Multifunctional CD4+ T cells correlate with active Mycobacterium tuberculosis infection

Th1 CD4⁺ T cells and their derived cytokines are crucial for protection against Mycobacterium tuberculosis. Using multiparametric flow cytometry, we have evaluated the distribution of seven distinct functional states (IFN‐γ/IL‐2/TNF‐α triple expressors, IFN‐γ/IL‐2, IFN‐γ/TNF‐α or TNF‐α/IL‐2 double expressors or IFN‐γ, IL‐2 or TNF‐α single expressors) of CD4⁺ T cells in individuals with latent M. tuberculosis infection (LTBI) and active tuberculosis (TB). We found that triple expressors, while detectable in 85–90%TB patients, were only present in 10–15% of LTBI subjects. On the contrary, LTBI subjects had significantly higher (12‐ to 15‐fold) proportions of IL‐2/IFN‐γ double and IFN‐γ single expressors as compared with the other CD4⁺ T‐cell subsets. Proportions of the other double or single CD4⁺ T‐cell expressors did not differ between TB and LTBI subjects. These distinct IFN‐γ, IL‐2 and TNF‐α profiles of M. tuberculosis‐specific CD4⁺ T cells seem to be associated with live bacterial loads, as indicated by the decrease in frequency of multifunctional T cells in TB‐infected patients after completion of anti‐mycobacterial therapy. Our results suggest that phenotypic and functional signatures of CD4⁺ T cells may serve as immunological correlates of protection and curative host responses, and be a useful tool to monitor the efficacy of anti‐mycobacterial therapy.     

Enrichment of IL‐17A+IFN‐γ+ and IL‐22+IFN‐γ+ T cell subsets is associated with reduction of NKp44+ILC3s in the terminal ileum of Crohn's disease patients

Crohn's disease (CD) is a chronic inflammatory condition of the human gastrointestinal tract whose aetiology remains largely unknown. Dysregulated adaptive immune responses and defective innate immunity both contribute to this process. In this study, we demonstrated that the interleukin (IL)‐17A⁺interferon (IFN)‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets accumulated specifically in the inflamed terminal ileum of CD patients. These cells had higher expression of Ki‐67 and were active cytokine producers. In addition, their proportions within both the IL‐17A‐producer and IL‐22‐producer populations were increased significantly. These data suggest that IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets might represent the pathogenic T helper type 17 (Th17) population in the context of intestinal inflammation for CD patients. In the innate immunity compartment we detected a dramatic alteration of both phenotype and function of the intestinal innate lymphoid cells (ILCs), that play an important role in the maintenance of mucosal homeostasis. In the inflamed gut the frequency of the NKp44^–CD117^–ILC1s subset was increased significantly, while the frequency of NKp44⁺ILC3s was reduced. Furthermore, the frequency of human leucocyte antigen D‐related (HLA‐DR)‐expressing‐NKp44⁺ILC3s was also reduced significantly. Interestingly, the decrease in the NKp44⁺ILC3s population was associated with an increase of pathogenic IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets in the adaptive compartment. This might suggest a potential link between NKp44⁺ILC3s and the IL‐17A⁺IFN‐γ⁺ and IL‐22⁺IFN‐γ⁺ T cell subsets in the terminal ileum of CD patients.     

A unique DNA methylation signature defines a population of IFN‐γ/IL‐4 double‐positive T cells during helminth infection

Th1 and Th2 cell fates are traditionally viewed as mutually exclusive, but recent work suggests that these lineages may be more plastic than previously thought. When isolating splenic CD4⁺ T cells from mice infected with the parasitic helminth Schistosoma mansoni, we observed a defined population of IFN‐γ/IL‐4 double‐positive cells. These IFN‐γ⁺IL‐4⁺ cells showed differences in DNA methylation at the Ifng and Il4 loci when compared with IFN‐γ⁺IL‐4^? (Th1) and IFN‐γ^?IL‐4⁺ (Th2) cells, demonstrating that they represent a distinct effector cell population. IFN‐γ⁺IL‐4⁺ cells also displayed a discrete DNA methylation pattern at a CpG island within the body of the Gata3 gene, which encodes the master regulator of Th2 identity. DNA methylation at this region correlated with decreased Gata3 levels, suggesting a possible role in controlling Gata3 expression. These data provide important insight into the molecular mechanisms behind the co‐existence of Th1 and Th2 characteristics.     

Functional deficits of pertussis-specific CD4(+) T cells in infants compared to adults following DTaP vaccination

Understanding the immune responses that explain why infants require multiple doses of pertussis vaccine to achieve protection against infection is a high priority. The objective of this study was to compare the function and phenotypes of antigen‐specific CD4⁺ T cells in adults (n = 12), compared to infants (n = 20), following vaccination with acellular pertussis (DTaP) vaccine. Peripheral blood mononuclear cells (PBMCs) were stimulated with pertussis toxoid (PT), pertactin (PRN) and filamentous haemagglutinin (FHA). Multi‐parameter flow cytometry was used to delineate CD4⁺ T cell populations and phenotypes producing interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐α and IL‐4. Based on surface CD69 expression, infants demonstrated activation of vaccine antigen‐specific CD4⁺ T cells similar to adults. However, among infants, Boolean combinations of gates suggested that type 1 (Th‐1) CD4⁺ T cell responses were confined largely to TNF‐α⁺IL‐2⁺IFN‐γ^‐ or TNF‐α⁺IL‐2^‐IFN‐γ^‐. A significantly lower percentage of polyfunctional T helper type 1 (Th1) responses (TNF‐α⁺IFN‐γ⁺IL‐2⁺) and type 2 (Th2) responses (IL‐4) were present in the infants compared to adults. Moreover, a significantly higher percentage of infants' functional CD4⁺ T cells were restricted to CD45RA^‐CCR7⁺CD27⁺ phenotype, consistent with early‐stage differentiated pertussis‐specific memory CD4⁺ T cells. We show for the first time that DTaP vaccination‐induced CD4⁺ T cells in infants are functionally and phenotypically dissimilar from those of adults.     

Multiple Antigen Peptide Containing B and T Cell Epitopes of F1 Antigen of Yersinia pestis Showed Enhanced Th1 Immune Response in Murine Model

Yersinia pestis is a facultative bacterium that can survive and proliferate inside host macrophages and cause bubonic, pneumonic and systemic infection. Apart from humoral response, cell‐mediated protection plays a major role in combating the disease. Fraction 1 capsular antigen (F1‐Ag) of Y. pestis has long been exploited as a vaccine candidate. In this study, F1‐multiple antigenic peptide (F1‐MAP or MAP)‐specific cell‐mediated and cytokine responses were studied in murine model. MAP consisting of three B and one T cell epitopes of F1‐antigen with one palmitoyl residue was synthesized using Fmoc chemistry. Mice were immunized with different formulations of MAP in poly DL‐lactide‐co‐glycolide (PLGA) microspheres. F1‐MAP with CpG oligodeoxynucleotide (CpG‐ODN) as an adjuvant showed enhanced in vitro T cell proliferation and Th1 (IL‐2, IFN‐γ and TNF‐α) and Th17 (IL‐17A) cytokine secretion. Similar formulation also showed significantly higher numbers of cytokine (IL‐2, IFN‐γ)‐secreting cells. Moreover, F1‐MAP with CpG formulation showed significantly high (P < 0.001) percentage of CD4⁺ IFN‐γ⁺ cells as compared to CD8⁺ IFN‐γ⁺ cells, and also more (CD4‐ IFN‐γ)⁺ cells secrete perforin and granzyme as compared to (CD8‐ IFN‐γ)⁺ showing Th1 response. Thus, the study highlights the importance of Th1 cytokine and existence of CD4⁺ and CD8⁺ immune response. This study proposes a new perspective for the development of vaccination strategies for Y. pestis that trigger T cell immune response.     

Increased natural killer cell subsets with inhibitory cytokines and inhibitory surface receptors in patients with recurrent miscarriage and decreased or normal subsets in kidney transplant recipients late post‐transplant

Patients with recurrent miscarriage (RM) show up‐regulated cytotoxic natural killer (NK) cells that are suspected to play a causal role in abortion. In the present study, we investigated counter‐regulating inhibitory mechanisms and compared the results in RM patients with those of healthy controls (HC), patients with end‐stage renal disease (ESRD) and kidney transplant recipients late post‐transplant (TX). NK, NK T and T cell subsets were analysed in the peripheral blood of 31 RM, 14 female ESRD and nine female TX patients as well as 21 female HC using eight‐colour fluorescence flow cytometry. Compared with HC, RM patients showed significantly higher absolute numbers of CD56⁺ NK cells co‐expressing the phenotype interferon (IFN)‐γR⁺, IL‐4⁺, transforming growth factor (TGF)‐β⁺, IL‐4⁺ human leucocyte antigen D‐related (HLA‐DR)⁺, TGF‐β⁺HLA‐DR⁺, IL‐4⁺TGF‐β⁺, IL‐4⁺TGF‐β^–, IFN‐γ⁺ and/or IL‐10^–IFN‐γ⁺ (all P ≤ 0·01), more IL‐17⁺CD56^bright (P = 0·028) NK cells and more CD56^dimCD16⁺ NK cells co‐expressing IFN‐γR, IFN‐γ, IL‐4 and/or TGF‐β (all P ≤ 0·01). When the same cell subsets were analysed in ESRD or TX patients, cytokine‐producing NK cell subsets were not significantly different from those of HC. RM patients showed significantly higher absolute numbers of CD158a⁺, CD158b⁺, CD158a^–CD158e⁺ (all P < 0·05), NKG2D⁺NKG2A⁺, NKG2D ⁺NKG2A^–, NKG2D⁺ and/or NKG2A⁺ (all P ≤ 0·01) CD56⁺ NK cells and higher CD158a⁺, CD158b⁺ (all P < 0·05), NKG2D⁺ and/or NKG2A⁺ (all P < 0·01) CD56^dim+CD16⁺ NK cells than HC. In contrast, ESRD patients had normal and TX recipients had lower CD158a⁺ and NKG2D⁺NKG2A^–CD56⁺ NK cells and lower CD158a⁺CD56^dim+CD16⁺ NK cells (all P < 0·05) than HC. RM patients have abnormally high circulating NK cells expressing inhibitory cytokines and inhibitory surface receptors which might contribute to the pathogenesis of RM.     

Multi‐functional flow cytometry analysis of CD4+ T cells as an immune biomarker for latent tuberculosis status in patients treated with tumour necrosis factor (TNF) antagonists

Although monitoring tuberculosis (TB) infection during long‐term treatment with tumour necrosis factor (TNF) antagonists is of great importance, no monitoring strategy has yet proved successful. Indeed, even the newly proposed interferon‐gamma release assays (IGRAs) are known to produce dynamic changes in IFN‐γ plasma levels, making them unreliable indicators of patients' pathological/clinical status. We used intracellular cytokine flow cytometry (ICCFC) to investigate the performance of multi‐functional CD4⁺ T cells producing IFN‐γ, interleukin (IL)‐2 and/or TNF in response to Mycobacterium tuberculosis‐specific antigens in subjects treated with TNF antagonists. Patients were classified into three groups based on their TB status before commencement of treatment and on IFN‐γ level fluctuations evaluated by IGRA during a 36‐month follow‐up period. The cytokine profile of M. tuberculosis‐specific CD4⁺ T cells showed that latent tuberculosis infection (LTBI) subjects had a higher frequency of double‐positive IFN‐γ⁺ IL‐2⁺ CD4⁺ T cells and triple‐positive IFN‐γ⁺ IL‐2⁺ TNF⁺ CD4⁺ T cells compared to those without LTBI, who showed IFN‐γ‐level fluctuations over time. In contrast, this latter group of patients showed similar proportions of cells producing IFN‐γ alone, IL‐2 alone and IL‐2 in combination with TNF in response to M. tuberculosis‐specific antigens. It therefore appears that patients with and without LTBI infection are characterized by different intracellular cytokine profiles. This is the first study evaluating ICCFC in patients treated with TNF antagonists, and suggests that multi‐functional analysis of CD4⁺ T cells could be useful for ruling out TB infection in patients classified at screening as LTBI‐negative but who show IGRA fluctuations under long‐term TNF antagonist treatment.     

IL‐18Rα‐deficient CD4+ T cells induce intestinal inflammation in the CD45RBhi transfer model of colitis despite impaired innate responsiveness

IL‐18 has been implicated in inflammatory bowel disease (IBD), however its role in the regulation of intestinal CD4⁺ T‐cell function remains unclear. Here we show that murine intestinal CD4⁺ T cells express high levels of IL‐18Rα and provide evidence that IL‐18Rα expression is induced on these cells subsequent to their entry into the intestinal mucosa. Using the CD45RB^hi T‐cell transfer colitis model, we show that IL‐18Rα is expressed on IFN‐γ⁺, IL‐17⁺, and IL‐17⁺IFN‐γ⁺ effector CD4⁺ T cells in the inflamed colonic lamina propria (cLP) and mesenteric lymph node (MLN) and is required for the optimal generation and/or maintenance of IFN‐γ‐producing cells in the cLP. In the steady state and during colitis, TCR‐independent cytokine‐induced IFN‐γ and IL‐17 production by intestinal CD4⁺ T cells was largely IL‐18Rα?dependent. Despite these findings however, IL‐18Rα?deficient CD4⁺ T cells induced comparable intestinal pathology to WT CD4⁺ T cells. These findings suggest that IL‐18‐dependent cytokine induced activation of CD4⁺ T cells is not critical for the development of T‐cell‐mediated colitis.     

CRTH2 expression on T cells in asthma

Mast cell‐derived prostaglandin D₂ (PGD₂) is the major prostanoid found within the airway of asthmatics immediately following allergen challenge. PGD₂ has been shown to have chemokinetic effects on eosinophils and T helper type 2 (Th2) cells in vitro. This occurs through the interaction of PGD₂ with the G‐protein‐coupled chemokine receptor homologous molecule expressed on Th2 lymphocytes (CRTH2). The expression of CRTH2 has been shown to be highly selective for Th2 cells. Using flow cytometry we have studied the expression of CRTH2 on T cells in blood and bronchoalveolar lavage fluid in asthmatics and normal subjects. CRTH2 expression was confined to a small percentage of blood T cells in asthmatics (1·8% ± 0·2) and normal (1·6% ± 0·2) subjects. CRTH2 was enriched significantly on interleukin (IL)‐4⁺/IL‐13⁺ T cells compared to interferon (IFN)‐γ⁺ T cells (P < 0·001). There was a small population of CRTH2⁺ T cells in the bronchoalveolar lavage (BAL) of asthmatics (2·3% ± 0·6) and normal subjects (0·3% ± 0·1), and there was a significant difference between the two groups (P < 0·05). There were similar amounts of PGD₂ in the BAL of asthma and normal subjects. Within paired blood–BAL samples from the same subject there was no increase in CRTH2⁺ T cells in the BAL compared to blood in asthmatics. Enrichment of CRTH2 on IL‐4⁺ and IL‐13⁺ T cells compared to IFN‐γ⁺ T cells was also seen in BAL from asthmatics (P < 0·001). CRTH2 is expressed preferentially by IL‐4⁺/IL‐13⁺ T cells compared to IFN‐γ⁺ T cells. However, given their small numbers they are unlikely to have a significant involvement in the pathogenesis of asthma. CRTH2 antagonism may not diminish T cell accumulation in the asthmatic lung.     

Recombinant Salmonella typhimurium outer membrane protein A is recognized by synovial fluid CD8 cells and stimulates synovial fluid mononuclear cells to produce interleukin (IL)‐17/IL‐23 in patients with reactive arthritis and undifferentiated spondyloarthropathy

In developing countries, one‐third of patients with reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) are triggered by Salmonella typhimurium. Synovial fluid mononuclear cells (SFMCs) of patients with ReA and uSpA proliferate to low molecular weight fractions (lmwf) of outer membrane proteins (Omp) of S. typhimurium. To characterize further the immunity of Omp of Salmonella, cellular immune response to two recombinant proteins of lmwf, OmpA and OmpD of S. typhimurium (rOmpA/D‐sal) was assessed in 30 patients with ReA/uSpA. Using flow cytometry, 17 of 30 patients' SF CD8⁺ T cells showed significant intracellular interferon (IFN)‐γ to Omp crude lysate of S. typhimurium. Of these 17, 11 showed significantly more CD8⁺CD69⁺ IFN‐γ T cells to rOmpA‐sal, whereas only four showed reactivity to rOmpD‐sal. The mean stimulation index was significantly greater in rOmpA‐sal than rOmpD‐sal [3·0 (1·5–6·5) versus 1·5 (1·0–2·75), P < 0·005]. Similarly, using enzyme‐linked immunospot (ELISPOT) in these 17 patients, the mean spots of IFN‐γ‐producing SFMCs were significantly greater in rOmpA‐sal than rOmpD‐sal [44·9 (3·5–130·7) versus 19·25 (6–41), P < 0·05]. SFMCs stimulated by rOmpA‐sal produced significantly more proinflammatory cytokines than rOmpD‐sal: IFN‐γ [1·44 (0·39–20·42) versus 0·72 (0·048–9·15) ng/ml, P < 0·05], interleukin (IL)?17 [28·60 (6·15–510·86) versus 11·84 (6·83–252·62) pg/ml, P < 0·05], IL‐23 [70·19 (15–1161·16) versus 28·25 (> 15–241·52) pg/ml, P < 0·05] and IL‐6 [59·78 (2·03–273·36) versus 10·17 (0·004–190·19) ng/ml, P < 0·05]. The rOmpA‐sal‐specific CD8⁺ T cell response correlated with duration of current synovitis (r = 0·53, P < 0·05). Thus, OmpA of S. typhimurium is a target of SF CD8⁺ T cells and drives SFMC to produce increased cytokines of the IL‐17/IL‐23 axis which contribute to the pathogenesis of Salmonella‐triggered ReA.     

Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women

Citation
Horká P, Jaro?ová R, Malí?ková K, Janatková I, Mare?ková H, Zima T, Kalousová M. Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women. Am J Reprod Immunol 2011; 65: 466–469 Problem To analyze the relation of the fertility and pregnancy of women of childbearing age to the intracellular (IC) production of tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), and interleukins 2 and 4 (IL‐2 and IL‐4). Method of study Intracellular cytokine production in peripheral blood CD3⁺ CD4⁺ lymphocytes was analyzed by flow cytometry in 185 women being treated for infertility and 50 fertile women of childbearing age. Results Infertile women have a significantly higher IC production of TNF‐α, IFN‐γ, IL‐2, and IL‐4 and higher ratios of TNF‐α/IL‐2, TNF‐α/IL‐4, and TNF‐α/IFN‐γ compared to the fertile women. Conclusion Cytokines produced by Th lymphocytes are important in orchestrating the immune response during conception, and Th‐cell dysregulation could be a reason for infertility.     

Dendritic cell‐based therapeutic vaccine elicits polyfunctional HIV‐specific T‐cell immunity associated with control of viral load

Efforts aimed at restoring robust immune responses limiting human immunodeficiency virus (HIV)‐1 replication therapeutically are warranted. We report that vaccination with dendritic cells generated ex vivo and loaded with HIV lipopeptides in patients (n = 19) on antiretroviral therapy was well tolerated and immunogenic. Vaccination increased: (i) the breadth of the immune response from 1 (1–3) to 4 (2–5) peptide‐pool responses/patient (p = 0.009); (ii) the frequency of functional T cells (producing at least two cytokines among IFN‐γ, TNF‐α, and IL‐2) from 0.026 to 0.32% (p = 0.002) and from 0.26 to 0.35% (p = 0.005) for CD4⁺ and CD8⁺ T cells, respectively; and (iii) the breadth of cytokines secreted by PBMCs upon antigen exposure, including IL‐2, IFN‐γ, IL‐21, IL‐17, and IL‐13. Fifty percent of patients experienced a maximum of viral load (VL) 1 log₁₀ lower than the other half following antiretroviral treatment interruption. An inverse correlation was found between the maximum of VL and the frequency of polyfunctional CD4⁺ T cells (p = 0.007), production of IL‐2 (p = 0.006), IFN‐γ (p = 0.01), IL‐21 (p = 0.006), and IL‐13 (p = 0.001). These results suggest an association between vaccine responses and a better control of viral replication. These findings will help in the development of strategies for a functional cure for HIV infection.     

Type I interferons contribute to experimental cerebral malaria development in response to sporozoite or blood‐stage Plasmodium berghei ANKA

Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T‐cell activation and type II IFN‐γ are required for Plasmodium berghei ANKA (PbA)‐induced murine experimental cerebral malaria (ECM), the role of type I IFN‐α/β in ECM development remains unclear. Here, we address the role of the IFN‐α/β pathway in ECM devel‐opment in response to hepatic or blood‐stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN‐γR1^?/? mice were fully resistant, IFNAR1^?/? mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN‐γR1^?/? mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1^?/? mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA‐induced brain sequestration of CXCR3⁺‐activated CD8⁺ T cells. This was associated with reduced expression of Granzyme B, IFN‐γ, IL‐12Rβ2, and T‐cell‐attracting chemokines CXCL9 and CXCL10 in IFNAR1^?/? mice, more so in the absence of IFN‐γR1. Therefore, the type I IFN‐α/β receptor pathway contributes to brain T‐cell responses and microvascular pathology, although it is not as essential as IFN‐γ for the development of cerebral malaria upon hepatic or blood‐stage PbA infection.     

Antigen-activated T cells induce IL-12p75 production from dendritic cells in an IFN-γ-independent manner

The addition of IL‐12p75 to naïve CD4⁺ T cells promotes their differentiation towards a T_H1‐type cytokine pattern. Dendritic cells stimulated by LPS generate IL‐12p75, but only if the environment also contains IFN‐γ. Thus, it appears that IFN‐γ is needed to start the response that will result in further production of IFN‐γ. We previously reported that paradoxically DCs produce IL‐12p75 only after engaging primed, but not naïve T cells. This study examines the mechanism by which primed T cells trigger IL‐12p75 secretion and asks whether this induction is also dependent on the presence of IFN‐γ. Here, we show that, in contrast to LPS, primed T cells induce IL‐12p75 in an IFN‐γ‐independent manner. Addition of rIFN‐γ to cocultures of naïve T cells with DCs did not induce IL‐12p75. Moreover, antigen‐activated CD4⁺ T cells from wild type or IFN‐γ‐deficient mice both initiated IL‐12p75 production from DCs. Surprisingly, we found that synergies between three T‐cell‐derived factors – CD40 Ligand, IL‐4 and GM‐CSF – were necessary and sufficient for IL‐12p75 production. These results suggest that there are at least two distinct pathways for IL‐12p75 production in vivo. Furthermore, the T‐cell‐dependent pathway of IL‐12p75 production employs molecules that are not classically associated with a T_H1‐type response.     

Galectin‐9 expands immunosuppressive macrophages to ameliorate T‐cell‐mediated lung inflammation

Galectin‐9 (Gal‐9) plays pivotal roles in the modulation of innate and adaptive immunity to suppress T‐cell‐mediated autoimmune models. However, it remains unclear if Gal‐9 plays a suppressive role for T‐cell function in non‐autoimmune disease models. We assessed the effects of Gal‐9 on experimental hypersensitivity pneumonitis induced by Trichosporon asahii. When Gal‐9 was given subcutaneously to C57BL/6 mice at the time of challenge with T. asahii, it significantly suppressed T. asahii‐induced lung inflammation, as the levels of IL‐1, IL‐6, IFN‐γ, and IL‐17 were significantly reduced in the BALF of Gal‐9‐treated mice. Moreover, co‐culture of anti‐CD3‐stimulated CD4 T cells with BALF cells harvested from Gal‐9‐treated mice on day 1 resulted in diminished CD4 T‐cell proliferation and decreased levels of IFN‐γ and IL‐17. CD11b⁺Ly‐6C^highF4/80⁺ BALF M? expanded by Gal‐9 were responsible for the suppression. We further found in vitro that Gal‐9, only in the presence of T. asahii, expands CD11b⁺Ly‐6C^highF4/80⁺ cells from BM cells, and the cells suppress T‐cell proliferation and IFN‐γ and IL‐17 production. The present results indicate that Gal‐9 expands immunosuppressive CD11b⁺Ly‐6C^high M? to ameliorate Th1/Th17 cell‐mediated hypersensitivity pneumonitis.     

Human phagosome processing of Mycobacterium tuberculosis antigens is modulated by interferon‐γ and interleukin‐10

Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen–MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon‐γ (IFN‐γ) and interleukin‐10 (IL‐10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide–MHC‐II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide–MHC‐II complexes in M. tuberculosis‐infected human monocyte‐derived macrophages (MDMs) using autologous M. tuberculosis‐specific CD4⁺ T cells. The MDMs were pre‐treated with either IFN‐γ or IL‐10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M.  tuberculosis ‐specific CD4⁺ T cells. Our results demonstrated that in MDMs pre‐treated with IFN‐γ, M. tuberculosis peptide–MHC‐II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN‐γ, the complexes were detected in the endosomal fractions. In MDMs pre‐treated with IL‐10, the M. tuberculosis peptide–MHC‐II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA‐DR at the cell surface only in the IFN‐γ‐treated MDMs, suggesting that IFN‐γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL‐10 favours the trafficking of Ag85B to vesicles that do not contain LAMP‐1. Therefore, IFN‐γ and IL‐10 play a role in the formation and trafficking of M. tuberculosis peptide–MHC‐II complexes.