Jonathan H. DeLong

Differentiation stage determines pathologic and protective allergen-specific CD4+ T cell outcomes during specific immunotherapy

BACKGROUND The main obstacle to elucidating the role of CD4(+) T cells in allergen-specific immunotherapy (SIT) has been the absence of an adequately sensitive approach to directly characterize rare allergen-specific T cells without introducing substantial phenotypic modifications by means of in vitro amplification. OBJECTIVE We sought to monitor, in physiological conditions, the allergen-specific CD4(+) T cells generated during natural pollen exposure and during allergy vaccination. METHODS Alder pollen allergy was used as a model for studying seasonal allergies. Allergen-specific CD4(+) T cells were tracked and characterized in 12 subjects with alder pollen allergy, 6 nonallergic subjects, and 9 allergy vaccine-treated subjects by using peptide-MHC class II tetramers. RESULTS Allergen-specific CD4(+) T cells were detected in all of the subjects with alder pollen allergy and nonallergic subjects tested. Pathogenic responses--chemoattractant receptor homologous molecule expressed on T(H)2 lymphocytes (CRTH2) expression and T(H)2 cytokine production--are specifically associated with terminally differentiated (CD27(-)) allergen-specific CD4(+) T cells, which dominate in allergic subjects but are absent in nonallergic subjects. In contrast, CD27(+) allergen-specific CD4(+) T cells are present at low frequencies in both allergic and nonallergic subjects and reflect classical features of the protective immune response with high expression of IL-10 and IFN-γ. Restoration of a protective response during SIT appears to be due to the preferential deletion of pathogenic (CD27(-)) allergen-specific CD4(+) T cells accompanied by IL-10 induction in surviving CD27(+) allergen-specific CD4(+) T cells. CONCLUSIONS Differentiation stage divides allergen-specific CD4(+) T cells into 2 distinct subpopulations with unique functional properties and different fates during SIT.

Specific immunotherapy modifies allergen-specific CD4+ T-cell responses in an epitope-dependent manner

BACKGROUND Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T-cell epitope level is critical for the design of new allergy vaccine strategies. OBJECTIVE We sought to characterize allergen-specific T-cell responses linked with allergy or peripheral tolerance and to determine how CD4(+) T-cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy. METHODS Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy, and phenotype of the DR04:01-restricted TGP-specific T-cell responses in 10 subjects with grass pollen allergy, 5 nonatopic subjects, and 6 allergy vaccine-treated subjects was determined by using an ex vivo peptide-MHC class II tetramer approach. RESULTS CD4(+) T cells in allergic subjects are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. Allergen-specific immunotherapy was associated with preferential deletion of allergen-specific TH2 cells and without a significant change in the frequency of TH1/TR1 cells. CONCLUSIONS Preferential allergen-specific TH2 cell deletion after repeated high-dose antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy.

Frequency of Epitope-Specific Naive CD4+ T Cells Correlates with Immunodominance in the Human Memory Repertoire

The frequency of epitope-specific naive CD4+ T cells in humans has not been extensively examined. In this study, a systematic approach was used to examine the frequency of CD4+ T cells that recognize the protective Ag of Bacillus anthracis in both anthrax vaccine-adsorbed vaccinees and nonvaccinees with HLA-DRB1*01:01 haplotypes. Three epitopes were identified that had distinct degrees of immunodominance in subjects that had received the vaccine. Average naive precursor frequencies of T cells specific for these different epitopes in the human repertoire ranged from 0.2 to 10 per million naive CD4+ T cells, which is comparable to precursor frequencies observed in the murine repertoire. Frequencies of protective Ag-specific T cells were two orders of magnitude higher in immunized subjects than in nonvaccinees. The frequencies of epitope-specific memory CD4+ T cells in vaccinees were directly correlated with the frequencies of precursors in the naive repertoire. At the level of TCR usage, at least one preferred Vβ in the naive repertoire was present in the memory repertoire. These findings implicate naive frequencies as a crucial factor in shaping the epitope specificity of memory CD4+ T cell responses.

Direct ex vivo analysis of allergen-specific CD4+ T cells

To the Editor: CD4+ T cells play essential roles in allergic sensitization and late-phase reactions. Previous studies identified epitopes within allergens such as the cat allergen Fel d 1.1-5 However, in some cases, HLA restrictions were not clearly defined. Recent studies have applied tetramers to define HLA-restricted epitopes, studying subjects with cat and other allergies.4 In general, previous reports analyzed single, previously characterized epitopes and, with 1 exception,4 analyzed cells after in vitro expansion, which can alter cell phenotypes. Recent advances in MHC class II tetramer analysis enhance the efficiency of epitope identification and provide the sensitivity to examine phenotypes without in vitro expansion.6,7 We applied these methods to study allergen specific CD4+ T-cell responses in subjects with cat allergy. To identify novel T-cell epitopes within Fel d 1, the Tetramer-Guided Epitope Mapping (TGEM) approach was applied to multiple subjects with allergy recruited with informed consent from the Virginia Mason Medical Center Allergy Clinic and Benaroya Research Institute. Overlapping peptides corresponding to both chains of Fel d 1 were pooled and used to stimulate T-cell cultures as described.6 Each peptide pool was loaded into purified class II molecules to generate tetramers.8 After 14 days, cultured cells were stained with corresponding pooled peptide loaded tetramers. Positive wells were stained again using tetramers loaded with single peptides (see this article’s Fig E1 in the Online Repository at www.jacionline.org). Applying this approach, we identified novel Fel d 1 epitopes for 6 HLA types (Table I). Binding predictions9 and experiments using shorter peptides defined minimal epitopes. Responses to these Fel d 1 peptides were absent in subjects without allergy (Fig E1). Table I Fel d 1 T cell epitopes To determine frequencies for Fel d 1 specific T cells, we used a direct enrichment protocol, essentially as described by Day et al.7 For 9 subjects with allergy and 5 subjects without allergy, 20 to 30 million uncultured PBMCs were stained with phycoerythrin tetramers for 100 minutes. Cells were then stained with surface anti-bodies (anti-CD3 fluorescein isothiocyanate and anti-CD4 allophycocyanin from eBioscience, San Diego, Calif; anti-CD14 peridinin-chlorophyll-protein [PerCP] and anti-CD19 PerCP from BD Pharmingen, San Diego, Calif), incubated with anti-phycoerythrin magnetic beads (Miltenyi Biotec, Auburn, Calif), washed, and enriched by using a magnetic column (Miltenyi Biotec). Bound, phycoerythrin-labeled cells were stained with ViaProbe (BD Bioscience, San Jose, Calif), flushed, collected, and enumerated by flow cytometry (FACS Caliber cytometer, BD Bioscience; FlowJo software, Tree Star, Ashland, Ore). Frequencies were calculated using the following formula Frequency=n/N where n designates the number of tetramer positive cells in the bound fraction and N designates the total number of CD4+ T cells in the sample. Fel d 1–specific CD4+ T-cell frequencies in subjects with allergy ranged from 1 in 7,000 to 1 in 300,000 (Fig 1, A). Frequencies in subjects without allergy were barely detectable (Fig 1, B). Frequency measurements were reproducible in that frequencies for samples from the same subjects obtained at least 2 months apart were not significantly different (P = .495, paired t test with Welch correction). FIG 1 Ex vivo staining of Fel d 1 CD4+ T cells with tetramers. A, PBMCs from subjects with cat allergy were stained with phycoerythrin-labeled Fel d 1 tetramers (Tet) followed by the anti-phycoerythrin bead enrichment protocol. Each panel shows a representative ... To determine the phenotype of Fel d 1–specific T cells, parallel samples were analyzed by direct enrichment, using antibodies against surface markers of interest instead of anti-CD3. This allowed direct phenotyping with minimal manipulation. Pheno-typing was carried out for 5 subjects with allergy (representative results shown in Fig 1, C). A comparison of the phenotype of Fel d 1–specific T cells and total CD4+ T cells for all 5 subjects is summarized in Fig 1, D. As shown, more than 90% of Fel d 1–specific T cells were CD45RO, CD28, CD62L, and CCR4–positive. Most cells were CXCR3 and CCR6–negative but showed heterogeneous expression of CRTH2 and CCR7. In comparison with total CD4+ cells, a higher percentage of Fel d 1–specific T cells expressed CCR4 and CRTH2, and a lower percentage expressed CXCR3. As an internal control, we examined the phenotype of influenza A–specific T cells (not shown). Influenza-specific T cells were CD45RO and CD28–positive, but were CXCR3-positive, CCR4-negative, and CRTH2-negative. Thus, allergen-specific T cells show a distinct phenotype compared with influenza-specific T cells and the total CD4+ T-cell population. In support of these phenotyping experiments, we conducted cytokine analysis of directly enriched Fel d 1–specific T cells using Miltenyi capture reagents after stimulation with tetramer, anti-CD28 (10 μg/mL), and anti-CD49d (2 μg/mL) for 4 hours at 37°C. Although 10% to 50% of the tetramer-positive cells secreted IL-5, no IFN-γ was detected (Fig 1, E). IL-5 secretion was limited to the tetramer positive population. To examine additional cytokines, cultured cells were reactivated by using plate-bound tetramer and soluble anti-CD28. Supernatants were harvested after 48 hours and assayed for cytokine content by using the Meso Scale Sector Imager, Gaithersburg, Md. In addition to IL-5, Fel d 1–specific T cells produced TH2 cytokines such as IL-13 and IL-4 (not shown). Previous reviews and studies have highlighted the attractive features of using MHC class II tetramers to simultaneously determine T-cell frequencies and phenotypes. Since the arrival of class II tetramers, technical advances have increased the utility of these reagents. First, the availability of additional alleles enables the study of larger segments of the population. Second, TGEM facilitates rapid identification of T-cell epitopes.6 Third, enrichment methods allow direct ex vivo detection of antigen-specific T cells.7 This study applied these methods to visualize and characterize Fel d 1–specific T cells in subjects with allergy. We identified epitopes restricted by multiple HLA-DR alleles. We observed frequencies ranging from 1 in 7,000 to 1 in 300,000 CD4+ T cells in subjects with allergy. In subjects without allergy, allergen-specific T-cell frequencies were near back-ground levels. However, tetramers may be limited in their ability to detect low-affinity cells. As such, tetramer measurements may underestimate T-cell frequencies in some cases. In contrast with many published studies, we have directly examined the phenotype of allergen-specific T cells. Our observations indicated that nearly all Fel d 1–specific T cells (ex vivo) exhibit a memory phenotype. Fel d 1–specific T cells showed heterogeneous expression of CCR7, a marker thought to be upregulated on central memory and downregulated on effector memory cells. A previous report observed enriched CCR4 expression by allergen-specific T cells.4 Our results were more dramatic in that almost all allergen-specific T cells were CCR4-positive (25%-30% of total CD4+ T cells were CCR4-positive). Expression of CCR4 is notable because CCR4 is a TH2 marker that has been associated with trafficking to nonlymphoid sites, including the skin and airway mucosa. Thus, high levels of CCR4 expression may lead to rapid recruitment into relevant sites for allergic immune responses. In contrast with CCR4 expression, the prostaglandin D2 receptor CRTH2 (another TH2 marker)10 was expressed at variable frequencies (17%-88%) among subjects with cat allergy. Although variable, these frequencies were always higher than total CD4+ T cells. Regardless of CRTH2 expression level, tetramer-based cytokine assays indicated high levels of IL-5 and low levels of IFN-γ. These cytokine results reinforced the surface phenotype results. Cytokine levels were robust, which is typical of effector T cells. The absence of CXCR3 and CCR6 expression indicates that these peripheral cells do not belong to TH1 or TH17 lineages. However, these lineages could be present within specific tissues or during stages of allergy that were not reflected in our samples. In conclusion, we used class II tetramers to rapidly define HLA-restricted epitopes within allergens and characterize allergen-specific T cells ex vivo. The application of tetramers to study allergen-specific CD4+ T cells may facilitate the development of peptide biologics for allergy treatment. Future studies using tetramers after immunotherapy should clarify the underlying mechanisms for allergen-specific immune tolerance.

A phenotypically and functionally distinct human TH2 cell subpopulation is associated with allergic disorders

A unique T helper cell signature in allergic patients isolates the pathogenic cells and provides a target for disease intervention. Defining damaging cells Although T helper type 2 (TH2) cells provide necessary protection from certain types of pathogens, they are also implicated in allergy pathogenesis. Until now, methods to distinguish pathogenic cells that are reactive to allergens from the rest of the TH2 population were very limited. Wambre et al. characterized a population of memory TH2 cells, termed TH2A, that were only found in allergic individuals. They were able to do so without the use of antigen-specific tetramers. These cells decreased in patients that benefited from allergen immunotherapy, indicating that targeting TH2A cells could disrupt allergic responses. Allergen-specific type 2 helper T (TH2) cells play a central role in initiating and orchestrating the allergic and asthmatic inflammatory response pathways. One major factor limiting the use of such atopic disease–causing T cells as both therapeutic targets and clinically useful biomarkers is the lack of an accepted methodology to identify and differentiate these cells from overall nonpathogenic TH2 cell types. We have described a subset of human memory TH2 cells confined to atopic individuals that includes all allergen-specific TH2 cells. These cells are terminally differentiated CD4+ T cells (CD27− and CD45RB−) characterized by coexpression of CRTH2, CD49d, and CD161 and exhibit numerous functional attributes distinct from conventional TH2 cells. Hence, we have denoted these cells with this stable allergic disease–related phenotype as the TH2A cell subset. Transcriptome analysis further revealed a distinct pathway in the initiation of pathogenic responses to allergen, and elimination of these cells is indicative of clinical responses induced by immunotherapy. Together, these findings identify a human TH2 cell signature in allergic diseases that could be used for response-monitoring and designing appropriate immunomodulatory strategies.

Grass-specific CD4(+) T-cells exhibit varying degrees of cross-reactivity, implications for allergen-specific immunotherapy.

Conceptually, allergic responses may involve cross‐reactivity by antibodies or T‐cells. While IgE cross‐reactivity among grass‐pollen allergens has been observed, cross‐reactivity at the allergen‐specific T‐cell level has been less documented. Identification of the patterns of cross‐reactivity may improve our understanding, allowing optimization of better immunotherapy strategies.

CD4+ T cells recognize unique and conserved 2009 H1N1 influenza hemagglutinin epitopes after natural infection and vaccination

Influenza A/California/4/2009 (H1N1/09) is a recently emerged influenza virus capable of causing serious illness or death in otherwise healthy individuals. Serious outcomes were most common in young adults and children, suggesting that pre-existing heterologous immunity may influence the severity of infection. Using tetramers, we identified CD4(+) T-cell epitopes within H1N1/09 hemagglutinin (HA) that share extensive homology with seasonal influenza and epitopes that are unique to H1N1/09 HA. Ex vivo tetramer staining revealed that T cells specific for conserved epitopes were detectable within the memory compartment, whereas T cells specific for unique epitopes were naive and infrequent prior to infection or vaccination. Following infection, the frequencies of T cells specific for unique epitopes were 11-fold higher, reaching levels comparable to those of T cells specific for immunodominant epitopes. In contrast, the frequencies of T cells specific for conserved epitopes were only 2- to 3-fold higher following infection. In general, H1HA-reactive T cells exhibited a memory phenotype, expressed CXCR3 and secreted IFN-γ, indicating a predominantly Th1-polarized response. A similar Th1 response was seen in vaccinated subjects, but the expansion of T cells specific for HA epitopes was comparatively modest after vaccination. Our findings indicate that CD4(+) T cells recognize both strain-specific and conserved epitopes within the influenza HA protein and suggest that naive T cells specific for HA epitopes undergo significant expansion, whereas memory T cells specific for the conserved epitopes undergo more restrained expansion.

Myeloid cell microsomal prostaglandin E synthase-1 fosters atherogenesis in mice

Significance Inhibitors of microsomal prostaglandin E synthase-1 (mPGES-1) are being developed as analgesics. Although global depletion of mPGES-1 suggests they will be less likely to confer a cardiovascular hazard than NSAIDs selective for inhibition of COX-2, mPGES-1–derived PGE2 may have contrasting effects on discrete cellular components of the vasculature. mPGES-1 inhibition may result in substrate rediversion to other PG synthases, the products of which differ between cell types and exert contrasting biologies. Here, myeloid cell mPGES-1, reflective of the macrophage enzyme, promotes atherogenesis, fostering inflammation and oxidative stress. By contrast, mPGES-1 in endothelial and vascular smooth muscle cells has no discernable effect. Selective targeting of macrophage mPGES-1 may conserve cardiovascular benefit while avoiding adverse effects related to enzyme blockade in other tissues. Microsomal prostaglandin E synthase-1 (mPGES-1) in myeloid and vascular cells differentially regulates the response to vascular injury, reflecting distinct effects of mPGES-1–derived PGE2 in these cell types on discrete cellular components of the vasculature. The cell selective roles of mPGES-1 in atherogenesis are unknown. Mice lacking mPGES-1 conditionally in myeloid cells (Mac-mPGES-1-KOs), vascular smooth muscle cells (VSMC-mPGES-1-KOs), or endothelial cells (EC-mPGES-1-KOs) were crossed into hyperlipidemic low-density lipoprotein receptor-deficient animals. En face aortic lesion analysis revealed markedly reduced atherogenesis in Mac-mPGES-1-KOs, which was concomitant with a reduction in oxidative stress, reflective of reduced macrophage infiltration, less lesional expression of inducible nitric oxide synthase (iNOS), and lower aortic expression of NADPH oxidases and proinflammatory cytokines. Reduced oxidative stress was reflected systemically by a decline in urinary 8,12-iso-iPF2α-VI. In contrast to exaggeration of the response to vascular injury, deletion of mPGES-1 in VSMCs, ECs, or both had no detectable phenotypic impact on atherogenesis. Macrophage foam cell formation and cholesterol efflux, together with plasma cholesterol and triglycerides, were unchanged as a function of genotype. In conclusion, myeloid cell mPGES-1 promotes atherogenesis in hyperlipidemic mice, coincident with iNOS-mediated oxidative stress. By contrast, mPGES-1 in vascular cells does not detectably influence atherogenesis in mice. This strengthens the therapeutic rationale for targeting macrophage mPGES-1 in inflammatory cardiovascular diseases.

IL-27 Limits Type 2 Immunopathology Following Parainfluenza Virus Infection

Respiratory paramyxoviruses are important causes of morbidity and mortality, particularly of infants and the elderly. In humans, a T helper (Th)2-biased immune response to these infections is associated with increased disease severity; however, little is known about the endogenous regulators of these responses that may be manipulated to ameliorate pathology. IL-27, a cytokine that regulates Th2 responses, is produced in the lungs during parainfluenza infection, but its role in disease pathogenesis is unknown. To determine whether IL-27 limits the development of pathogenic Th2 responses during paramyxovirus infection, IL-27-deficient or control mice were infected with the murine parainfluenza virus Sendai virus (SeV). Infected IL-27-deficient mice experienced increased weight loss, more severe lung lesions, and decreased survival compared to controls. IL-27 deficiency led to increased pulmonary eosinophils, alternatively activated macrophages (AAMs), and the emergence of Th2 responses. In control mice, IL-27 induced a population of IFN-γ+/IL-10+ CD4+ T cells that was replaced by IFN-γ+/IL-17+ and IFN-γ+/IL-13+ CD4+ T cells in IL-27-deficient mice. CD4+ T cell depletion in IL-27-deficient mice attenuated weight loss and decreased AAMs. Elimination of STAT6 signaling in IL-27-deficient mice reduced Th2 responses and decreased disease severity. These data indicate that endogenous IL-27 limits pathology during parainfluenza virus infection by regulating the quality of CD4+ T cell responses and therefore may have therapeutic potential in paramyxovirus infections.

Interferon-related secretome from direct interaction between immune cells and tumor cells is required for upregulation of PD-L1 in tumor cells

PD-L1, also known as CD274, plays a vital role in tumor cell related immune escape. It can be expressed on the cell surface of many solid tumors (Brahmer et al., 2012) and inhibits T cell proliferation and cytokine production by binding to the Tcell surface receptor programmed death 1 (PD-1) or B7-1 (McClanahan et al., 2015). In 2013, targeting PD-1/ PD-L1 signaling for cancer immunotherapy was selected as the No.1 scientific breakthrough of the year by the editors of Science. Interferons (IFNs) are a group of pleiotropic cytokines, demonstrated anti-viral, anti-tumor, and immune regulatory functions (York et al., 2015). Type I interferon binds a heterodimeric receptor composed of IFNAR1 and IFNAR2. This activates a canonical JAK/STAT signaling pathway that ultimately induces a set of interferon-stimulated genes to exert its biological activity (Ejlerskov et al., 2015). Recently, PD-L1 was reported to be downstream of IFN signaling in human oral squamous carcinoma, melanoma, and human acute myeloid leukemia blast cells (Chen et al., 2012; Furuta et al., 2014; Kronig et al., 2014). The tumor microenvironment plays an important role in tumor growth and metastasis. Different components of the tumor microenvironment such as T cells, B cells, NK cells, dendritic cells, mast cells, granulocytes, Treg cells, myeloid derived suppressor cells (MDSC), and tumor associated macrophages (TAM) are recruited by different pathways (Joyce and Fearon, 2015). Tumor cells have been shown to upregulate PD-L1 after interacting with infiltrating immune cells (Cho et al., 2011; Hou et al., 2014), but the mechanism by which this occurs is not well understood. In this study, we found that PD-L1 upregulation in tumors was dependent on direct interaction with immune cells and was driven by a secreted factor such as type I interferon after cell-cell contact. Previous studies have demonstrated a positive correlation between tumor-infiltrating immune cells and elevated PD-L1 expression in tumor cells, but the mechanism by which this occurs is poorly understood. To investigate this, we co-cultured murine B16F10 melanoma cells with syngeneic splenocytes for 48 h. In addition, to determine whether direct cell contact is required for immune cell-mediated PD-L1 expression, the two types of cells were separated by a transwell-membrane that blocked their direct cell-cell interactions. Furthermore, another condition was tested in which B16F10 cells and immune cells were co-cultured in the plate and B16F10 cells were cultured in the transwell insert (Fig. 1A). Then the non-adherent immune cells were removed and B16F10 cells were harvested and analyzed for PD-L1 expression by flow cytometry. PD-L1 was more highly expressed in B16F10 cells that were co-cultured with splenocytes than in those cultured alone (Fig. 1B). However, PD-L1 expression was not increased in B16F10 cells separated from the splenocytes by a transwell membrane. We also found that a B16F10-splenocyte co-culture was able to induce PD-L1 in tumor cells separated from the co-culture by a transwell membrane (Fig. 1B). These effects were also observed in PD-L1 mRNA level changes by qPCR (Fig. 1C). These results suggested that active factors were secreted into the supernatant after the direct cell-cell interaction that was able to induce PD-L1 expression in tumor cells. To identify whether the regulation of PD-L1 was indeed driven by a secreted factor, B16F10 cells and splenocytes were co-cultured for 48 h. The supernatant was collected and centrifuged, and then used to treat B16F10 cells independently. The corresponding supernatant derived from B16F10 cells and splenocytes alone was also used to treat B16F10 cells as control groups (Fig. 1D). After 24 h, B16F10 cells treated with supernatant from the co-culture expressed more PD-L1 than cells treated with supernatant from the control mono-cultures (Fig. 1E and 1F). In addition, co-cultures of B16F10 cells with bone marrow (BM)-derived cells (Fig. 1G) or lymph node (LN)-derived cells also upregulated PD-L1 expression (Fig. 1H). To determine whether a similar effect would be seen in other types of cancer cells, additional studies on MC38 and Hepa1-6 cells