Innate IFN‐γ promotes development of experimental autoimmune encephalomyelitis: A role for NK cells and M1 macrophages

The role of IFN‐γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN‐γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN‐γ during development of EAE. Depletion of NK cells or neutralization of IFN‐γ delayed the onset of EAE and was associated with reduced infiltration of IL‐17⁺ and GM‐CSF⁺ T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)‐immunized IFN‐γ^?/? mice failed to induce EAE, despite producing IL‐17 and GM‐CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen‐4 (VLA‐4) expression and failed to infiltrate the CNS. Addition of recombinant IFN‐γ to immune cells from the IFN‐γ^?/? mice activated M1 macrophages and restored VLA‐4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti‐VLA‐4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN‐γ‐producing T cells are not required for development of EAE, but NK cell‐derived IFN‐γ has a key role in promoting M1 macrophage expansion and VLA‐4‐mediated migration of encephalitogenic T cells into the CNS.     

Basophils are potent antigen‐presenting cells that selectively induce Th2 cells

Basophils and mast cells are important effector cells in helminth‐infected host and IgE‐mediated allergic inflammation. Although they have the same progenitors, basophils and mast cells complete their terminal differentiation in the bone marrow and peripheral tissues, respectively, and only basophils circulate in the blood. Although it is recognized that basophils are important for Th2 responses, and it is also well established that IL‐4 is required for Th2 differentiation from naïve CD4⁺ T cells, the nature of the cells that produce “early” IL‐4, remained elusive until recently. Three groups independently demonstrated that basophils are the predominant APC in inducing Th2 response against helminth parasites and allergens. Basophils express MHC class II and CD80/86, have the potential to take‐up and process protein Ag (particularly Ag–IgE complex) and to present peptide in the context of MHC class II, and to produce IL‐4. These Ag‐pulsed basophils induce the development of Th2 cells both in vitro and in vivo. Thus, basophils contribute to Th2/IgE response by the production of IL‐4 and presentation of MHC class II/peptide complex to naïve CD4⁺ T cells, in contrast to the Th1‐inducing action of DC. In this review, we summarize what is known regarding basophil function in allergy and parasite infection, examine the novel Ag‐presenting function of basophils and discuss potential clinical implications of this finding.     

STAT of the union: Dynamics of distinct tumor‐associated macrophage subsets governed by STAT1

The tumor stroma has long been ignored as therapeutic target, but it has become clear that several stromal cell types play a nonredundant role during tumor progression. In particular, macrophages possess the capacity to stimulate tumor growth and metastasis via multiple mechanisms. In this issue of the European Journal of Immunology, a study by Tymoszuk et al. Eur. J. Immunol. 2014. 44: 2247–2262 demonstrates that both monocyte recruitment and local macrophage proliferation determines the tumor‐associated macrophage (TAM) pool size in HER2/Neu‐driven mammary carcinomas. These tumors contain two main TAM subsets—MHC class II (MHC‐II)^loF4/80^hi and MHC‐II^hiF4/80^lo—similar to what was observed in other tumor models. Interestingly, only the MHC‐II^loF4/80^hi subset is largely absent in a STAT1‐deficient background. STAT1 induces the expression of CSF‐1, which in turn drives TAM proliferation and possibly also the M2 gene signature of MHC‐II^loF4/80^hi TAM. Conversely, STAT1 deficiency upregulates M2 gene expression in MHC‐II^hiF4/80^lo TAM, demonstrating that both TAM subsets are differentially regulated, probably as a consequence of their distinct intratumoral localization. In this Commentary, we place these findings in the context of current knowledge and propose new avenues for future research.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B‐cell responses

Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169⁺ macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood‐borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169⁺ macrophages in mice resulted in strong, isotype‐switched, high‐affinity Ab production and the preferential induction and long‐term persistence of Ag‐specific GC B cells and follicular Th cells. In agreement with these observations, CD169⁺ macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B‐cell responses. Our results identify CD169⁺ macrophages as promoters of high‐affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses.     

Leishmania-infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells

CD4⁺ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the 14.07 macrophage cell line (H-2^k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4⁺ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4⁺ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4⁺ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.     

Th1 cytokines are more effective than Th2 cytokines at licensing anti‐tumour functions in CD40‐activated human macrophages in vitro

CD40 agonists are showing activity in early clinical trials in patients with advanced cancer. In animal models, CD40 agonists synergise with T‐cell‐activating therapies to inhibit tumour growth by driving tumour macrophage repolarisation from an immunosuppressive to a Th1 immunostimulatory, tumouricidal phenotype. We therefore tested the hypothesis that T‐cell‐derived cytokines license anti‐tumour functions in CD40‐activated human macrophages. CD40 ligand (CD40L) alone activated macrophages to produce immunosuppressive IL‐10, in a similar fashion to bacterial LPS, but failed to promote anti‐tumour functions. The Th1 cytokine IFN‐γ optimally licensed CD40L‐induced macrophage anti‐tumour functions, inducing a switch from IL‐10 to IL‐12p70 production, promoting macrophage‐mediated Th1 T‐cell skewing and enhancing tumouricidal activity. We found that even the Th2 cytokines IL‐4 and IL‐13 promoted IL‐12p70 production (albeit without inhibiting IL‐10 production) and enhanced Th1 T‐cell skewing by CD40L‐activated macrophages. However, IL‐4 and IL‐13 did not enhance tumouricidal activity in CD40L‐activated macrophages. Thus, while both Th1 and Th2 cytokines biased macrophages to a Th1 immunostimulatory phenotype, only Th1 cytokines promoted tumouricidal activity in CD40L‐activated macrophages. The presence of tumour‐infiltrating Th1 or Th2 cells might therefore be predictive for patient response to CD40 agonism.     

Dexamethasone promotes tolerance in vivo by enriching CD11cloCD40lo tolerogenic macrophages

We previously showed that antigen immunization in the presence of the immunosuppressant dexamethasone (a strategy we termed “suppressed immunization”) could tolerize established recall responses of T cells. However, the mechanism by which dexamethasone acts as a tolerogenic adjuvant has remained unclear. In the present study, we show that dexamethasone enriches CD11c^loCD40^lo macrophages in a dose‐dependent manner in the spleen and peripheral lymph nodes of mice by depleting all other CD11c⁺CD40⁺ cells including dendritic cells. The enriched macrophages display a distinct MHC class II (MHC II)^loCD86^hi phenotype. Upon activation by antigen in vivo, CD11c^loCD40^lo macrophages upregulate IL‐10, a classic marker for tolerogenic antigen‐presenting cells, and elicit a serum IL‐10 response. When presenting antigen in vivo, these cells do not elicit recall responses from memory T cells, but rather stimulate the expansion of antigen‐specific regulatory T cells. Moreover, the depletion of CD11c^loCD40^lo macrophages during suppressed immunization diminishes the tolerogenic efficacy of the treatment. These results indicate that dexamethasone acts as a tolerogenic adjuvant partly by enriching the CD11c^loCD40^lo tolerogenic macrophages.     

Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells

Summary: Th3 CD4⁺ regulatory cells were identified during the course of investigating mechanisms associated with oral tolerance. Different mechanisms of tolerance are induced following oral antigen administration, including active suppression, clonal anergy and deletion. Low doses favor active suppression whereas high doses favor anergy/deletion. Th3 regulatory cells form a unique T‐cell subset which primarily secretes transforming growth factor (TGF)‐β, provides help for IgA and has suppressive properties for both Th1 and Th2 cells. Th3 type cells are distinct from the Th2 cells, as CD4⁺ TGF‐β‐secreting cells with suppressive properties have been generated from interleukin (IL)‐4‐deficient animals. In vitro differentiation of Th3 cells from Th precursors from T‐cell antigen receptor (TCR) transgenic mice is enhanced by culture with TGF‐β, IL‐4, IL‐10, and anti‐IL‐12. Th3 CD4⁺ myelin basic protein regulatory clones are structurally identical to Th1 encephalitogenic clones in TCR usage, MHC restriction and epitope recognition, but produce TGF‐β with various amounts of IL‐4 and IL‐10. Because Th3 regulatory cells are triggered in an antigen‐specific fashion but suppress in an antigen‐non‐specific fashion, they mediate “bystander suppression” when they encounter the fed autoantigen at the target organ. In vivo induction of Th3 cells and low dose oral tolerance is enhanced by oral administration of IL‐4. Anti‐CD86 but not anti‐CD80 blocks the induction of Th3 cells associated with low dose oral tolerance. Th3 regulatory cells have been described in other systems (e.g. recovery from experimental allergic encephalomyelitis) but may be preferentially generated following oral antigen administration due to the gut immunologic milieu that is rich in TGF‐β and has a unique class of dendritic cells. CD4⁺CD25⁺ regulatory T‐cell function also appears related to TGF‐β.     

CD206‐positive myeloid cells bind galectin‐9 and promote a tumor‐supportive microenvironment

In patients with metastatic melanoma, high blood levels of galectin‐9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin‐9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin‐9 with macrophages. We aimed to determine whether galectin‐9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor‐supportive. It was determined that incubation of CD68⁺ macrophages with galectin‐9 or anti‐CD206 blocked target binding and that both CD206 and galectin‐9 were detected by immunoprecipitation of cell lysates. CD206 and galectin‐9 had a binding affinity of 2.8 × 10^?7 m . Galectin‐9 causes CD206⁺ macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP‐1), but less macrophage‐derived chemokine (MDC). Galectin‐9 had no effect on classical monocyte subsets, but caused expansion of the non‐classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin‐9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin‐9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti‐tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Sculpting MHC class II–restricted self and non‐self peptidome by the class I Ag‐processing machinery and its impact on Th‐cell responses

It is generally assumed that the MHC class I antigen (Ag)‐processing (CAP) machinery — which supplies peptides for presentation by class I molecules — plays no role in class II–restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition, TAP deficiency or ERAAP deficiency led to dramatically altered T helper (Th)‐cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4⁺ T‐cell repertoire, or both underlay the above finding. We found that TAP deficiency and ERAAP deficiency dramatically altered the quality of class II‐associated self peptides suggesting that the CAP machinery impacts class II–restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4⁺ T‐cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of WT animals resulting in altered CD4⁺ T‐cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II–restricted peptidome, impacting the CD4⁺ T‐cell repertoire, and ultimately altering Th‐cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II–restricted epitopes that would otherwise be capable of eliciting functional Th‐cell responses.     

GM‐CSF therapy inhibits chronic graft‐versus‐host disease via expansion of regulatory T cells

Regulatory T cells (Tregs) attenuate excessive immune responses, making their expansion beneficial in immune‐mediated diseases, including allogeneic bone marrow transplantation associated with graft‐versus‐host disease (GVHD). In addition to interleukin‐2, Tregs require T‐cell receptor and costimulatory signals from antigen‐presenting cells, such as DCs, for their optimal proliferation. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) increases DC number and may promote DC‐dependent Treg proliferation. Here, we demonstrate that GM‐CSF treatment increases CD4⁺CD8^– DCs, which are associated with Treg expansion. In a mouse model of chronic GVHD (cGVHD), GM‐CSF therapy expanded Tregs, protected against the development of skin GVHD, and regulated both Th1 and Th17 responses in the peripheral lymph nodes, resulting in an attenuation of skin cGVHD. Notably, the expanded Tregs were instrumental to GM‐CSF‐mediated cGVHD inhibition, which was dependent upon an increased ratio of Tregs to conventional T cells rather than augmentation of suppressive function. These data suggest that GM‐CSF induces Treg proliferation by expanding CD4⁺CD8^? DCs, which in turn regulate alloimmune responses in a cGVHD mouse model. Thus, GM‐CSF could be used as a therapeutic DC modulator to induce Treg expansion and to inhibit excessive alloimmune responses in immune‐related diseases.     

TREM‐2 Promotes Macrophage‐Mediated Eradication of Pseudomonas aeruginosa via a PI3K/Akt Pathway

Triggering receptor expressed on myeloid cells 2 (TREM‐2) is a cell surface receptor abundantly expressed on myeloid lineage cells such as macrophages and dendritic cells. It is reported that TREM‐2 functions as an inflammatory inhibitor in macrophages and dendritic cells. However, the role of TREM‐2 in bacterial killing remains unclear. This study explored the role of TREM‐2 in bacterial eradication of Pseudomonas aeruginosa (PA), a Gram‐negative bacterium which causes various opportunistic infections. Phagocytosis assay assessed by flow cytometry suggested that TREM‐2 was not involved in the uptake of PA by macrophages, while bacterial plate count data showed that TREM‐2 was required for macrophage‐mediated intracellular killing of PA. Moreover, our results demonstrated that TREM‐2 promoted macrophage killing by enhancing reactive oxygen species (ROS), but not nitric oxygen (NO) production. Treatment with N‐acetylcysteine, a ROS scavenger, diminished the TREM‐2‐mediated intracellular killing of PA. To further investigate the underlined mechanisms of TREM‐2‐promoted bacterial killing, we examined the activation of downstream mitogen‐activated protein kinases and PI3K/Akt pathway. Western blot data showed that silencing of TREM‐2 inhibited phosphorylation of Akt, but not ERK, JNK or P38. In addition, pretreatment with PI3K active product PIP3 DiC16 reversed the elevation of intracellular bacterial load in TREM‐2‐silenced macrophages, while PI3K inhibitor wortmannin restored the decline of bacterial load in TREM‐2‐overexpressed macrophages. These data together suggested that the TREM‐2‐mediated bacterial killing is dependent on the activation of PI3K/Akt signalling, which may provide a better understanding of the host antibacterial immune defence.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

T‐bet protects against exacerbation of schistosome egg‐induced immunopathology by regulating Th17‐mediated inflammation

C57BL/6 mice infected with Schistosoma mansoni naturally develop mild CD4⁺ T‐cell‐mediated immunopathology characterized by small hepatic granulomas around parasite eggs. However, immunization with soluble egg Ag in CFA markedly exacerbates the lesions by inducing a potent proinflammatory environment with high levels of IFN‐γ and IL‐17, which are signature cytokines of distinct Th1‐ versus Th17‐cell lineages. To determine the relative role of these subsets in disease exacerbation, we examined mice deficient in T‐bet (T‐bet^?/?), which is required for Th1 differentiation and IFN‐γ production. We now report that immunization with soluble egg Ag in CFA caused a significantly greater enhancement of egg‐induced hepatic immunopathology in T‐bet^?/? mice compared with WT controls, and analysis of their granulomas disclosed a higher proportion of activated DC and CD4⁺ T cells, as well as a marked influx of neutrophils. The absence of IFN‐γ in the T‐bet^?/? mice correlated with a marked increase in IL‐23p19, IL‐17 and TNF‐α in granulomas and MLN. In contrast, T‐bet^?/? mice had lower levels of IL‐4, IL‐5 and IL‐10 and a reduction in FIZZ1 and FoxP3 expression, suggesting diminished regulatory activity, respectively, by alternatively activated macrophages and Treg. These findings demonstrate that T‐bet‐dependent signaling negatively regulates Th17‐mediated immunopathology in severe schistosomiasis.     

Impaired NK‐cell education diminishes resistance to murine CMV infection

Ly49G2 (G2⁺) NK cells mediate murine (M)CMV resistance in MHC D^k‐expressing mice. Bone marrow transplantation (BMT) studies revealed that G2⁺ NK cell‐mediated MCMV resistance requires D^k in both hematopoietic and nonhematopoietic cells. As a Ly49G2 ligand, D^k in both cell lineages may contribute to lysis of virus‐infected cells. Alternatively, cellular differences in self‐MHC D^k may have affected NK‐cell education, and consequently NK cell‐mediated viral clearance. We investigated the D^k‐licensing effect on BM‐derived NK cells in BMT recipients by analyzing cytokines, cytotoxicity and MCMV resistance. In BMT recipients with lineage‐restricted D^k, G2⁺ NK‐cell reactivity and cytotoxicity was diminished in comparison to BMT recipients with self‐MHC in all cells. Reduced G2⁺ NK‐mediated MCMV resistance in BMT recipients with lineage‐restricted self‐MHC indicates that licensing of G2⁺ NK cells is related to NK‐cell reactivity and viral control. Titrating donor BM with self‐MHC‐bearing hematopoietic cells, as well as adoptive transfer of mature G2⁺ NK cells into BMT recipients with self‐MHC in non‐hematopoietic cells only, enhanced NK‐cell licensing and rescued MCMV resistance. This disparate self‐MHC NK‐cell education model would suggest that inadequately licensed NK cells corresponded to inefficient viral sensing and clearance.     

JNK2 modulates the CD1d‐dependent and ‐independent activation of iNKT cells

Invariant natural killer T (iNKT) cells play critical roles in autoimmune, anti‐tumor, and anti‐microbial immune responses, and are activated by glycolipids presented by the MHC class I‐like molecule, CD1d. How the activation of signaling pathways impacts antigen (Ag)‐dependent iNKT cell activation is not well‐known. In the current study, we found that the MAPK JNK2 not only negatively regulates CD1d‐mediated Ag presentation in APCs, but also contributes to CD1d‐independent iNKT cell activation. A deficiency in the JNK2 (but not JNK1) isoform enhanced Ag presentation by CD1d. Using a vaccinia virus (VV) infection model known to cause a loss in iNKT cells in a CD1d‐independent, but IL‐12‐dependent manner, we found the virus‐induced loss of iNKT cells in JNK2 KO mice was substantially lower than that observed in JNK1 KO or wild‐type (WT) mice. Importantly, compared to WT mice, JNK2 KO mouse iNKT cells were found to express less surface IL‐12 receptors. As with a VV infection, an IL‐12 injection also resulted in a smaller decrease in JNK2 KO iNKT cells as compared to WT mice. Overall, our work strongly suggests JNK2 is a negative regulator of CD1d‐mediated Ag presentation and contributes to IL‐12‐induced iNKT cell activation and loss during viral infections.     

Macrophage‐mediated psoriasis can be suppressed by regulatory T lymphocytes

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis‐like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro‐inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis‐like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF‐mediated psoriasis, ihTNFtg mice were crossbred into RAG1^KO mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1^KO or wild‐type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild‐type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3‐positive cells into RAG1^KO or wild‐type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro‐inflammatory activity of macrophages, which are the major immune effector cells in hTNF‐mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Artificial human antigen‐presenting cells are superior to dendritic cells at inducing cytotoxic T‐cell responses

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T‐cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I–peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T‐cell response, we generated artificial antigen‐presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic‐co‐glycolic) acid nanoparticles (PLGA‐NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen‐specific CTLs were then induced using either peptide‐loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen‐specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA‐A2‐positive tumour antigen‐bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA‐NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.     

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.