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Blockade of surface co-inhibitory receptor programmed cell death-1 (PD-1; CD279) has been established as an important immunotherapeutic approach to treat malignancies. On a cellular level, PD-1 is demonstrated to be of particular importance in inhibiting differentiation and effector function of cytotoxic Tc1 cells (CTLs). Nevertheless, the role of PD-1 in modulating interleukin (IL)-17-producing CD8+ T-cells (Tc17 cells), which generally display suppressed cytotoxic nature, is not well understood. To evaluate the impact of PD-1 in Tc17 responses, we examined its functioning using different in vitro and in vivo models. Upon activation of CD8+ T-cells in Tc17 environment, we found that PD-1 was rapidly expressed on the surface of CD8+ T-cells and triggered a T-cell-internal mechanism that inhibited the expression of IL-17 and Tc17-supporting transcription factors pSTAT3 and RORγt. Expression of type17-polarising cytokine IL-21 and the receptor for IL-23 were also suppressed. Intriguingly, adoptively transferred, PD-1-/- Tc17 cells were highly efficient in rejection of established B16 melanoma in vivo and displayed Tc1 like characteristics ex vivo. When using IL-17A-eGFP reporter mice for in vitro fate tracking, IL-17A-eGFP expressing cells lacking PD-1 signaling upon re-stimulation with IL-12 quickly acquired Tc1 characteristics such as IFN-γ, and granzyme B expression, implicating lineage independent upregulation of CTL-characteristics that are needed for tumor control. In line with plasticity characteristics, absence of PD-1 signaling in Tc17 cells increased the expression of the stemness and persistence-associated molecules TCF1 and BCL6. Thus, PD-1 plays a central role in the specific suppression of Tc17 differentiation and its plasticity in relation to CTL-driven tumor rejection, which provides further explanation as to why the blockade of PD-1 is such an efficient therapeutic target for inducing tumor rejection.

Atopic diseases in childhood are a major burden worldwide and there is still a lack of knowledge about treatable causes. In industrialized countries such as Germany, almost every second child is sensitized to at least one common allergen. Recent studies show that although the predisposition to allergies is inherited, the adaptive immune system of neonates and infants follows a developmental trajectory and whether an allergy actually occurs depends also on timing of allergen exposure including diet as well as environmental factors. New recommendations are far from being rigid of allergen avoidance; it is rather moving toward conditions that stand for more biodiversity. The observation that introduction of peanuts or eggs early in life significantly reduced the development of a later allergy will change our recommendations for the introduction of complementary foods. This is consistent with the hygiene hypothesis that early provocation shapes the developing immune system so that it reacts appropriately. Therefore, promoting the development of tolerance is at the heart of sensible allergy prevention - and this begins with the last trimester of pregnancy. In light of this concept, actual recommendations are discussed.

The monoclonal antibody against CTLA-4, Ipilimumab, is a first-in-class immune-checkpoint inhibitor approved for treatment of advanced melanoma in adults but not extensively studied in children. In light of the fact that the immune response early in life differs from that of adults, we have applied a human in vitro model stimulating CD4+ T-cells from neonates, children (1–5 years), and adults antigen-specifically with Staphylococcus aureus (S. aureus) for assessment of CTLA-4 blockade early in life. We show that T-cell proliferation as well as frequencies of antigen-specific T-cells (CD40L+CD4+) were enhanced in neonatal T-cells upon CTLA-4 blockade showing a larger variance within the group (F-test p

The origin of human T-cell responses against fungal pathogens early in life is not clearly understood. Here, we show that antifungal T-cell responses are vigorously initiated within the first years of life against lysates and peptides of Candida albicans or Aspergillus fumigatus, presented by autologous monocytes. The neonatal responding T-cell pool consists of 20 different TCR-Vβ families, whereas infant and adult pools display dramatically less variability. Although we demonstrate no bias for anti-fungal IL-4 expression early in life, there was a strong bias for anti-fungal IL-17 production. Of note, only T-cells from neonates and infants show an immediate co-expression of multiple cytokines. In addition, only their T-cells co-express simultaneously transcription factors T-bet and RORγt in response to fungi and subsequently their target genes IL-17 and IFNγ. Thus, T-cells of neonates and infants are predetermined to respond quickly with high plasticity to fungal pathogens, which might give an excellent opportunity for therapeutic interventions.

Although CD8+ T cells that produce IL-17 (Tc17 cells) have been linked to host defense, Tc17 cells show reduced cytotoxic activity, which is the characteristic function of CD8+ T cells. Here, we show that CTLA-4 enhances the frequency of IL-17 in CD8+ T cells, indicating that CTLA-4 (CD152) specifically promotes Tc17 differentiation. Simultaneous stimulation of CTLA-4+/+ and CTLA-4−/− T cells in cocultures and agonistic CTLA-4 stimulation unambiguously revealed a cell-intrinsic mechanism for IL-17 control by CTLA-4. The quality of CTLA-4-induced Tc17 cells was tested in vivo, utilizing infection with the facultative intracellular bacterium Listeria monocytogenes (LM). Unlike CTLA-4+/+ Tc17 cells, CTLA-4−/− were nearly as efficient as Tc1 CTLA-4+/+ cells in LM clearance. Additionally, adoptively transferred CTLA-4−/− Tc17 cells expressed granzyme B after rechallenge, and produced Tc1 cytokines such as IFN-γ and TNF-α, which strongly correlate with bacterial clearance. CTLA-4+/+ Tc17 cells demonstrated a high-quality Tc17 differentiation program ex vivo, which was also evident in isolated IL-17-secreting Tc17 cells, with CTLA-4-mediated enhanced upregulation of Tc17-related molecules such as IL-17A, RORγt, and IRF-4. Our results show that CTLA-4 promotes Tc17 differentiation that results in robust Tc17 responses. Its inactivation might therefore represent a central therapeutic target to enhance clearance of infection.

As the blockade of inhibitory surface-molecules such as CTLA-4 on T cells has led to recent advances in antitumor immune therapy, there is great interest in identifying novel mechanisms of action of CD8+ T cells to evoke effective cytotoxic antitumor responses. Using in vitro and in vivo models, we investigated the molecular pathways underlying the CTLA-4-mediated differentiation of IL-17-producing CD8+ T cells (Tc17 cells) that strongly impairs cytotoxicity. Our studies demonstrate that Tc17 cells lacking CTLA-4 signaling have limited production of STAT3-target gene products such as IL-17, IL-21, IL-23R and RORγt. Upon re-stimulation with IL-12, these cells display fast downregulation of Tc17 hallmarks and acquire Tc1 characteristics such as IFNγ and TNF-α co-expression, which is known to correlate with tumor control. Indeed, upon adoptive transfer, these cells were highly efficient in the antigen-specific rejection of established OVA-expressing B16 melanoma in vivo. Mechanistically, in primary and re-stimulated Tc17 cells, STAT3 binding to the IL-17 promoter was strongly augmented by CTLA-4, associated with less binding of STAT5 and reduced relative activation of STAT1 which is known to block STAT3 activity. Inhibiting CTLA-4-induced STAT3 activity reverses enhancement of signature Tc17 gene products, rendering Tc17 cells susceptible to conversion to Tc1-like cells with enhanced cytotoxic potential. Thus, CTLA-4 critically shapes the characteristics of Tc17 cells by regulating relative STAT3 activation, which provides new perspectives to enhance cytotoxicity of antitumor responses.

Tc17 cells are known to express low IFN-γ and granzyme B, resulting in diminished cytotoxicity. Adoptive transfer into Ag-bearing hosts converts them partially towards IFN-γ producing phenotype retaining Tc17 characteristics like increased persistence of survival. Nevertheless molecular mechanisms involved in Tc17 lineage plasticity and stability are yet to be identified. CTLA-4, a homologue to co-stimulatory molecule CD28, diminishes cytotoxicity of Tc1 cells by selective downregulation of GranzymeB, Eomes, and IFN-γ production, but its impact on Tc17 differentiation and plasticity is not known. CTLA-4+/+ and −/− CD8+ T cells were stimulated under Tc17 conditions. Tc1 and Tc17 characteristics in these cells were analysed by flow cytometry, western blot and real-time-PCR. For plasticity studies, cells were re-stimulated and analyzed for expression of Tc17 and Tc1 characteristics. Listeria Monocytogenes(LM) infection model and B16 melanoma model were used to investigate the effects of CTLA-4 on Tc17 cell mediated clearance of infection and controlling tumor growth. CTLA-4 showcased a cell intrinsic ability to enhance Tc17 differentiation program, by sustaining the levels of IL-17 inducing factors like STAT3, RORγ T and IRF4. Adoptively transferred Tc17 cells lacking CTLA-4 signal were more effective in clearing LM infection and controlling tumor growth. Re-stimulated CTLA-4 incompetent Tc17 cells revealed that higher cytotoxicity of these cells is due to their enhanced plasticity nature to develop Tc1 characteristics. Inactivation of CTLA-4 could be a unique target for Tc17 cells, to enhance lineage plasticity and develop Tc1 characteristics with increased persistence of survival and immunity.