cells recently exposed to IL‐15. Taken together, CD127^hi effluxer CD8⁺ T cells represent a novel population of early memory T cells resident in BM with properties required for long‐lived memory.     

CD27 costimulation contributes substantially to the expansion of functional memory CD8+ T cells after peptide immunization

Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long‐lived memory cells. The costimulatory receptor, CD27, is essential for optimal T‐cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8⁺ T‐cell reactivation remains controversial. We have used OVA and anti‐CD40 to establish a memory CD8⁺ T‐cell population and report here that their secondary expansion, driven by peptide and anti‐CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8⁺ T‐cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector‐ and central‐memory CD8⁺ T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8⁺ T‐cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.     

鼻咽癌细胞中表达的人白细胞介素18(hIL-18)对鼻咽癌患者外周血 CD8+T细胞细胞毒活性增强作用的研究

目的探讨人白细胞介素18在鼻咽癌细胞中的表达能否提高鼻咽癌病人外周血CD8+T细胞的杀伤活性以及作用途径.方法构建人白细胞介素18的真核表达载体[pcDNA3.1(-)/hIL-18,转染人鼻咽癌细胞株SUNE;以转染的SUNE细胞和未转染的SUNE细胞为靶细胞,以鼻咽癌患者外周血的CD8+T细胞为效应细胞,混合培养12 h,用LDH释放法测定CD8+T细胞的细胞毒活性;用免疫组化法测定混合培养物中CD8+T细胞上Perlorin、Fas-L、Granzyme B的表达.结果所构建的真核表达载体在鼻咽癌细胞中能高效表达hIL-18;ELISA法测得转染阳性细胞培养上清液中hIL-18的含量为(85±10)pg/ml,而未转染的SUNE细胞的培养上清液中hIL-18的含量低于5 pg/ml.将表达hIL-18的SUNE细胞与鼻咽癌患者外周血CD8+T细胞混合培养12 h后,CD8+T细胞的细胞毒活性显著增强(P＜0.001),尤其是当效应细胞/靶细胞为101时,CD8+T细胞对转染的SUNE细胞的裂解率高达46.7%,而CD8+T细胞对未转染的SUNE细胞的裂解率仅为4.6%.免疫组化法表明,这种杀伤活性的增强与CD8+T细胞表达Perforin有关,而与Fas-L和Granzyme B途径无关.结论hIL-18能显著增强鼻咽癌患者外周血CD8+T细胞的体外杀伤活性,这种杀伤活性与CD8+T细胞表达Perlorin有关.     

CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells

We studied the contribution of CD4 T cell help to survival and competitive fitness of CD8 memory T cells specific for influenza virus nucleoprotein. In agreement with recent studies, the optimal generation of functional memory CD8 T cells required CD4 help, although long-term maintenance of resting CD8 memory T cells did not absolutely depend on the presence of CD4 T cells. Nonetheless, CD4 T cells were essential during differentiation of CD8 memory T cells to imprint on them the capacity to compete effectively with other memory T cells. CD8 memory cells generated with help survived better in secondary polyclonal hosts, and co-transfer into lymphopenic hosts together with "un-helped" CD8 memory cells showed improved homeostatic expansion of CD8 memory cells that had been generated with CD4 help. Therefore, the requirement for CD4 help in CD8 T cell memory extends to homeostatic parameters that ensure the maintenance and competitive fitness of memory clones.     

Interleukin-15 selectively expands CD57+ CD28- CD4+ T cells, which are increased in active rheumatoid arthritis

Proinflammatory cytokines as well as CD4(+) T cells play critical roles in the pathogenesis of rheumatoid arthritis (RA). Recently, an increase of CD57(+) or CD28(-)CD4(+) T cells was demonstrated in RA, although the mechanism of the increase of these T cells is unclear. In this study, we first examined the relationship between CD57(+)CD4(+) T cells and CD28(-)CD4(+) T cells and found CD57(+)CD28(-)CD4(+) T cells, but neither CD57(+)CD28(+) nor CD57(-)CD28(+) cells, expanded in the peripheral blood of active RA. In vitro experiments revealed that CD57(+)CD28(-)CD4(+) T cells selectively expanded in response to IL-15. Furthermore IL-15-stimulated CD57(+)CD28(-)CD4(+) T cells induced TNF-alpha production from monocytes. These results suggest that CD57(+)CD28(-)CD4(+) T cells are involved in the pathogenesis of RA by responding to IL-15.     

Heterogeneity of CD4+ memory T cells: Functional modules for tailored immunity

Phenotypic and functional heterogeneity is the hallmark of effector and memory T cells. Upon antigenic stimulation, naïve CD4⁺ T cells make choices to become effector Th1, Th2 or Th17 cells, or even Treg. In addition to differences in cytokine repertoire, effector CD4⁺ T cells exhibit diversity in homing, such as migration to lymph node follicles to help B cells versus migration to inflamed tissues. Upon clearance of the antigen, two major types of memory T cells remain: central memory cells, which patrol lymphoid organs, and effector memory cells that act as sentinels in peripheral tissues such as the skin and the gut. Here, we review our current understanding of CD4⁺ T‐cell lineage heterogeneity and flexibility, with emphasis on the human system, and propose an organization of effector and memory T cells based on distinct functional modules.     

A strict requirement of interleukin-4 for interleukin-4 induction in antigen-stimulated human memory T cells

The role of interleukin-4 (IL-4) in the induction of IL-4 in mouse T cells is well established, but conflicting results have been reported with anti-CD3-primed human T cells and T cell clones. Therefore, IL-4 regulation was investigated in short-term cultured human T cells primed in vitro with either a superantigen or a hapten, nickel sulfate (NiSO₄), for 3 days and expanded with IL-2 for another 5 days. Under these conditions, antigen-specific IL-4 producing T cells were generated in 35 / 40 cultures. Priming for IL-4 production was abrogated in all cultures by anti-IL-4 antibody or soluble IL-4 receptor (sIL-4R). Primed T cells that were IL-4⁻ when cultured with IL-2 only developed an IL-4 producing phenotype when primed and expanded in the presence of exogenous IL-4. T cells primed in the presence of either endogenous or exogenous IL-4 produced 10–200-fold more IL-4 than T cells primed in the presence of anti-IL-4 antibody or sIL-4R. While IL-4 induction was absolutely dependent on IL-4, neither endogenous nor exogenous IL-4 influenced IFN-γ synthesis. Most importantly, IL-4 induced and sIL-4R abolished priming for IL-4 production even in NiSO₄-specific memory T cells from sensitized individuals. Thus, IL-4 induction in antigen-specific human memory T cell populations absolutely required IL-4. The IL-4 pathway of memory T cells retained a remarkable plasticity in sensitized individuals.     

Individual T cells hold unexpected clues to the nature of anergy and memory

The primary interest of our laboratory is understanding how the signals that a T cell receives influence its behavior during an immune response. Recently, we have focused our attention on the examination of T cell responses at an individual cell level. This article outlines our approach, presents our initial findings, and discusses the implications of these findings relative to our current models of T cell activation.     

Responses to human rhinovirus in CD45 T cell subsets isolated from tonsil

Isotypes of CD45 have been used extensively as markers of memory and naive populations of T cells in peripheral blood. In this study, T cells were isolated from human tonsil and their proliferative response against human rhinovirus was measured. Unexpectedly, equivalent responses were found among the CD4⁺CD45RA⁺ and CD4⁺CD45RO⁺ populations of T cells. This response requires MHC class II-positive antigen-presenting cells. The time course of the T cell response in vitro was that of a classical recall response, and no proliferative response to the virus could be detected in human cord blood. These results suggest that tonsils contain a significant population of CD45RA⁺ memory cells. The presence of this population may reflect ongoing stimulation with this common infectious agent, and the anatomical location of the T cells within the major lymphoid organ draining the naso-pharyngeal epithelial surface.     

CD28 co-stimulation is intact and contributes to prolonged ex vivo survival of hyporesponsive synovial fluid T cells in rheumatoid arthritis

In rheumatoid arthritis (RA), T cells in the inflamed joint are considered to play a crucial role in the pathogenesis. However, despite the fact that synovial T cells have an activated memory phenotype, they are functionally suppressed upon combined CD3 and CD28 stimulation. Here, we analyzed the contribution of both CD3 and CD28 to the hyporesponsiveness of synovial T cells in RA. In contrast to the low CD3 responsiveness of synovial fluid (SF) T cells compared to peripheral blood (PB) T cells, the CD28 co-stimulatory response was observed to be unaffected. Hyporesponsiveness of SF T cells has previously been associated with decreased levels of intracellular glutathione (GSH), an antioxidant and regulator of the intracellular redox state. Treatment of SF T cells with N-acetylcysteine, an antioxidant and replenisher of GSH, selectively improved CD3-induced responses, while leaving CD28 respon siveness unaffected. These data show that the CD3 pathway is highly sensitive to intracellular GSH alterations, whereas CD28 responsiveness is relatively refractory. Furthermore, in support for a functional role of CD28 co-stimulation, it was demonstrated that CD28 ligationacted in synergy with the IL-2 receptor γ chain signaling cytokine IL-15 in the enhancement of the ex vivo survival of SF T cells. These data indicate that CD28 co-stimulatory capacity of SF T cells, in contrast to CD3 stimulation, remains intact despite an altered intracellular redox state. Thereby, CD28 stimulation may contribute to the persistence of T cells at the site of inflammation, which might be of relevance in the pathogenesis of RA.