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.     

Depletion of CD4+CD25+ T cells switches the whey‐allergic response from immunoglobulin E‐ to immunoglobulin free light chain‐dependent

Background Symptoms of allergy are largely attributed to an IgE‐mediated hypersensitivity response. However, a considerable number of patients also exhibit clinical features of allergy without detectable systemic IgE. Previous work showed that Ig‐free light chains (IgLC) may act as an alternate mechanism to induce allergic responses. CD4⁺CD25⁺ T cells are crucial in the initiation and regulation of allergic responses and compromised function might affect the response to allergens. Objective To examine the contribution of CD4⁺CD25⁺ T cells and IgLC towards the whey‐allergic response. Methods Mice were sensitized orally with whey using cholera toxin as an adjuvant. CD25⁺ T cells were depleted in vivo using a CD25 mAb. The acute allergic skin response to whey and ex vivo colon reactivity was measured in the presence or absence of F991, a specific inhibitor of IgLC. Serum whey‐specific antibodies and IgLC in serum and mesenteric lymph node (MLN) supernatants were measured. Depletion of CD4⁺CD25⁺ T cells was confirmed in the spleen. Results Anti‐CD25 treatment strongly reduced whey‐specific antibody levels and resulted in a partial depletion of effector T cells and a major depletion of Foxp3⁺ regulatory T cells. Surprisingly, despite the abolished specific IgE response, the acute allergic skin response to whey was not affected. IgLC levels were enhanced in the serum and MLN supernatants of CD25‐depleted sensitized mice. F991 inhibited the acute skin response and colon hyperreactivity in anti‐CD25‐treated mice, indicating that these responses were mainly IgLC dependent. Conclusions Depletion of CD4⁺CD25⁺ T cells resulted in a switch from an IgE‐ to an IgLC‐dependent acute skin response and functional hyperresponsiveness of the colon. Our data suggest that CD25⁺ T cells play a crucial role in balancing cow's milk allergy between IgE and IgE‐independent responses and both mechanisms might play a role in allergic responses to the same allergen. Cite this as: B. C. A. M. van Esch, B. Schouten, B. R. J. Blokhuis, G. A. Hofman, L. Boon, J. Garssen L. M. J. Knippels, L. E. M. Willemsen and F. A. Redegeld, Clinical & Experimental Allergy, 2010 (40) 1414–1421.     

Phenotypic analysis of perennial airborne allergen‐specific CD4+ T cells in atopic and non‐atopic individuals

Background Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4⁺ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. Objective To determine the frequency, differentiation phenotype and function of circulating Fel d 1‐specific CD4⁺ T cells in adult individuals with severe persistent AD in comparison with healthy controls. Methods Using HLA class II tetrameric complexes based on a HLA‐DPB1^*0401‐restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL‐4 and IFN‐γ ELISpots. Results Ex vivo Fel d 1‐specific DPB1^*0401‐restricted CD4⁺ T cells in both atopics and non‐atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL‐4 production from the cells derived from atopics, which correlated with disease severity. Conclusions and Clinical Relevance Circulating Fel d 1‐specific DPB1^*0401‐restricted CD4⁺ T cells in both atopic and non‐atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals. Cite this as: L. R. Crack, H. W. Chan, T. McPherson and G. S. Ogg, Clinical & Experimental Allergy, 2011 (41) 1555–1567.     

Role of Treg in immune regulation of allergic diseases

Allergy is a Th2‐mediated disease that involves the formation of specific IgE antibodies against innocuous environmental substances. The prevalence of allergic diseases has dramatically increased over the past decades, affecting up to 30% of the population in industrialized countries. The understanding of mechanisms underlying allergic diseases as well as those operating in non‐allergic healthy responses and allergen‐specific immunotherapy has experienced exciting advances over the past 15 years. Studies in healthy non‐atopic individuals and several clinical trials of allergen‐specific immunotherapy have demonstrated that the induction of a tolerant state in peripheral T cells represent a key step in healthy immune responses to allergens. Both naturally occurring thymus‐derived CD4⁺CD25⁺FOXP3⁺ Treg and inducible type 1 Treg inhibit the development of allergy via several mechanisms, including suppression of other effector Th1, Th2, Th17 cells; suppression of eosinophils, mast cells and basophils; Ab isotype change from IgE to IgG4; suppression of inflammatory DC; and suppression of inflammatory cell migration to tissues. The identification of the molecules involved in these processes will contribute to the development of more efficient and safer treatment modalities.     

Allergen‐specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression

Background A sensitive measurement of low numbers of intracellular cytokine‐expressing antigen‐specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy. Objective To evaluate the applicability of this method for measuring changes in cytokine production by allergen‐specific T cells in a clinical trial setting. Methods Ex vivo ragweed‐specific CD154 and intracellular cytokine expression were evaluated using a subset of subjects in an environmental chamber study of allergic rhinitis immunotherapy. PBMC were collected and cryopreserved from Amb a 1‐immunostimulatory oligodeoxynucleotide conjugate (AIC)‐treated (n=17) and placebo‐treated (n=15) ragweed‐allergic subjects both after pre‐ and post‐treatment ragweed exposures. In vitro allergen‐stimulated CD3⁺CD4⁺CD154⁺ T cell intracellular IL‐4, IL‐5, IL‐13, and IFN‐γ expression were evaluated by flow cytometry. Results Compared with the T helper type 2 (Th2) cytokine expression measured after pre‐treatment ragweed exposures, placebo‐treated subjects demonstrated a significantly elevated ragweed‐ and Amb a 1‐specific T cell IL‐4 and IL‐13 co‐expression (P=0.005 and P=0.022, respectively) and a significantly elevated ragweed‐specific IL‐5 expression (P<0.001) following post‐treatment ragweed exposures. In contrast, AIC‐treated subjects demonstrated no increases in allergen‐specific Th2 cytokine expression following post‐treatment ragweed exposures. IFN‐γ expression remained low and un‐changed in both groups. Subject reported total nasal symptom scores demonstrated modest but significant correlations with Amb a 1‐ and ragweed‐stimulated intracellular Th2 cytokine responses. Conclusion Combined CD154 and intracellular cytokine staining in PBMC can be used to sensitively monitor changes in antigen‐specific T cell subset frequencies in clinical studies. Antigen‐specific cytokine expression moderately correlated with the reported levels of allergic symptoms. Trial Registration NCT00537355 Cite this as: J. D. Campbell, P. Buchmann, S. Kesting, C. R. Cunningham, R. L. Coffman and E. M. Hessel, Clinical & Experimental Allergy, 2010 (40) 1025–1035.     

The impact of HLA class I and EBV latency‐II antigen‐specific CD8+ T cells on the pathogenesis of EBV+ Hodgkin lymphoma

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein–Barr virus (EBV) latency‐II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV⁺cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA‐A*02 is protective in EBV⁺cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA‐A*02^– versus HLA‐A*02⁺ EBV⁺cHL patients, suggesting that LMP2A‐specific CD8⁺ T cell anti‐tumoral immunity may be relatively ineffective in HLA‐A*02^– EBV⁺cHL. To ascertain the impact of HLA class I on EBV latency antigen‐specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV⁺cHL, the magnitude of ex‐vivo LMP1/2A‐specific CD8⁺ T cell responses was elevated in HLA‐A*02⁺ patients. Furthermore, in a controlled in‐vitro assay, LMP2A‐specific CD8⁺ T cells from healthy HLA‐A*02 heterozygotes expanded to a greater extent with HLA‐A*02‐restricted compared to non‐HLA‐A*02‐restricted cell lines. In an extensive analysis of HLA class I‐restricted immunity, immunodominant EBNA3A/3B/3C‐specific CD8⁺ T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A‐specific responses were confined largely to HLA‐A*02. Our results demonstrate that HLA‐A*02 mediates a modest, but none the less stronger, EBV‐specific CD8⁺ T cell response than non‐HLA‐A*02 alleles, an effect confined to EBV latency‐II antigens. Thus, the protective effect of HLA‐A*02 against EBV⁺cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency‐II antigen‐specific CD8⁺ T cell hierarchies.     

Blomia tropicalis allergen 5 (Blo t 5) T‐cell epitopes and their ability to suppress the allergic immune response

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T‐cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T‐cell epitopes identified using the interferon‐γ ELISPOT assay with 15‐mer overlapping peptides. C57BL/6 mice were sensitized with bone‐marrow‐derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H‐2^b restricted epitopes (Bt5_76–90 and Bt5_106–115) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5‐pulsed BMDC and challenged with intranasal Blo t 5 Bt5_76–90 and Bt5_106–115, peptide‐specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin‐5 and interleukin‐13. Intradermal administration of synthetic peptides encoding the identified T‐cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T‐cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T‐cell response in a murine model of allergic airway inflammation.     

Depletion of CD4+CD25+CD127lo regulatory T cells does not increase allergen‐driven T cell activation

Background It has been suggested that allergic diseases are caused by defective suppression of allergen‐specific Th2 cells by CD4⁺CD25⁺ regulatory T cells. However, such studies have been hampered by the difficulty in distinguishing regulatory T cells from CD25‐expressing activated T cells. Recently, it was shown that conventional T cells expressed high levels of CD127, whereas regulatory T cells were CD127^lo, allowing discrimination between these distinct T cell subpopulations. Objective The aim of this study was to study whether the putative regulatory subset defined as CD4⁺CD25⁺CD127^lo was involved in grass pollen‐reactive T cell responses. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from allergic donors and non‐atopic controls out of season. Grass pollen‐induced cytokine production and proliferation were compared in cultures of undepleted cells and cells depleted of CD4⁺CD25⁺, CD4⁺CD25⁺CD127^hi or CD4⁺CD25⁺CD127^lo T cells. Results Undepleted cell cultures from allergic patients showed significantly increased proliferation and Th2 cytokine production compared with non‐atopic controls. Depletion of all CD25⁺ T cells did not increase cytokine production or proliferation, and more importantly, no increase in Th2 cytokine production or proliferation was observed in cell cultures depleted of CD4⁺CD25⁺CD127^lo cells (putative regulatory T cells) compared with undepleted PBMCs in both the allergic and the non‐atopic group. Conclusion Our study showed that T cells from grass pollen‐allergic patients and non‐atopic controls responded very differently to grass pollen extract, but this difference could not be explained by differences in regulatory T cell function. Further studies are needed to understand the importance of regulatory T cells in allergy.     

Ligation of TLR7 on CD19+CD1dhi B cells suppresses allergic lung inflammation via regulatory T cells

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B‐cell population is defined as IL‐10‐producing CD19⁺CD1d^hi cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19⁺CD1d^hi B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19⁺CD1d^hi B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL‐10 reporter, Il10^?/? and Tlr7^?/‐ mice, we formally demonstrate that TLR7 ligation of CD19⁺CD1d^hi B cells increases their capacity to produce IL‐10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7‐elicited CD19⁺CD1d^hi B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3⁺ T regulatory cells in allergen‐sensitized mice, we show that that TLR7‐elicited CD19⁺CD1d^hi B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL‐10‐producing CD19⁺CD1d^hi B cells, which can suppress allergic lung inflammation via T regulatory cells.     

Distinct activation phenotype of a highly conserved novel HLA‐B57‐restricted epitope during dengue virus infection

Variation in the sequence of T‐cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T‐cell responses during second heterologous infections. We identified a highly conserved, novel, HLA‐B57‐restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57‐NS1_26–34‐specific CD8⁺ T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer‐positive T‐cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57‐NS1_26–34‐specific and other DENV epitope‐specific CD8⁺ T cells, as well as total CD8⁺ T cells, expressed an activated phenotype (CD69⁺ and/or CD38⁺) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope‐specific CD8⁺ T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen‐specific T‐cell activation.     

The CD4+ T‐cell epitope‐binding register is a critical parameter when generating functional HLA‐DR tetramers with promiscuous peptides

Detection of CD4⁺ T cells specific for tumor‐associated antigens is critical to investigate the spontaneous tumor immunosurveillance and to monitor immunotherapy protocols in patients. We investigated the ability of HLA‐DR^*1101 multimers to detect CD4⁺ T cells specific for three highly promiscuous MAGE‐A3 derived peptides: MAGE‐A3_191–205 (p39), MAGE‐A3_281–295 (p57) and MAGE‐A3_286–300 (p58). Tetramers stained specific CD4⁺ T cells only when loaded with p39, although all peptides activated the specific T cells when presented by plastic‐bound HLA‐DR^*1101 monomers. This suggested that tetramer staining ability was determined by the mode rather than the affinity of peptide binding to HLA‐DR^*1101. We hypothesized that peptides should bear a single P1 anchor residue to bind all arms of the multimer in a homogeneous register to generate peptide‐HLA‐DR conformers with maximal avidity. Bioinformatics analysis indicated that p39 contained one putative P1 anchor residue, whereas the other two peptides contained multiple ones. Designing p57 and p58 analogues containing a single anchor residue generated HLA‐DR^*1101 tetramers that stained specific CD4⁺ T cells. Producing HLA‐DR^*1101 monomers linked with the optimized MAGE‐A3 analogues, but not with the original epitopes, further improved tetramer efficiency. Optimization of CD4⁺ T‐cell epitope‐binding registers is thus critical to generate functional HLA‐DR tetramers.     

Presence of HLA‐DR Molecules and HLA‐DRB1 mRNA in Circulating CD4+ T Cells

The human major histocompatibility complex class II isotype HLA‐DR is currently used as an activation marker for T cells. However, whether an endogenous protein expression or a molecular acquisition accounts for the presence of HLA‐DR on T cells remains undetermined and still controversial. To further characterize this phenomenon, we compared several aspects of the presence of the HLA‐DR protein to the presence of associated mRNA (HLA‐DRB1), focusing on human T cells from peripheral blood of healthy individuals. Using a flow cytometric approach, we determined that the HLA‐DR observed on CD4⁺ T cells was almost exclusively cell surface‐associated, while for autologous CD19⁺ B cells, the protein could be located in the plasma membrane as well as in the cytoplasm. Moreover, negligible expression levels of HLA‐DRB1 were found in CD4⁺ T cells, using an HLA‐DRB1 allele‐specific qPCR assay. Finally, the presence of HLA‐DR was not confined to activated CD4⁺ and CD8⁺ T cells, as evaluated by the co‐expression of CD25. The functional role of the HLA‐DR molecule on T cells remains enigmatic; however, this study presents evidence of fundamental differences for the presence of HLA‐DR on T cells from HLA‐DR in the context of antigen‐presenting cells, which is a well‐known phenomenon. Although an inducible endogenous protein expression cannot be excluded for the T cells, our findings suggest that a re‐evaluation of the HLA‐DR as a T cells activation marker is warranted.     

Inhaled allergen-driven CD1c up-regulation and enhanced antigen uptake by activated human respiratory-tract dendritic cells in atopic asthma

Background Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory‐tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD1 antigen presentation by hRTDC may contribute to asthma pathogenesis. Objective To investigate the influence of hRTDC on the balance between atopy and allergic asthma in human subjects and to determine whether CD1 expression by hRTDC is modulated during asthmatic inflammation. Methods Sputum cells were induced from steroid‐naïve, allergen‐challenged and allergen‐naïve subjects (atopic asthmatics, atopic non‐asthmatics and non‐atopic controls). hRTDC were identified using monoclonal antibody labelling and analysis by flow cytometry. Results hRTDC stained HLA‐DR⁺ (negative for markers of other cell lineages) were predominantly myeloid and comprised ∼0.5% of viable sputum cells. Sputum cells were potent stimulators of allogeneic CD4⁺ naïve T cells and enrichment/depletion experiments correlated stimulatory potency with DC numbers. Sputum contained cells that exhibited typical dendritic morphology when analysed by electron microscopy. Myeloid hRTDC were endocytically active, but uptake of FITC‐dextran was enhanced in cells from asthmatics (P<0.001). Despite their increased endocytic capacity, asthmatic myeloid hRTDC appeared mature and expressed increased levels of maturation markers (P<0.05–P<0.001), CD1c, CD1d and langerin (P<0.05). CD1c expression by asthmatic myeloid hRTDC was enhanced upon in vivo allergen challenge (three to ninefold within 24 h; P<0.05). CD11c⁻CD123^high hRTDC were only detected in asthmatic sputum and were increased in number following allergen challenge. Conclusion Despite limited cell numbers, it proved possible to analyse human RTDC in induced sputum, providing evidence that increased antigen uptake and enhanced CD1 presentation by activated hRTDC may contribute to allergic airway disease. CD1 presentation by hRTDC in atopic asthma may therefore constitute a novel target for future intervention strategies.     

Induction of allergic inflammation in the lungs of sensitized sheep after local challenge with house dust mite

Background Previous sheep models of asthma are based on sheep sensitized to nematode (Ascaris) allergens and these have been used to evaluate the physiological and pharmacological effects of potential anti‐asthma agents. The immunological mechanisms associated with the allergic response in sheep lungs has not been examined in detail. Objective To develop an experimental sheep model of allergic lung inflammation based on a relevant major human allergen, house dust mite, and to define the immunological features of the allergic response in this model. Methods Sheep immunized subcutaneously with solubilized house dust mite extract were given a single bronchial challenge with house dust mite. Bronchoalveolar lavage (BAL) and peripheral blood leucocytes were collected before and after challenge for flow cytometry, and tissue samples were taken post‐mortem (48 h post‐challenge) for histology and immunohistochemical analyses. Results Immunizations with 50 μg house dust mite induced an allergen‐specific IgE response in 50 to 60% of sheep (allergic sheep), with higher antigen doses increasing specific IgG₁ but not IgE. Lung challenge of allergic sheep with house dust mite led to the initial recruitment of neutrophils (at 6 h post‐challenge) followed by eosinophils and activated lymphocytes into the lung tissue and BAL, similar to the late‐phase allergic response seen in human asthma. Eosinophil recruitment peaked at 48 h post‐challenge, representing 10 to 33% of BAL leucocytes in allergen‐challenged allergic sheep compared to 0 to 3% in allergen‐challenged control (naïve) sheep. Lymphocytes recovered from the lung after allergen challenge were enriched for CD4⁺ T cells and were more activated than lymphocytes in blood. There was significant down‐regulation of CD62L (L‐selectin) and CD49d (VLA‐4) expression after allergen challenge on BAL eosinophils and lymphocytes compared to blood. In addition, VCAM‐1 (ligand for VLA‐4) was up‐regulated on blood vessels of allergen‐challenged lungs. Eosinophils, CD4⁺ T cells and CD45R⁺ B cells were the most prominent leucocytes found in lung tissue 48 h after allergen challenge. Conclusion This study demonstrates, for the first time, the ability of house dust mite to induce allergic responses in sheep lungs. This novel sheep model of allergic lung inflammation using relevant human allergens, exhibits similarities to human asthmatic disease and will be a useful tool for studies of the immunological and physiological mechanisms of allergic asthma.     

Staphylococcus aureus convert neonatal conventional CD4+ T cells into FOXP3+ CD25+ CD127low T cells via the PD‐1/PD‐L1 axis

The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4⁺ T cells for the induction of CD25⁺ CD127^low Treg cells in vitro. The proportion of circulating CD25⁺ CD127^low Treg cells and their expression of FOXP3, Helios and CTLA‐4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet‐stained non‐Treg cells from cord or peripheral blood from adults were co‐cultured with autologous CD25⁺ CD127^low Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25⁺ CD127^low Treg cells than adults, but these cells were Helios⁺ and CTLA‐4⁺ to a higher extent than in adults. FOXP3⁺ CD25⁺ CD127^low T cells were induced mainly in neonatal CellTrace‐stained non‐Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25⁺ CD127^low T cells only if sorted naive CD45RA⁺ non‐Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25⁺ CD127^low T cells from S. aureus‐stimulated cultures were still suppressive. Finally, blocking PD‐L1 during stimulation reduced the induction of FOXP3⁺ CD25⁺ CD127^low T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios⁺ Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4⁺ T cells into FOXP3⁺ CD25⁺ CD127^low Treg cells via the PD‐1/PD‐L1 axis.     

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.     

Lack of Mycobacterium tuberculosis–specific interleukin‐17A–producing CD4+ T cells in active disease

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb‐specific CD4⁺ T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL‐17A, IFN‐γ, TNF‐α, and IL‐2 by Mtb‐specific CD4⁺ T cells was assessed both directly ex vivo and following in vitro antigen‐specific T‐cell expansion. Unlike for extracellular bacteria, Mtb‐specific CD4⁺ T‐cell responses lacked immediate ex vivo IL‐17A effector function in both LTBI and TB individuals. Furthermore, Mtb‐specific Th17 cells were absent in BALs, while extracellular bacteria‐specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb‐specific CD4⁺ T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL‐17A following Mtb‐specific T‐cell expansion. Finally, IL‐17A acquisition by Mtb‐specific CD4⁺ T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb‐specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.