Glatiramer acetate attenuates the pro‐migratory profile of adhesion molecules on various immune cell subsets in multiple sclerosis

An altered expression pattern of adhesion molecules (AM) on the surface of immune cells is a premise for their extravasation into the central nervous system (CNS) and the formation of acute brain lesions in multiple sclerosis (MS). We evaluated the impact of glatiramer acetate (GA) on cell‐bound and soluble AM in the peripheral blood of patients with relapsing–remitting MS (RRMS). Fifteen patients treated de novo with GA were studied on four occasions over a period of 12 months. Surface levels of intracellular cell adhesion molecule (ICAM)‐1, ICAM‐3, lymphocyte function‐associated antigen (LFA)‐1 and very late activation antigen (VLA)‐4 were assessed in T cells (CD3⁺CD8⁺, CD3⁺CD4⁺), B cells, natural killer (NK) cells, natural killer T cells (NK T) and monocytes by five‐colour flow cytometry. Soluble E‐selectin, ICAM‐1, ICAM‐3, platelet endothelial cell adhesion molecule (PECAM)‐1, P‐selectin and vascular cell adhesion molecule (VCAM)‐1 were determined with a fluorescent bead‐based immunoassay. The pro‐migratory pattern in RRMS was verified by comparison with healthy controls and was characterized by up‐regulation of LFA‐1 (CD3⁺CD4⁺ T cells, B cells), VLA‐4 (CD3⁺CD8⁺ T cells, NK cells), ICAM‐1 (B cells) and ICAM‐3 (NK cells). Effects of GA treatment were most pronounced after 6 months and included attenuated levels of LFA‐1 (CD3⁺CD4⁺) and VLA‐4 (CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NK T, monocytes). Further effects included lowering of ICAM‐1 and ICAM‐3 levels in almost all immune cell subsets. Soluble AM levels in RRMS did not differ from healthy controls and remained unaltered after GA treatment. The deregulated pro‐migratory expression profile of cell‐bound AM is altered by GA treatment. While this alteration may contribute to the beneficial action of the drug, the protracted development and unselective changes indicate more secondary immune regulatory phenomena related to these effects.     

Programmed death‐1+ T cells inhibit effector T cells at the pathological site of miliary tuberculosis

Optimal T cell activation is vital for the successful resolution of microbial infections. Programmed death‐1 (PD‐1) is a key immune check‐point receptor expressed by activated T cells. Aberrant/excessive inhibition mediated by PD‐1 may impair host immunity to Mycobacterium tuberculosis infection, leading to disseminated disease such as miliary tuberculosis (MTB). PD‐1 mediated inhibition of T cells in pulmonary tuberculosis and TB pleurisy is reported. However, their role in MTB, particularly at the pathological site, remains to be addressed. The objective of this study was to investigate the role of PD‐1–PD‐ligand 1 (PD‐L1) in T cell responses at the pathological site from patients of TB pleurisy and MTB as clinical models of contained and disseminated forms of tuberculosis, respectively. We examined the expression and function of PD‐1 and its ligands (PD‐L1–PD‐L2) on host immune cells among tuberculosis patients. Bronchoalveolar lavage‐derived CD3 T cells in MTB expressed PD‐1 (54·2 ± 27·4%, P ≥ 0·0009) with significantly higher PD‐1 ligand‐positive T cells (PD‐L1: 19·8 ± 11·8%; P ≥ 0·019, PD‐L2: 12·6 ± 6·2%; P ≥ 0·023), CD19⁺ B cells (PD‐L1: 14·4 ± 10·4%; P ≥ 0·042, PD‐L2: 2·6 ± 1·43%; not significant) and CD14⁺ monocytes (PD‐L1: 40·2 ± 20·1%; P ≥ 0·047, PD‐L2: 22·4 ± 15·6%; P ≥ 0·032) compared with peripheral blood (PB) of MTB and healthy controls. The expression of PD‐1 was associated with a diminished number of cells producing effector cytokines interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, interleukin (IL)?2 and elevated apoptosis. Locally accumulated T cells were predominantly PD‐1⁺–PD‐L1⁺, and blocking this pathway restores the protective T cell response. We conclude that M. tuberculosis exploits the PD‐1 pathway to evade the host immune response by altering the T helper type 1 (Th1) and Th2 balance at the pathological site of MTB, thereby favouring disease dissemination.     

A three-cell model for activation of naïve T helper cells

It is generally assumed that the activation of naïve T helper (Th) cells is the result of a two‐cell interaction between the Th cell and a dendritic cell (DC) and that three signals are required. Signal one or stimulation is the recognition by the T‐cell receptor (TCR) of antigenic peptides presented by major histocompatibility complex (MHC) class II molecules. Signal two or co‐stimulation is mainly provided by the triggering of CD28 on the T cell by CD80 and CD86 molecules on the DC. Signal three or polarization directs T‐cell differentiation into various effector phenotypes such as Th1 and Th2. Both signals, two and three, are often assumed to result from the binding of microbial products or endogenous molecular danger signals to germline‐encoded receptors such as toll‐like receptors (TLR) on the DC. However, recent data challenge this two‐cell model by revealing that Th1 polarization requires the presence of interferon‐γ (IFN‐γ) provided by a third cell. I propose here a three‐cell model for naïve Th‐cell activation. In this model, delivery of signal three by the DC is dependent on help provided by other innate immune cells such as NK cells, NK T cells, γδ T cells, mast cells, eosinophils and basophils. The rationale behind this model is that the innate immune system has been designed by evolution to select an appropriate class of immune response to protect the host.     

Klebsiella pneumoniae‐triggered DC recruit human NK cells in a CCR5‐dependent manner leading to increased CCL19‐responsiveness and activation of NK cells

Besides their role in destruction of altered self‐cells, NK cells have been shown to potentiate T‐cell responses by interacting with DC. To take advantage of NK–DC crosstalk in therapeutic DC‐based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK‐cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5‐dependent manner. An additional mechanism of DC‐induced NK‐cell recruitment is characterized by the induction of CCR7 expression on CD56^dimCD16⁺ NK cells after physical contact with membrane fraction of K. pneumoniae‐matured DC, resulting in an enhanced migratory responsiveness to the lymph node‐associated chemokine CCL19. Bacterial fragment‐matured DC do not only mediate NK‐cell migration but also meet the prerequisites needed for augmentation of NK‐cell cytotoxicity and IFN‐γ production, the latter of which contributes to Th1 polarization.     

Natural killer cells become tolerogenic after interaction with apoptotic cells

NK cells are effectors in innate immunity and also participate in immunoregulation through the release of TGF‐β1 and lysis of activated/autoreactive T cells. Apoptotic cells (AC) have been shown to induce tolerogenic properties in innate immune cells, including macrophages and dendritic cells, but not NK cells. In this study, we demonstrated that after interaction with AC, NK cells released TGF‐β1, which in turn suppressed the production of IFN‐γ by NK cells upon IL‐12 and IgG activation. We further identified phosphatidylserine as a potential target on AC for the NK cells, as phosphatidylserine could stimulate NK cells to release TGF‐β1, which in turn suppressed CD4⁺ T‐cell proliferation and activation. Moreover, AC‐treated NK cells displayed cytotoxicity against autologous‐activated CD4⁺ T cells by upregulating NKp46. This lysis occurred in part through the NKp46‐vimentin pathway, as activated CD4⁺ T cells expressed vimentin on the cell surface and blocking of vimentin or NKp46, but not other NK‐cell receptors, significantly suppressed the NK‐cell cytotoxicity. We report here a novel interaction between NK cells and AC, resulting in the tolerogenic properties of NK cells required for immune contraction.     

Functional exhaustion of CD4+ T cells induced by co‐stimulatory signals from myeloid leukaemia cells

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T‐cell exhaustion. T‐cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co‐stimulatory molecules to T‐cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia‐derived co‐stimulatory signals on CD4⁺ T helper (Th) cell exhaustion, which may limit anti‐tumour immunity. Here, CD86 and inducible T‐cell co‐stimulator ligand (ICOS‐LG) co‐stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co‐cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro‐generated exhausted Th cells were defined by up‐regulation of programmed cell death 1 (PD‐1), cytotoxic T‐lymphocyte antigen 4 (CTLA‐4), lymphocyte activation gene 3 (LAG3) and T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3) inhibitory receptors. They were reluctant to proliferate upon re‐stimulation and produced reduced amounts of interleukin‐2 (IL‐2), tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ). Nonetheless, IL‐2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co‐stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD‐1, TIM‐3 and/or LAG3 was identified together with CD86⁺ and/or ICOS‐LG⁺ myeloid blasts. Collectively, co‐stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.     

NK cells modulate the lung dendritic cell‐mediated Th1/Th17 immunity during intracellular bacterial infection

The impact of the interaction between NK cells and lung dendritic cells (LDCs) on the outcome of respiratory infections is poorly understood. In this study, we investigated the effect and mechanism of NK cells on the function of LDCs during intracellular bacterial lung infection of Chlamydia muridarum in mice. We found that the naive mice receiving LDCs from C. muridarum‐infected NK‐cell‐depleted mice (NK‐LDCs) showed more serious body weight loss, bacterial burden, and pathology upon chlamydial challenge when compared with the recipients of LDCs from infected sham‐treated mice (NK+LDCs). Cytokine analysis of the local tissues of the former compared with the latter exhibited lower levels of Th1 (IFN‐γ) and Th17 (IL‐17), but higher levels of Th2 (IL‐4), cytokines. Consistently, NK‐LDCs were less efficient in directing C. muridarum‐specific Th1 and Th17 responses than NK+LDCs when cocultured with CD4⁺ T cells. In NK cell/LDC coculture experiments, the blockade of NKG2D receptor reduced the production of IL‐12p70, IL‐6, and IL‐23 by LDCs. The neutralization of IFN‐γ in the culture decreased the production of IL‐12p70 by LDCs, whereas the blockade of TNF‐α resulted in diminished IL‐6 production. Our findings demonstrate that NK cells modulate LDC function to elicit Th1/Th17 immunity during intracellular bacterial infection.     

Natural killer cells support the induction of protective immunity during dendritic cell-mediated vaccination against Leishmania major

Dendritic cell (DC)‐mediated vaccination against Leishmania major induces a parasite‐specific T helper 1 (Th1) response and long‐lasting protective immunity in susceptible mice. As the cytokine interleukin‐12 required for induction of this Th1 response is not derived from the transferred DC, but has to be produced by the vaccinated host, we examined cross‐presentation of transferred DC via resident DC of the host and cross‐activation with natural killer (NK) cells as mechanisms supporting the induction of protective immunity after DC‐mediated vaccination. Co‐culture with DC that had been conditioned ex vivo by loading with L. major lysate and stimulation with CpG‐containing oligodeoxynucleotides did not result in the activation of naive DC in vitro. Furthermore, L. major antigen from conditioned DC was not cross‐presented to a significant extent in vivo. In contrast, co‐culture of DC with NK cells led to cross‐activation of both cell populations with induction of interferon‐γ, which was dependent on the activation status of the conditioned DC. Transient depletion of NK cells during vaccination of L. major‐susceptible mice with conditioned DC resulted in reduced protection. Our findings indicate that cross‐presentation of conditioned DC after DC‐based vaccination against L. major plays a minor role in the induction of protective immunity. However, we demonstrated for the first time that the capacity of DC to mediate protection against L. major is supported by cross‐activation with NK cells of the host and NK‐cell‐derived interferon‐γ.     

The pro‐Th2 cytokine IL‐33 directly interacts with invariant NKT and NK cells to induce IFN‐γ production

IL‐33 has recently been identified as a cytokine endowed with pro‐Th2 functions, raising the question of its effect on invariant natural killer T cell (iNKT), which are potent IL‐4 producers. Here, we report a two‐fold increase of iNKT‐cell counts in spleen and liver after a 7‐day treatment of mice with IL‐33, which results from a direct effect, given that purified iNKT cells express the T1/ST2 receptor constitutively and respond to IL‐33 by in vitro expansion and functional activation. Conversely to the expected pro‐Th2 effect, IL‐33 induced a preferential increase in IFN‐γ rather than IL‐4 production upon TCR engagement that depended on endogenous IL‐12. Moreover, in combination with the pro‐inflammatory cytokine IL‐12, IL‐33 enhanced IFN‐γ production by both iNKT and NK cells. Taken together these data support the conclusion that IL‐33 can contribute as a co‐stimulatory factor to innate cellular immune responses.     

Regulation of immune responses by CD1d-restricted natural killer T cells

Natural killer T (NKT) cells are a unique subset of T lymphocytes that share receptor structures and properties with conventional T lymphocytes and natural killer (NK) cells. NKT cells are specific for glycolipid antigens such as the marine sponge-derived agent α-galactosylceramide (α-GalCer) presented by the major histocompatibility complex (MHC) class I-like molcule CD1d. My laboratory has evaluated the function of NKT cells by generating and analyzing CD1d-deficient mice. These studies showed that CD1d expression is required for NKT cell development, but not absolutely necessary for the generation of polarized T helper (Th) cell responses. Further, we have studied the in vivo response of NKT cells toα-GalCer stimulation and the capacity of α-GalCer to modulate innate and adaptive immune responses. Our results revealed that, quickly following administration of α-GalCer, NKT cells expand and produce cytokines, trans-activate a variety of innate and adaptive immune cells, and promote Th2 responses that are capable of suppressing Th1-dominant autoimmunity. Our findings indicate that NKT cells play a regulatory role in the immune response and that specific activation of these cells may be exploited for therapeutic purposes.     

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.     

Functional plasticity and robustness are essential characteristics of biological systems: Lessons learned from KLRG1‐deficient mice

Killer cell lectin‐like receptor G1 (KLRG1) receptor is considered to be a marker of terminally differentiated NK and T cells and is strongly induced by viral and other infections. KLRG1 is a C‐type lectin‐like inhibitory receptor, which interacts with members of the cadherin family of molecules leading to the inhibition of T‐ and NK‐cell function. A study in this issue of the European Journal of Immunology addresses the role of KLRG1 in the maturation and differentiation of NK and T cells in vivo. Using KLRG1‐deficient mice generated by homologous recombination, the study reveals that KLRG1 is dispensable for NK‐ and CD8⁺ T‐cell differentiation and function in vivo. This interesting finding is discussed in this Commentary in light of the plasticity and robustness of immune response mechanisms.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

Ganglioside-exposed dendritic cells inhibit T-cell effector function by promoting regulatory cell activity

Tumour pathogenesis is characterized by an immunosuppressive microenvironment that limits the development of effective tumour‐specific immune responses. This is in part the result of tumour‐dependent recruitment and activation of regulatory cells, such as myeloid‐derived suppressor cells and regulatory T cells in the tumour microenvironment and draining lymph nodes. Shedding of gangliosides by tumour cells has immunomodulatory properties, suggesting that gangliosides may be a critical factor in initiating an immunosuppressive microenvironment. To better define the immunomodulatory properties of gangliosides on antigen‐specific T‐cell activation and development we have developed an in vitro system using ganglioside‐treated murine bone‐marrow‐derived dendritic cells to prime and activate antigen‐specific CD4⁺ T cells from AND T‐cell receptor transgenic mice. Using this system, ganglioside treatment promotes the development of a dendritic cell population characterized by decreased CD86 (B7‐2) expression, and decreased interleukin‐12 and interleukin‐6 production. When these cells are used as antigen‐presenting cells, CD4 T cells are primed to proliferate normally, but have a defect in T helper (Th) effector cell development. This defect in Th effector cell responses is associated with the development of regulatory T‐cell activity that can suppress the activation of previously primed Th effector cells in a contact‐dependent manner. In total, these data suggest that ganglioside‐exposed dendritic cells promote regulatory T‐cell activity that may have long‐lasting effects on the development of tumour‐specific immune responses.