Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.     

Enhancing alloreactivity does not restore GVHD induction but augments skin graft rejection by CD4⁺ effector memory T cells

Graft-versus-host disease (GVHD) caused by donor T cells attacking recipient tissues is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (alloSCT). Studies have shown that effector memory T (T(EM) ) cells do not cause GVHD but are capable of immune functions post-transplant, including graft-versus-leukemia (GVL) effects, but the reasons for this are unclear. In mice, the T(EM) pool may have a less diverse T-cell receptor (TCR) repertoire than naive T (T(N) ) cells with fewer alloreactive clones. We therefore tested whether enhancing the alloreactivity of T(EM) cells would restore their ability to cause GVHD. In an MHC-matched system, alloreactive T(EM) cells were created by transferring GVHD effector cells into syngeneic recipients and allowing conversion to T(EM) cells. Upon retransfer to freshly transplanted recipients, these cells caused only mild GVHD. Similarly, in an MHC-mismatched system, T(EM) cells with a proven increased precursor frequency of alloreactive clones only caused limited GVHD. Nonetheless, these same cells mounted strong in vitro alloresponses and caused rapid skin graft rejection. T(EM) cells created from CD4(+) T cells that had undergone lymphopenia-induced proliferation (LIP) also caused only mild GVHD. Our findings establish that conversion to T(EM) cells significantly reduces GVHD potency, even in cells with a substantially enhanced alloreactive repertoire.     

Nef-specific CD45RA+ CD8+ T cells secreting MIP-1β but not IFN-γ are associated with nonprogressive HIV-1 infection

Background

Long-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease.

Results

We analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gamma^neg MIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gamma^neg MIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease.

Conclusion

The novel antigen-specific CD45RA+ IFN-gamma^neg MIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.     

IFN-γ induced by IL-12 administration prevents diabetes by inhibiting pathogenic IL-17 production in NOD mice

Interleukin 12 (IL-12) is a pivotal Th1-associated cytokine and a potent immunoregulatory molecule. However, the role of IL-12 in inducing immune tolerance that prevents insulitis and inhibits type 1 diabetes (T1D) remains unknown. The aim of this study was to investigate whether intermittent administration of IL-12 could prevent the development of T1D in nonobese diabetic (NOD) mice. We examined whether IL-12 treatment prevented diabetes by injecting different doses of IL-12 into NOD mice and compared the incidence of diabetes and insulitis in NOD mice with the incidence in control mice. Furthermore, we investigated the potential mechanisms of IL-12-mediated prevention of diabetes and insulitis. The expression of pro-inflammatory and immunoregulatory cytokines was measured before and following therapeutic administration of IL-12 in NOD mice. Our data demonstrated that both the absolute number and the function of DCs were impaired in NOD mice and that the levels of the Th17-associated pro-inflammatory cytokines, IL-1β, IL-6 and IL-23, were elevated in NOD mice compared with age-matched BALB/c and C57BL/6 mice. However, treatment of NOD mice with IL-12 suppressed insulitis and increased the number of healthy islets, and the levels of IL-17, IL-1β, IL-6 and IL-23 were significantly decreased. Moreover, IL-12 treatment of NOD mice induced the secretion of IFN-γ, a potent inhibitor of Th17 cells. These data indicated that intermittent administration of IL-12 prevented diabetes by inducing IFN-γ, suppressing the pathogenic IL-17-producing cells and reducing the expression of Th17-associated pro-inflammatory cytokines. Our results suggest a promising strategy for the treatment of human T1D and other Th17 cell-mediated autoimmune diseases.     

Antigen-presenting cells recruited by Brugia malayi induce Th2 differentiation of naïve CD4(+) T cells

A key feature of nematode infection is a bias towards a type 2 immune response. To investigate the role that antigen-presenting cells (APC) may play in promoting this bias, we used adherent peritoneal exudate cells (PEC) recruited in response to the filarial nematode Brugia malayi, to stimulate na?ve T cells from pigeon cytochrome c (PCC)-specific TCR transgenic (PCC-tg) mice. Although the proliferation of PCC-tg T cells was inhibited by parasite- induced PEC during primary stimulation, they proliferated normally upon secondary stimulation and were not rendered anergic. However, PCC-tg T cells primed by suppressive APC differentiated into IL-4-producing Th2 cells upon secondary stimulation instead of IFN-gamma-producing Th1 cells, as has been previously described. Studies with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled cells indicated that Th2 differentiation was associated with the inhibition of (or failure to stimulate) IFN-gamma production during primary stimulation. Interestingly, blocking antibodies against TGF-beta (but not IL-10) restored the differentiation of IFN-gamma-producing Th1 cells. Identical results with CFSE-labeled cells were obtained using purified IL-4-dependent F4/80(+) macrophages. These data indicate that T cells exposed to parasite-induced alternatively activated macrophages are driven towards Th2 differentiation. This may be an important factor in the Th2 bias that accompanies nematode infection.     

Nef-mediated MHC class I down-regulation unmasks clonal differences in virus suppression by SIV-specific CD8 T cells independent of IFN-γ and CD107a responses

CD8⁺ T lymphocytes (CTL) play a role in controlling HIV/SIV infection. CTL antiviral activity is dependent on recognition of antigenic peptides associated with MHC class I molecules on infected target cells, and CTL activation can be impaired by Nef-mediated down-regulation of MHC class I molecules. We tested the ability of a series of rhesus macaque CD8⁺ T-cell clones specific for the SIV Gag CM9 peptide to suppress SIV infection of autologous CD4⁺ T cells. We used a set of SIV_mac239 viruses with either wild-type Nef or Nef mutations that impair MHC class I down-regulation. All CTL clones efficiently suppressed virus replication in cells infected with mutant viruses with altered Nef function, phenotypically MHC class I^high or MHC class I^intermediate. However, the ability of the clones to suppress virus replication was variably reduced in the presence of wild-type Nef (MHC class I^low) despite the observations that all CTL clones showed similar IFN-γ responses to titrated amounts of cognate peptide as well as to SIV-infected cells. In addition, the CTL clones showed variable CD107a (CTL degranulation marker) responses that did not correlate with their capacity to suppress virus replication. Thus, the clonal differences are not attributable to TCR avidity or typical effector responses, and point to a potential as yet unknown mechanism for CTL-mediated suppression of viral replication. These data emphasize that current assays for evaluating CTL responses in infected or vaccinated individuals do not fully capture the complex requirements for effective CTL-mediated control of virus replication.     

CD4(+) T cells defined by their Vβ T cell receptor expression are associated with immunoregulatory profiles and lesion size in human leishmaniasis

Leishmaniasis is caused by infection with the protozoan parasite, Leishmania, that parasitizes human cells, and the cellular immune response is essential for controlling infection. In order to measure the host T cell response to Leishmania infection, we have measured the expansion, activation state and functional potential of specific T cells as identified by their T cell receptor Vβ region expression. In a group of cutaneous leishmaniasis (CL) patients, we evaluated these characteristics in nine different T cell subpopulations as identified by their Vβ region expression, before and after specific Leishmania antigen stimulation. Our results show: (1) an increase in CD4(+) T cells expressing Vβ 5·2 and Vβ 24 in CL compared to controls; (2) a Leishmania antigen-induced increase in CD4(+) T cells expressing Vβ 5·2, 11, 12 and 17; (3) a profile of previous activation of CD4(+) Vβ 5·2-, 11- and 24-positive T cells, with higher expression of CD45RO, HLA-DR, interferon-γ, tumour necrosis factor-α and interleukin-10 compared to other Vβ-expressing subpopulations; (4) a positive correlation between higher frequencies of CD4(+) Vβ5·2(+) T cells and larger lesions; and (5) biased homing of CD4(+) T cells expressing Vβ 5·2 to the lesion site. Given that CL disease involves a level of pathology (ulcerated lesions) and is often followed by long-lived protection and cure, the identification of specific subpopulations active in this form of disease could allow for the discovery of immunodominant Leishmania antigens important for triggering efficient host responses against the parasite, or identify cell populations most involved in pathology.     

CD4+CD25+Foxp3+IFN-γ+ human induced T regulatory cells are induced by interferon-γ and suppress alloresponses nonspecifically

Interferon-γ (IFN-γ)-producing CD3(+)CD4(+)CD25(+)Foxp3(+) peripheral blood lymphocytes (PBL) are more frequently detectable in patients with good than in patients with impaired long-term kidney graft function, suggesting an immunoregulatory role of this induced T regulatory (iTreg) subtype. Herein, the in vitro function of separated CD3(+)CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL that were induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin or alloantigenic stimulation was investigated using cell coculture techniques and flow cytometry. CD4(+)CD25(+)Foxp3(+) PBL with intracellular IFN-γ production increased to 26% in cell cultures stimulated with PMA/ionomycin for 6 hours. Recombinant IFN-γ augmented and anti-IFN-γ monoclonal antibody blocked induction of CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL, suggesting their IFN-γ-dependent induction. In addition, CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL produced immunosuppressive interleukin (IL)-10, transforming growth factor-β, and IL-4 intracellularly and expressed both IFN-γ and IFN-γ receptors (CD119) on the cell surface, allowing separation of CD4(+)CD25(+)IFN-γ(+) PBL with 98% purity. Addition of enriched CD4(+)CD25(+)IFN-γ(+) PBL to autologous PMA/ionomycin stimulated PBL decreased blast formation (p < 0.05), indicating suppression of cell proliferation by CD4(+)CD25(+)IFN-γ(+) PBL. CD4(+)CD25(+)IFN-γ(+) PBL separated from primary mixed leukocyte cultures (MLC) and added to autologous or third-party secondary MLC suppressed allogeneic T-cell activation nonspecifically (p < 0.05). We conclude that CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL are induced by IFN-γ, making them sensors for IFN-γ and initial immune responses. Circulating CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL could suppress allogeneic T-cell responses in patients and may be involved in inhibition of the posttransplant alloresponse.     

Potent induction of IFN-γ production from cord blood NK cells by the stimulation with single-stranded RNA

Natural killer (NK) cells play important roles in the innate immunity against viral infections. Although newborn infants are more susceptible to severe and recurrent viral infections than adults, the precise role of NK cells in the innate immunity against viral infections during neonatal period is not known. To clarify the functional characteristics of cord blood (CB) NK cells, we examined the capacity of CB NK cells to produce interferon gamma (IFN-γ) in response to the Toll-like receptor (TLR) ligands. We found that NK cells produced a large amount of IFN-γ by the stimulation with ssRNA, a TLR8 ligand, in the presence of interleukin-2 (IL-2), Interferon alpha (INF-α), and monocytes. Surprisingly, CB NK cells produced higher amount of IFN-γ than adult peripheral blood NK cells in this condition. IL-12 produced from monocytes by the stimulation with ssRNA was indispensable for the production of IFN-γ by NK cells. NK cells in cooperation with other innate immune cells may play more important role during the neonatal period than in adults in the host defense against viral infections by high capacity of IFN-γ production to compensate immature acquired immunity.     

Depletion of pro-inflammatory CD161(+) double negative (CD3(+)CD4(-)CD8(-)) T cells in AIDS patients is ameliorated by expansion of the γδ T cell population

In this present investigation, flow cytometry was utilized to evaluate 13 healthy controls and 31 HIV-1 infected patients who had advanced to the AIDS stage of infection (CD4 count below 200 cells/mm(3)), for the expression of CD161 on CD3(+) double negative (DN) (CD3(+)CD4(-)CD8(-)) T cells, CD4(+) T cells, CD8(+) T cells and γδ T cells. The observed depletion of CD161(+) T cells from peripheral circulation was due primarily to the loss of CD4(+)CD161(+) T cells; as these cells represented 8.67±0.74% of the total healthy control peripheral T cell population, while the CD4(+)CD161(+) T cells of the AIDS group represented only 3.35±0.41% (p=<0.0001) of the total peripheral T cell population. We have also shown here that the DN T cell population was more than doubled in the AIDS group, with the DN T cell population expanding from 3.29±0.45% of the healthy control peripheral T cell population to 8.64±1.16% (p=0.0001) of the AIDS group peripheral T cell population. By evaluating the expression of CD161 on the surface of the DN T cells we showed that within the healthy control group, 47.4±4.99% of the DN T cells were positive for the expression of CD161, while only 26.4±3.54% (p=0.002) of the AIDS group's DN T cells expressed CD161. Despite CD161 expression being halved on the DN T cells of the AIDS group, when we compared the total peripheral T cell percentage of CD161(+) DN T cells between the healthy control group and the AIDS group, there was no statistical difference. Even though only 26.4% DN T cells within the AIDS group were positive for CD161(+), the overall DN T cell population had expanded to such an extent that there was no statistical difference between the groups with regard to CD161(+) DN T cells as a percentage of the total peripheral T cell population. Furthermore, we showed that within the DN T cell population, there was an approximate 2:1 ratio of γδ to αβ T cells, and this ratio was maintained in both the healthy control group and the AIDS group. While evaluating γδ T cells we also discovered that CD8(+) γδ T cells were expanded from 0.62±.09% of the healthy control peripheral T cell population to 5.01±.88% (p=<0.0001) of the peripheral T cell population of the AIDS group; and that this population of CD8(+) γδ T cells underwent the same reduction in percentage of cells expressing CD161(+), further demonstrated that the phenomenon of CD161(+) percentage reduction and compensatory increase in total cell population was affecting the entire circulating γδ T cell population.     

NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8⁺ T cells

Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8(+) T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α(+) DCs were particularly efficient at sensing bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8(+) T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during bacterial immunity.     

Thymic but not splenic CD8⁺ DCs can efficiently cross-prime T cells in the absence of licensing factors

Cross‐presentation is an important mechanism to elicit both immune defenses and tolerance. Although only a few DC subsets possess the machinery required for cross‐presentation, little is known about differences in cross‐presenting capabilities of DCs belonging to the same subpopulation but localized in different lymphoid organs. In this study, we demonstrate that steady‐state thymic CD8⁺ DCs can efficiently cross‐prime naïve CD8⁺ T cells in the absence of costimulation. Surprisingly, cross‐priming by splenic CD8⁺ DCs was dependent on licensing factors such as GM‐CSF. In the absence of GM‐CSF, antigen–MHC‐class‐I complexes were detected on thymic but not on splenic CD8⁺ DCs, indicating that the cross‐presentation capacity of the thymic subpopulation was higher. The observed cross‐priming differences between thymic and splenic CD8⁺ DCs did not correlate with differential antigen capture or costimulatory molecules found on the surface of DCs. Moreover, we did not detect overall impairment of antigen presentation, as peptide‐loaded splenic CD8⁺ DCs were able to induce CD8⁺ T‐cell proliferation. The observation that thymic CD8⁺ DCs are more efficient than splenic CD8⁺ DCs in T‐cell cross‐priming in the absence of licensing factors indicates that the requirements for efficient antigen presentation differ between these cells.     

Intracellular production of IL-2, IL-4 and IFN-γ by peripheral blood CD3+ cells in intermittent allergic rhinitis

Background: Allergic inflammation is mainly driven by type 2 T helper cells. The aim was to assess the changes in production of type 1 and 2 cytokines by CD3⁺ T cells dependent on natural exposure to allergens in subjects with intermittent allergic rhinitis (IAR) and in non-atopic subjects.Material: A total of 13 patients with IAR and 13 healthy non-atopics were recruited into the study. 11 patients with IAR were examined during the grass pollen season and 11 patients outside the season, 9 of them were assessed on both occasions.Methods: A flow cytometric assessment of intracellular expression of IL-2, IL-4 and IFN- by CD3⁺ cells was performed. For statistical analysis non-parametric tests were used.Results: A tendency to decreased production of IL-4 outside the season was observed (6.94% [3.42–13.33] in season vs. 2.06% [0.7–3.6] out of season). The production of IL-4 was higher in the rhinitic group in the season than in the control group (1.93% [1.07–4.97], p = 0.0034) and production of IL-2 was higher both in and outside the season (9.1% [3.94–15.09] and 10.0% [4.79–25.35] vs. 3.64% (2.64–5.03), p = 0.037 and 0.045, respectively). IL-4/IL-2 and IL-4/IFN- ratios were higher in the IAR group in the season than outside the season.Conclusion: A tendency towards a switch from a predominant type 2 response during natural allergen exposure to its suppression outside the season was found, together with a stable type 1 response.Received 22 July 2004; returned for revision 27 August 2004; accepted by M. J. Parnham 2 November 2004     

Circulating IFN-γ producing CD4+ T cells and IL-17A producing CD4+ T cells,HLA-shared epitope and ACPA may characterize the clinical response to therapy in rheumatoid arthritis patients

This study analyzed the association between peripheral distributions of helper T cell subsets, HLA shared-epitope (SE), anti-cyclic citrullinated peptide antibody (ACPA) and clinical response to therapy in rheumatoid arthritis (RA) patients. Frequencies of IFN-γ-producing CD4+T (Th1) and IL-17A-producing CD4+T (Th17) cells were determined by flow cytometry in 167 patients (114 cases with good-response (GR) and 53 poor-response (PR) based on DAS28). HLA-DRB1 alleles for patients and 150 healthy controls were determined by PCR-SSP. We observed that 65.2% of RA patients were SE⁺, 63.4%ACPA⁺, 43.7%SE⁺ACPA⁺ and 14.9% were SE⁻ACPA⁻. Higher significantly proportions of Th1 and Th17 cells were found in RA patients than controls (P < 0.05) as well as in the SE⁺ or ACPA⁺RA patients compared to SE⁻ and ACPA⁻ patients. Increased frequencies of both Th subsets were found in SE⁺ACPA⁺ versus SE⁻ACPA⁻ patients (P < 0.001) and in the PR versus GR group (P < 0.001). We showed significant differences for Th cells frequencies between SE⁺ and SE⁻ patients in both groups, and between ACPA⁺ and ACPA⁻ cases in the PR group. Our findings suggest a close link between Th1 and Th17 cells proportions and HLA-SE/ACPA in the RA patients and remarkably in the PR group which could be indicative for the importance of immune monitoring for evaluation of response to therapy.     

IL-17/IFN-γ double producing CD8+ T (Tc17/IFN-γ) cells: a novel cytotoxic T-cell subset converted from Tc17 cells by IL-12

It has been reported that IFN-γ-producing CD8(+) T (Tc1) cells express cytotoxic molecules such as perforin and granzyme B to exhibit higher cytotoxicity against tumor cells compared with Tc2 cells. However, the critical role of IL-17-producing CD8(+) T (Tc17)-cell subsets in tumor immunity remains unclear. Tc17 cells differentiated from naive CD8(+) T cells did not possess cytotoxic molecules and exhibited no strong cytotoxicity. However, when Tc17 effector cells were further cultured with IL-12, they converted into IFN-γ-producing Tc17 cells, which mainly consisted of IL-17/IFN-γ double-producing cells (Tc17/IFN-γ). IL-12-converted Tc17 cells also acquired cytotoxic function in addition to IFN-γ producibility. Moreover, they showed strong anti-tumor activity both in vitro and in vivo as well as Tc1 cells. Among four distinct subsets in IL-12-converted Tc17 cell populations, the isolated Tc17/IFN-γ cells exhibited cytotoxicity as well as IFN-γ-producing Tc1-like cells. Thus, we first indicate direct evidence that Tc17/IFN-γ cells, which were plastically converted from non-cytotoxic Tc17 cells by IL-12, exhibited strong anti-tumor activity as well as Tc17 cell-derived Tc1-like cells.