Allergen‐specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4⁺ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4⁺CD45RO⁺) and naïve (CD4⁺CD45RA⁺) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2_127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2_127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2_127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2_127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.     

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP. Objective Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation. Methods CD11c⁺DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4⁺T cells. Results CD11c⁺ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4⁺ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4⁺ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone. Conclusions These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.     

CD40 ligand‐expressing recombinant vaccinia virus promotes the generation of CD8+ central memory T cells

Central memory CD8⁺ T cells (T_CM) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8⁺ T_CM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8⁺ T_CM‐cell generation is challenging, and usually requires CD4⁺ CD40L⁺ T‐cell “help” during the priming of naïve CD8⁺ T cells. We have generated a replication incompetent CD40 ligand‐expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8⁺ T cells into T_CM specific for viral and tumor‐associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild‐type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4⁺ T cells, a single “in vitro” stimulation of naïve CD8⁺ T cells by rVV40L‐infected nonprofessional CD14⁺ antigen presenting cells promotes the rapid generation of viral or tumor associated antigen‐specific CD8⁺ T cells displaying T_CM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8⁺ mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

Emergence of a distinct HIV‐specific IL‐10‐producing CD8+ T‐cell subset with immunomodulatory functions during chronic HIV‐1 infection

Interleukin‐10 (IL‐10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL‐10 is upregulated throughout HIV‐1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL‐10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8⁺ T cells with a CD25^neg FoxP3^neg phenotype contributes substantially to IL‐10 production in response to HIV‐1 gag stimulation. The frequencies of gag‐specific IL‐10‐ and IFN‐γ‐producing T cells in ART‐naïve subjects were strongly correlated and the majority of these IL‐10⁺ CD8⁺ T cells co‐produced IFN‐γ; however, patients with a predominant IL‐10⁺/IFN‐γ^neg profile showed better control of viraemia. Depletion of HIV‐specific CD8⁺ IL‐10⁺ cells from PBMCs led to upregulation of CD38 on CD14⁺ monocytes together with increased IL‐6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL‐10⁺ population and was also induced by exposure of monocytes to HIV‐1 in vitro. Production of IL‐10 by HIV‐specific CD8⁺ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.     

Origins and functional basis of regulatory CD11c+CD8+ T cells

Previously, we showed that CD11c defines a novel subset of CD8⁺ T cells whose in vivo activity is therapeutic for arthritis; however, the mechanisms directing their development, identity of their precursors, and basis of their effector function remain unknown. Here, we show that the novel subset develops from CD11c^surface?CD8⁺ T cells and undergoes robust expansion in an antigen‐ and 4‐1BB (CD137)‐dependent manner. CD11c⁺CD8⁺ T cells exist in naïve mice (<3%) with limited suppressive activity. Once activated, they suppress CD4⁺ T cells in vivo and in vitro. Suppression of CD4⁺ by CD11c⁺CD8⁺ T cells is related to an increase in IDO activity induced in competent cells via the general control non‐derepressible‐2 pathway. CD11c⁺CD8⁺ T cells are refractory to the effect of IDO but constrict in a novel 1‐methyl D ,L ‐tryptophan‐dependent mechanism resulting in reversal of their suppressive effects. Thus, our data uncover, for the first time, the origin, development, and basis of the suppressive function of this novel CD11c⁺CD8⁺ T‐cell subpopulation that has many signature features of Treg.     

Direct recognition of LPS by human but not murine CD8+ T cells via TLR4 complex

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8⁺ T cells are also capable of doing so. We report here that naïve human CD8⁺ T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8⁺ T cells can then secrete high concentrations of IFN‐γ, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergise with IL‐12 to polarise the CD8⁺ T cells into cytotoxic T‐cell 1 (Tc1) that produce IFN‐γ but not IL‐4, with or without TCR activation. Moreover, CD8⁺CD45RO⁺ memory T cells constitutively expressed TLR4 and markedly enhanced IFN‐γ production when stimulated with LPS. In contrast, activated murine CD8⁺ T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8⁺ T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8⁺ T cells.     

A CD40/CD40L feedback loop drives the breakdown of CD8+ T‐cell tolerance following depletion of suppressive CD4+ T cells

Dendritic cells (DCs) are the key APCs not only for the priming of naïve T cells, but also for the induction and maintenance of peripheral T‐cell tolerance. We have recently shown that cognate interactions between Foxp3⁺ Tregs and steady‐state DCs are crucial to maintain the tolerogenic potential of DCs. Using DIETER mice, which allow the induction of antigen presentation selectively on DCs without altering their maturation status, we show here that breakdown of CD8⁺ T‐cell tolerance, which ensues after depletion of suppressive CD4⁺ T cells, is driven by a positive feedback loop in which autoreactive CD8⁺ T cells activate DCs via CD40. These data identify ligation of CD40 on DCs as a stimulus that promotes autoreactive T‐cell priming when regulatory T‐cell suppression fails and suggest that feedback from autoreactive T cells to DCs may contribute to the well‐documented involvement of CD40 in many autoimmune diseases.     

TLR5 functions as an endocytic receptor to enhance flagellin-specific adaptive immunity

Innate immune activation via TLR induces dendritic cell maturation and secretion of inflammatory mediators, generating favorable conditions for naïve T‐cell activation. Here, we demonstrate a previously unknown function for TLR5, namely that it enhances MHC class‐II presentation of flagellin epitopes to CD4⁺ T cells and is required for induction of robust flagellin‐specific adaptive immune responses. Flagellin‐specific CD4⁺ T cells expanded poorly in TLR5‐deficient mice immunized with flagellin, a deficiency that persisted even when additional TLR agonists were provided. Flagellin‐specific IgG responses were similarly depressed in the absence of TLR5. In marked contrast, TLR5‐deficient mice developed robust flagellin‐specific T‐cell responses when immunized with processed flagellin peptide. Surprisingly, the adaptor molecule Myd88 was not required for robust CD4⁺ T‐cell responses to flagellin, indicating that TLR5 enhances flagellin‐specific CD4⁺ T‐cell responses in the absence of conventional TLR signaling. A requirement for TLR5 in generating flagellin‐specific CD4⁺ T‐cell activation was also observed when using an in vitro dendritic cell culture system. Together, these data uncover an Myd88‐independent function for dendritic cell TLR5 in enhancing the presentation of peptides to flagellin‐specific CD4⁺ T cells.     

In CD28‐costimulated human naïve CD4+ T cells,I‐κB kinase controls the expression of cell cycle regulatory proteins via interleukin‐2‐independent mechanisms

Stimulation of naïve CD4⁺ T cells through engagement of the T‐cell receptor (TCR) and the CD28 co‐receptor initiates cell proliferation which critically depends on interleukin (IL)‐2 secretion and subsequent autocrine signalling via the IL‐2 receptor. However, several studies indicate that in CD28‐costimulated T cells additional IL‐2‐independent signals are also required for cell proliferation. In this study, using a neutralizing anti‐human IL‐2 antibody and two selective, structurally unrelated, cell‐permeable I‐κB kinase (IKK) inhibitors, BMS‐345541 and PS‐1145, we show that in human naïve CD4⁺ T cells stimulated through a short engagement of the TCR and the CD28 co‐receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin‐dependent kinase 2 (CDK2) and the stability of the F‐box protein S‐phase kinase‐associated protein 2 (SKP2) and its co‐factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL‐2‐independent 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.     

Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

Tracking antigen‐specific CD4+ T cells throughout the course of chronic Leishmania major infection in resistant mice

Primary Leishmania major infection typically produces cutaneous lesions that not only heal but also harbor persistent parasites. While the opposing roles of CD4⁺ T‐cell‐derived IFN‐γ and IL‐10 in promoting parasite killing and persistence have been well established, how these responses develop from naïve precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex class II (pMHCII) tetramers to investigate the endogenous, parasite‐specific primary CD4⁺ T‐cell response to L. major in mice resistant to infection. Maximal frequencies of IFN‐γ⁺ CD4⁺ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL‐10⁺ CD4⁺ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type‐1 (Th1) cells, not Foxp3⁺ regulatory T cells, were the chief producers of IL‐10 and were not exhausted. Therefore, tracking antigen‐specific CD4⁺ T cells revealed that IL‐10 production by Th1 cells is not due to persistent T‐cell antigen receptor stimulation, but rather driven by early antigen encounter at the site of infection.     

Mapping of the lingual immune system reveals the presence of both regulatory and effector CD4+ T cells

Background Sublingual immunotherapy (SLIT) is safe and reduces both symptoms and medication requirements in patients with type I respiratory allergies. Nonetheless, immune mechanisms underlying SLIT need to be further documented. Objective A detailed characterization of the lingual immune system was undertaken in mice, to investigate the presence of tolerogenic and pro‐inflammatory mechanisms. Methods Immune cells were characterized in lingual tissues from BALB/c mice using immunohistology and flow cytometry. Resident CD4⁺ T cells were sorted and toll‐like receptor (TLR) expression profiles as well as functional characterization were assessed by RT‐PCR, T cell suppressive assays and cytokine gene expression, respectively. Results Eosinophils and mast cells were only detected in submucosal tissues. No NK, NK‐T, γ/δ, CD8⁺ T cells, nor B‐lymphocytes were detected. Potential antigen presenting cells include various subsets of dendritic cells (CD207⁺ Langerhans cells, CD11b⁺CD11c⁺ myeloid cells and 120G8⁺ plasmacytoid DCs) together with F4/80⁺ macrophages. Noteworthy, both CD103^? and CD103⁺ CD4⁺ T cells expressing TLR2 and TLR4 receptors are present along the lamina propria, in vicinity of myeloid CD11b⁺CD11c^± dendritic cells. Such resident lingual CD4⁺ T lymphocytes comprise both suppressive T cells as well as cells with memory/effector functions (i.e. expressing IFNγ, IL4, IL10 and IL17 genes following stimulation), irrespective of the presence of the mucosal addressing marker CD103. Conclusion The sublingual route is pertinent to induce antigen‐specific tolerance, due to (i) limited numbers of pro‐inflammatory cells, rather located in submucosal tissues, (ii) co‐localization of APCs and resident CD4⁺ T cells with regulatory functions. Since the oral immune system can also elicit pro‐inflammatory effector responses, the cytokine milieu in which allergens are presented by sublingual APCs needs to be controlled during immunotherapy (e.g. with adjuvants) in order to favour tolerance over inflammation.     

Tumor‐specific CD4+ T cells maintain effector and memory tumor‐specific CD8+ T cells

Immunotherapies that augment antitumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor‐specific CD4⁺ T cells enhance CD8⁺ T‐cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of tyrosinase‐related protein 1‐specific CD4⁺ transgenic T cells‐CD4⁺ T cells and pmel‐CD8⁺ T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p ≤ 0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8⁺ T cells with tumor‐specific cytokine expression. When combined with CD4⁺ T cells, transfer of total (naïve and effector) or effector CD8⁺ T cells were highly effective, suggesting CD4⁺ T cells can help mediate therapeutic effects by maintaining function of activated CD8⁺ T cells. In addition, CD4⁺ T cells had a pronounced effect in the early posttransfer period, as their elimination within the first 3 days significantly (p < 0.001) reduced therapeutic efficacy. The CD8⁺ T cells recovered from mice treated with both CD8⁺ and CD4⁺ T cells had decreased expression of PD‐1 and PD‐1‐blockade enhanced the therapeutic efficacy of pmel‐CD8 alone, suggesting that CD4⁺ T cells help reduce CD8⁺ T‐cell exhaustion. These data support combining immunotherapies that elicit both tumor‐specific CD4⁺ and CD8⁺ T cells for treatment of patients with cancer.     

CD4+CD25+ Treg induction by an HSP60‐derived peptide SJMHE1 from Schistosoma japonicum is TLR2 dependent

Chronic schistosome infection results in the suppression of host immune responses, allowing long‐term schistosome survival and restricting pathology. Current theories suggest that Treg play an important role in this regulation. However, the mechanism of Treg induction during schistosome infection is still unknown. The aim of this study was to determine the mechanism behind the induction of CD4⁺CD25⁺ T cells by Schistosoma japonicum HSP60 (SjHSP60)‐derived peptide SJMHE1 as well as to elucidate the cellular and molecular basis for the induction of CD4⁺CD25⁺ T cells during S. japonicum infection. Mice immunized with SJMHE1 or spleen and LN cells from naïve mice pretreated with SJMHE1 in vitro all displayed an increase in CD4⁺CD25⁺ T‐cell populations. Release of IL‐10 and TGF‐β by SJMHE1 stimulation may contribute to suppression. Adoptively transferred SJMHE1‐induced CD4⁺CD25⁺ T cells inhibited delayed‐type hypersensitivity in BALB/c mice. Additionally, SJMHE1‐treated APC were tolerogenic and induced CD4⁺ cells to differentiate into suppressive CD4⁺CD25⁺ Treg. Furthermore, our data support a role for TLR2 in SJMHE1‐mediated CD4⁺CD25⁺ Treg induction. These findings provide the basis for a more complete understanding of the S. japonicum–host interactions that contribute to host homeostatic mechanisms, preventing an excessive immune response.