Cord blood CD34+ cells cultured with FLT3L, stem cell factor, interleukin-6, and IL-3 produce CD11c+CD1a-/c- myeloid dendritic cells

Methods that allow expansion of myeloid dendritic cells (MDCs) from CD34(+) cells are potentially important for boosting anti-leukemic responses after cord blood (CB) hematopoietic stem cell transplantation (HSCT). We showed that the combination of early-acting cytokines FLT3-ligand (FL), stem cell factor (SCF), interleukin (IL)-3, and IL-6 supported the generation of CD11c(+)CD16() CD1a()/c() MDCs from CB CD34(+) cells or CB myeloid precursors. Early-acting cytokine-derived MDCs were maintained within the myeloid CD33(+)CD14()CD15() precursors with a mean of 4 x 10(6) cells generated from 1-4 x 10(4) CB CD34(+) cells or myeloid precursors after 2 weeks. After 8-12 days of culture the MDCs expressed higher levels of HLA-DR antigen but lower levels of CD40 and CD86 antigen, compared to adult blood MDCs. At this stage of differentiation, the early-acting cytokine-derived MDCs had acquired the ability to induce greater allogeneic T cell proliferation than monocytes or granulocytes derived from same culture. Early-acting cytokine-derived MDCs exposed to the cytokine cocktail (CC) comprising IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and prostaglandin E (PGE)-2, upregulated the surface co-stimulatory molecules CD40 and CD86 and enhanced allogeneic T cell proliferation, as is characteristic of MDCs maturation. The reliable production of MDCs from CB CD34(+) cells provides a novel way to study their lineage commitment pathway(s) and also a potential means of enriching CB with MDCs to improve prospects for DC immunotherapy following CB HSCT.     

Human CD4+ central and effector memory T cells produce IL-21: effect on cytokine-driven proliferation of CD4+ T cell subsets

IL-21 regulates certain functions of T cells, B cells, NK cells and dendritic cells. Although activated CD4(+) T cells produce IL-21, data identifying the specific CD4(+) T cell subsets that produce IL-21 are conflicting. In a previous study, mouse IL-21 message was detected in T(H)2, whereas human IL-21 (hIL-21) message was found in both T(H)1 and follicular helper T cells. To identify the IL-21-secreting cell populations in human, we established a hybridoma cell line producing an anti-hIL-21 mAb. Intracellular hIL-21-staining experiments showed that hIL-21 was mainly expressed in activated CD4(+) central memory T cells and in activated CD4(+) effector memory T cells, but not in activated CD4(+) naive T cells. Moreover, IL-21 was produced upon activation by some IFN-gamma-producing T(H)1-polarized cells and some IL-17-producing T(H)17-polarized cells, but not by IL-4-producing T(H)2-polarized cells. These results suggest that specific CD4(+) T cell populations produce IL-21. In the functional analysis, we found that IL-21 significantly enhanced the cytokine-driven proliferation of CD4(+) helper T cells synergistically with IL-7 and IL-15 without T cell activation stimuli. Taken together, IL-21 produced from CD4(+) memory T cells may have a supportive role in the maintenance of CD4(+) T cell subsets.     

Rescue of memory CD8+ T cell reactivity in peptide/TLR9 ligand immunization by codelivery of cytokines or CD40 ligation

The capability of cellular immune components to rapidly recall upon challenge in most situations decides the efficacy of a vaccine. Here, we show that immunization of mice with SSIEFARL peptide (immunodominant epitope in glycoprotein B of herpes simplex virus type 1, aa498-505) combined with TLR9 ligand in the absence of helper CD4(+) T cell activation generates a functionally impaired CD8(+) T cell memory response. Codelivery of IL-12, IL-15, or anti-CD40 together with MHC class-I-restricted peptide combined with TLR9 ligand at inception of immunization resulted in generation of memory CD8(+) T cells that were several fold less compromised than immunization with peptide alone. Furthermore, administration of either plasmid DNA encoding IL-15 or anti-CD40 mAb but not rIL-12 during the memory phase restored the reactivity of memory CD8(+) T cells. Moreover, the rescued CD8(+) T cells preserved their cytotoxic capability and were able to clear a recombinant vaccinia virus encoding glycoprotein B of HSV. Our results indicate that good memory CD8(+) T cell response to peptide immunization can be achieved by using costimulatory procedures at the time of priming or recall immunization.     

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.     

The development and functions of CD4(+) T cells expressing a transgenic TCR specific for an MHC-I-restricted tumor antigenic epitope

It has been reported that the ratio of CD4(+) to CD8(+) T cells has no bias in a few class I major histocompatibility complex (MHC-I)-restricted T-cell receptor (TCR)-transgenic mice specific for alloantigens or autoantigens, in which most CD4(+) T cells express an MHC-I-restricted TCR. In this study, we further showed that more than 50% of CD4(+) T cells in MHC-I-restricted P1A tumor antigen-specific TCR (P1ATCR)-transgenic mice could specifically bind to MHC-I/P1A peptide complex. P1A peptide could stimulate the transgenic CD4(+) T cells to proliferate and secrete both type 1 helper T cell and type 2 helper T cell cytokines. The activated CD4(+) T cells also showed cytotoxicity against P1A-expressing tumor cells. The analysis of TCR α-chains showed that these CD4(+) T cells were selected by co-expressing endogenous TCRs. Our results show that CD4(+) T cells from P1ATCR transgenic mice co-expressed an MHC-I-restricted transgenic TCR and another rearranged endogenous TCRs, both of which were functional.     

Interleukin-7 signalling is sufficient to phenotypically and functionally prime human CD4 naive T cells

Interleukin-7 (IL-7) is produced by bone marrow and lymphoid stromal cells and is involved in the synthesis, survival and homeostasis of T cells. These attributes are the basis for current strategies to utilize IL-7 as an immune modulator for several clinical conditions to replenish depleted T-cell numbers. Because we had previously determined that IL-7 can induce potent human immunodeficiency virus replication in the otherwise non-permissive CD4(+) naive T-cell compartment, we evaluated here the impact of IL-7 on the phenotype and functional potential of naive CD4(+) T cells in an attempt to understand the mechanism of this induction. We demonstrate that IL-7 mediated the up-regulation of CD25, CD95 and human leucocyte antigen-DR, while it did not alter the expression of CD45RO, CD69, CD40, or CD154. Examination of the cytokine profile of IL-7-treated naive T cells using a Type1/Type2 Proteome Array indicated a remarkable IL-7-mediated induction of interferon-gamma production, while the other cytokines evaluated (IL-2, IL-12, tumour necrosis factor-alpha, IL-4, IL-5, IL-10 and IL-13) were not affected. Intracellular staining of IL-7-treated naive T cells for interferon-gamma verified the Proteome data. IL-7 did not induce cell cycle proliferation of naive CD4(+) T cells, as evaluated by 7-AAD/pyronin immunostaining and carboxyfluorescein diacetate succinimidyl ester dye tracking. IL-7 treatment of naive CD4(+) T cells induced their ability to prime monocytes, as was indicated by induction of CD80 and CD86 expression on monocytes cocultured with IL-7-treated naive CD4(+) T cells. Collectively, these data indicate that IL-7 signalling is sufficient to phenotypically and functionally prime human CD4(+) naive T cells independent of antigen stimulation.     

Schistosoma japonicum egg antigens stimulate CD4 CD25 T cells and modulate airway inflammation in a murine model of asthma

A number of epidemiological and clinical studies have suggested an inverse association between allergy and helminth infection, such as Schistosomiasis. Therefore, we hypothesize that Schistosoma japonicum egg antigens, a type of native antigen, can induce production of CD4(+) CD25(+) T cells with regulatory activity, modulating airway inflammation and inhibiting asthma development. The frequency of CD4(+) CD25(+) T cells was determined by flow cytometry for mice treated with ovalbumin (OVA), CD25(+) depletion/OVA, schistosome egg antigens, schistosome egg antigens/OVA and for control mice. The ability of CD25(+) T cells from these mice to suppress T-cell proliferation and cytokine production was investigated both in vivo and in vitro. Results showed that the CD4(+) CD25(+) T cells of OVA-treated mice exhibited impaired control of dysregulated mucosal T helper 2 responses compared to the controls (P < 0.05). Depletion of CD25(+) cells accelerated OVA-induced airway inflammation and increased the expression of interleukin (IL)-5 and IL-4. Treatment with schistosome egg antigens increased the number and suppressive activity of CD4(+) CD25(+) T cells, which made IL-10, but little IL-4. In a murine model of asthma, S. japonicum egg antigens decreased the expression of Th2 cytokines, relieved antigen-induced airway inflammation, and inhibited asthma development. Thus, we provided evidence that S. japonicum egg antigens induced the production of CD4(+) CD25(+) T cells, resulting in constitutive immunosuppressive activity and inhibition of asthma development. These results reveal a novel form of protection against asthma and suggest a mechanistic explanation for the protective effect of helminth infection on the development of allergy.     

Differential expression of Ly6C and T-bet distinguish effector and memory Th1 CD4(+) cell properties during viral infection

CD4(+) T?cells differentiate into multiple effector types, but it is unclear how they form memory T?cells during infection in?vivo. Profiling virus-specific CD4(+) T?cells revealed that effector cells with T helper 1 (Th1) or T follicular helper (Tfh) cell characteristics differentiated into memory cells, although expression of Tfh cell markers declined over time. In contrast to virus-specific effector CD8(+) T?cells, increased IL-7R expression was not a reliable marker of CD4(+) memory precursor cells. However, decreased Ly6C and T-bet (Tbx21) expression distinguished a subset of Th1 cells that displayed greater longevity and proliferative responses to secondary infection. Moreover, the gene expression profile of Ly6C(lo)T-bet(int) Th1 effector cells was virtually identical to mature memory CD4(+) T?cells, indicating early maturation of memory CD4(+) T?cell features in this subset during acute viral infection. This study provides a framework for memory CD4(+) T?cell development after acute viral infection.     

Non-redundant role for IL-7R signaling for the survival of CD8+ memory T cells

IL-7 and IL-15 are important cytokines for CD8 memory T cells. However, the extent that IL-7 is essential for CD8 T cell memory remains unclear because blocking IL-7 in vivo results in near complete inhibition of T cell development with the few mature T cells exhibiting functional abnormalities. To bypass this complication, CD8 memory development was examined utilizing a mouse model where transgenic IL-7Ralpha was selectively expressed in the thymus of IL-7Ralpha(-/-) mice. T cell development was corrected but the resulting peripheral T cells were essentially IL-7 non-responsive. Activation of IL-7R-defective OT-I CD8(+) T cells with OVA(257-264) and IL-2 readily yielded CTL. Upon further culture with IL-15, these CTL expressed phenotypic and functional properties of central memory-like cells. Thus, IL-7R-defective CD8(+) T cells do not exhibit intrinsic defects in effector or memory development. When IL-7R-defective OT-I CTL were adoptively transferred into normal or IL-15(-/-) recipient mice in a non-inflammatory setting, they converted into memory-like cells, but did not persist, which was even more striking in IL-15(-/-) recipients. This poor persistence was rescued after expression of transgenic Bcl-2 in IL-7R-defective OT-I T cells. Collectively, these data indicate that IL-7 is non-redundantly required for the survival of CD8 memory T cells.     

gamma(c) cytokines provide multiple homeostatic signals to naive CD4(+) T cells

Cytokines signaling through receptors sharing the common gamma chain (gamma(c)), including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, are critical for the generation and peripheral homeostasis of B, T and NK cells. To identify unique or redundant roles for gamma(c) cytokines in naive CD4(+) T cells, we compared monoclonal populations of CD4(+) T cells from TCR-Tg mice that were gamma(c) (+), gamma(c) (-), CD127(-/-) or CD122(-/-). We found that gamma(c) (-) naive CD4(+) T cells failed to accumulate in the peripheral lymphoid organs and the few remaining cells were characterized by small size, decreased expression of MHC class I and enhanced apoptosis. By over-expressing human Bcl-2, peripheral naive CD4(+) T cells that lack gamma(c) could be rescued. Bcl-2(+) gamma(c) (-) CD4(+) T cells demonstrated enhanced survival characteristics in vivo and in vitro, and could proliferate normally in vitro in response to antigen. Nevertheless, Bcl-2(+) gamma(c) (-) CD4(+) T cells remained small in size, and this phenotype was not corrected by enforced expression of an activated protein kinase B. We conclude that gamma(c) cytokines (primarily but not exclusively IL-7) provide Bcl-2-dependent as well as Bcl-2-independent signals to maintain the phenotype and homeostasis of the peripheral naive CD4(+) T cell pool.     

Interleukin-2 rescues helpless effector CD8+ T cells by diminishing the susceptibility to TRAIL mediated death

CD8(+) T cells primed in the absence of CD4(+) T cell help are programmed to produce TRAIL, which results in Death receptor (DR5) mediated apoptosis upon restimulation. Here, we studied whether these 'helpless' effector CD8(+) T cells are consigned to an apoptotic fate or whether their helpless program can be altered by inflammatory or growth cytokines. We found that helpless CD8(+) T cells regained their full proliferative and functional capacity only when IL-2 was added to cell cultures, while IL-7 and IL-15, two common gamma chain cytokines associated with CD8(+) T cell homeostasis and memory, could only partly restore secondary expansion in helpless CD8(+) T cells. Recovery of functional CD8(+) T cell immunity by IL-2 was concomitant with induction of IL2Rα (CD25) expression, downregulation of TRAIL, and the upregulation of anti-apoptotic molecules Bcl-2 and FLIP. The addition of IL-2 to helpless CD8(+) T cells also interfered with DR5-mediated apoptosis induction, indicating that IL-2 affects several components of the TRAIL-DR5 pathway. Collectively, these data demonstrate that the helpless phenotype is not fixed, and that IL-2R signaling at the time of reactivation can play an important role in restoring CD8(+) T cell function.     

Comparison of allergen-stimulated dendritic cells from atopic and nonatopic donors dissecting their effect on autologous naive and memory T helper cells of such donors

BACKGROUND: Because of their production of IL-12, mature dendritic cells (DC) are potent inducers of T(H)1 responses. However, recent reports have demonstrated that DCs can also induce T(H)2 differentiation. OBJECTIVE: In the current study we investigated which immune response is induced by DCs in naive CD45RA(+) or memory CD45R0(+) CD4(+) T cells from atopic individuals (patients with grass pollen, birch pollen, or house dust mite allergy) compared with nonatopic control subjects. METHODS: Immature DCs, generated from peripheral blood monocytes from atopic and nonatopic donors, were pulsed with the respective allergen and fully matured. Then the mature DCs were cocultured in vitro with autologous naive (CD45RA(+)) and memory (CD45R0(+)) CD4(+) T cells and cytokine and IgE production were measured by ELISA. RESULTS: After the second restimulation with allergen-pulsed DCs, naive as well as memory autologous CD4(+) T cells from atopic but not from nonatopic donors showed an enhanced production of the T(H)2-type cytokines IL-4, IL-5, and IL-10, resulting in an increased IgE production, whereas IFN-gamma production and proliferation were not different. IL-12 production and surface marker expression of DCs derived from atopic and nonatopic donors did not differ and addition of neutralizing anti-IL-12 mAbs did not increase IL-4 but diminished IFN-gamma production. CONCLUSION: These data indicate that mature DCs are able to induce naive and activate allergen-specific T helper cells to produce T(H)2 cytokines if the T cells are derived from atopic donors. This phenomenon is not due to diminished IL-12 production by DCs of atopic donors.     

Overexpression of IL-21 promotes massive CD8+ memory T cell accumulation

The ability of IL-21 to promote in vitro T cell survival led us to investigate its biological activity in vivo. We report that overexpression of IL-21 in transgenic mice drives CD8(+) memory T cell accumulation with a concomitant reduction in naive T cell numbers. These memory T cells are functional, given their ability to rapidly produce IFN-gamma and proliferate following stimulation. Since the homeostasis of naive and memory T cells is controlled by cytokines, we evaluated whether IL-21 influences cytokine receptor expression. We show that IL-21 inhibits IL-7R expression on naive T cells in vitro, suggesting impaired IL-7-mediated naive T cell survival in IL-21-transgenic mice. In contrast, IL-7R expression on CD4(+) memory T cells is not affected, allowing their IL-7-dependent survival in IL-21-transgenic mice. Although IL-21 decreases IL-7R expression on CD8(+) memory T cells, this has no impact on their survival since their maintenance in the T cell pool is IL-7-independent. Rather, we demonstrate that CD8(+) memory T cells are receptive to IL-21 survival signals allowing for their accumulation in IL-21-transgenic mice. This study identifies new roles for IL-21 in T cell homeostasis and in the regulation of T cell responses to cytokines.     

Interleukin-15 stimulates macrophages to activate CD4+ T cells: a role in the pathogenesis of rheumatoid arthritis?

Interleukin-15 (IL-15) is a proinflammatory cytokine that is overexpressed in rheumatoid arthritis (RA), a disease characterized by activation of monocytes/macrophages (MPhi), and by expansion of autoreactive CD4(+) T cells. We hypothesized that IL-15 plays a major role for this expansion of CD4(+) T cells and modulates the phenotype of monocytes/MPhi and their interaction with CD4(+) T cells. Here, we show that IL-15 enhances the proliferation of CD4(+) T cells from patients with RA in peripheral blood mononuclear cell cocultures. To further dissect the underlying mechanisms, we employed MPhi from IL-15(-/-) or IL-15 transgenic mice. These were induced to differentiate or were stimulated with IL-15. Here we show that addition of IL-15 during differentiation of MPhi (into 'IL-15MPhi') and overexpression of IL-15 by MPhi from IL-15(tg) mice leads to increased levels of major histocompatibility complex class II expression. This resulted in enhanced stimulation of antigen-specific CD4(+) T cells in vitro and was accompanied by reduced messenger RNA expression in MPhi for immunosuppressive SOCS3. The proliferation rates of IL-15MPhi and IL-15(tg)MPhi were high, which was reflected by increased p27(Kip1) and reduced p21(Waf1) levels. In view of high serum and synovial levels of IL-15 in patients with RA, our data suggest the possibility that this excess IL-15 in RA may stimulate monocytes/MPhi to activate the characteristic autoreactive CD4(+) T cells in RA.     

Phenotypic heterogeneity of antigen-specific CD4 T cells under different conditions of antigen persistence and antigen load

The factors responsible for the phenotypic heterogeneity of memory CD4 T cells are unclear. In the present study, we have identified a third population of memory CD4 T cells characterized as CD45RA(+)CCR7(-) that, based on its replication history and the homeostatic proliferative capacity, was at an advanced stage of differentiation. Three different phenotypic patterns of memory CD4 T cell responses were delineated under different conditions of antigen (Ag) persistence and load using CD45RA and CCR7 as markers of memory T cells. Mono-phenotypic CD45RA(-)CCR7(+) or CD45RA(-)CCR7(-) CD4 T cell responses were associated with conditions of Ag clearance (tetanus toxoid-specific CD4 T cell response) or Ag persistence and high load (chronic HIV-1 and primary CMV infections), respectively. Multi-phenotypic CD45RA(-)CCR7(+), CD45RA(-)CCR7(-) and CD45RA(+)CCR7(-) CD4 T cell responses were associated with protracted Ag exposure and low load (chronic CMV, EBV and HSV infections and HIV-1 infection in long-term nonprogressors). The mono-phenotypic CD45RA(-)CCR7(+) response was typical of central memory (T(CM)) IL-2-secreting CD4 T cells, the mono-phenotypic CD45RA(-)CCR7(-) response of effector memory (T(EM)) IFN-gamma-secreting CD4 T cells and the multi-phenotypic response of both IL-2- and IFN-gamma-secreting cells. The present results indicate that the heterogeneity of different Ag-specific CD4 T cell responses is regulated by Ag exposure and Ag load.     

Frequencies and role of regulatory T cells in patients with (pre)malignant cervical neoplasia

Oncogenic human papillomavirus (HPV)-infection is crucial for developing cervical cancer and its precursor lesions [cervical intraepithelial neoplasia (CIN)]. Regulatory T cells (T(regs)) might be involved in the failure of the immune system to control the development of HPV-induced cancer. We investigated frequencies, phenotype and activity of T(regs) in patients with cervical neoplasia. CIN and cervical cancer patients showed increased CD4(+)/CD25(high) T cell frequencies in peripheral blood and CD4(+) T cell fraction. These CD4(+)/CD25(high) T cells represent T(regs) as demonstrated by their low proliferation rate, low interferon (IFN)-gamma/interleukin (IL)-10 ratio, high expression of CD45RO, GITR, CTLA-4, forkhead box P3 (FoxP3) and low CD45RA expression. Moreover, in HPV16(+) cervical cancer patients, in-vitro depletion of CD25(+) T cells resulted in increased IFN-gamma T cell responses against HPV16 E6- and E7 peptides. Thus, increased frequencies of T(regs) in cervical cancer patients may indeed suppress HPV-specific immunity. Longitudinal analysis of CD4(+)/CD25(high) T cell frequencies in patients showed a modest decline 1 year after curative surgery or chemoradiation. This study demonstrates increased frequencies and suppressive activity of T(regs) in cervical cancer. These results imply that T(regs) may suppress the immune control of cervical neoplasia and furthermore that suppression of immunity by T(regs) will be another hurdle to overcome in therapeutic immunization strategies against cervical neoplasia.     

Homeostasis of V alpha 14i NKT cells

CD1d-reactive natural killer T (NKT) cells with an invariant V alpha 14 rearrangement (V alpha 14i) are a distinct subset of T lymphocytes that likely have important immune-regulatory functions. Little is known regarding the factors responsible for their peripheral survival. Using alpha-galactosylceramide-containing CD1d tetramers to detect V alpha 14i NKT cells, we show here that the expansion of V alpha 14i NKT cells in lymphopenic mice was not dependent on CD1d expression and was unaffected by the presence of host NKT cells. Additionally, we found that IL-15 was important in the expansion and/or survival of V alpha 14i NKT cells, with IL-7 playing a lesser role. These results demonstrate that the homeostatic requirements for CD1d-restricted NKT cells, which are CD4(+) or CD4(-)CD8(-), resemble those of CD8(+) memory T cells. We propose that this expansion and/or survival in the periphery of V alpha 14i NKT cells is affected by competition for IL-15, and that IL-15-requiring cells-such as NK cells and CD8(+) memory cells-may define the V alpha 14i NKT cell niche.     

IL-15 enhances the function and inhibits CD95/Fas-induced apoptosis of human CD4+ and CD8+ effector-memory T cells

Although the effect of IL-15 has been described on murine cells in vitro and in vivo, its effect on human memory CD8(+) T cells is not well characterized. We show here that IL-15 preferentially enhances the activation and effector function of human effector-memory CD45RA(-)CD62L(-) and CD45RA(+)CD62L(-) CD4(+) and CD8(+) T cells in both healthy and HIV-infected individuals. We find that IL-15 increases 2- to 5-fold both the activation and secretion of the effector cytokines IFN-gamma and tumor necrosis factor (TNF)-alpha by anti-CD3-stimulated purified CD4(+) and CD8(+) T cells and peripheral blood mononuclear cells from healthy and HIV-infected individuals. Furthermore, IL-15 potently inhibits CD95/Fas-induced apoptosis of the effector-memory CD4(+) and CD8(+) T cells from HIV-infected individuals. These findings suggest that in addition to being a growth and survival factor for memory CD8(+) T cells, IL-15 is also a potent activator of human effector-memory CD8(+) T cells both in healthy and in HIV-infected individuals.