Characterizing the immune responses of those who survived or succumbed to COVID-19: Can immunological signatures predict outcome?

BACKGROUND: Immunodeficiency has pivotal role in the pathogenesis of coronavirus disease 2019 (COVID-19). Several studies have indicated defects in the immune system of COVID-19 patients at different disease stages. Therefore, this study investigated whether alters in immune responses of COVID-19 patients may be considered as predicting factors for disease outcome. METHODS: The percentages of innate and adoptive immune cells in the recovered and dead patients with COVID-19, and healthy subjects were determined by flow cytometry. The levels of pro- and anti-inflammatory cytokines and other immune factors were also measured by enzyme-linked immunosorbent assay. RESULTS: At the first day of hospitalization, the frequencies of CD56dim CD16+ NK cells and CD56bright CD16dim/- NK cells in patients who died during treatment were significantly increased compared to recovered and healthy individuals (P < 0.0001). The recovered and dead patients had a significant increase in monocyte number in comparison with healthy subjects (P < 0.05). No significant change was observed in Th1 cell numbers between the recovered and dead patients while Th2, Th17 cell, and Treg percentages in death cases were significantly lower than healthy control and those recovered, unlike exhausted CD4 + and CD8 + T cells and activated CD4 + T cells (P < 0.0001-0.05). The activated CD8 + T cell was significantly higher in the recovered patients than healthy individuals (P < 0.0001-0.05). IL-1α, IL-1ß, IL-6, and TNF-α levels in patients were significantly increased (P < 0.0001-0.01). However, there were no differences in TNF-α and IL-1ß levels between dead and recovered patients. Unlike TGF-ß1 level, IL-10 was significantly increased in recovered patients (P < 0.05). Lymphocyte numbers in recovered patients were significantly increased compared to dead patients, unlike ESR value (P < 0.001-0.01). CRP value in recovered patients significantly differed from dead patients (P < 0.001). CONCLUSION: Changes in frequencies of some immune cells and levels of some immune factors may be considered as predictors of mortality in COVID-19 patients.

Follicular helper T cells in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by the dysfunction of immune system and consequently the destruction of insulin-producing ß cells. In past decades, numerous studies have uncovered that CD4+ T cell subsets are critical in the pathogenesis of T1D, manifesting that type 1 T helper (Th1) and Th17 cells are pathogenic, while regulatory T (Treg) cells and Th2 cells are protective. More recently, the pathogenic role of another subset, follicular helper T (Tfh) cells that essentially regulate germinal center (GC) formation and humoral responses, has also been demonstrated in T1D and many other autoimmune diseases. In this review, we summarize the evidence for the aberrant differentiation and function of Tfh cells in T1D, and also discuss the underlying mechanisms. A better understanding on the pathogenic role of Tfh cells in T1D will inspire the design of potential therapeutic strategies to target this subset in the future.

Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation.

Overproduction of cytokines by T helper 2 (Th2) cells in the lung is thought to be a cause of asthma. Here we report that innate lymphocytes termed lung natural helper (LNH) cells are a T cell-independent source of Th2 cell-type cytokines in protease allergen-treated lungs. LNH (Lin(-)Sca-1(+)c-kit(+/lo)CD25(+)CD127(+)) cells, when stimulated by IL-33 plus IL-2, IL-7, or thymic stroma lymphopoietin (TSLP), produced large amounts of IL-5 and IL-13. Intranasal administration of protease allergen papain induced eosinophil infiltration and mucus hyperproduction in the lung of wild-type and Rag1(-/-) mice, but not in Rag2(-/-)Il2rg(-/-) mice that lack LNH cells. LNH cell depletion inhibited papain-induced airway inflammation in Rag1(-/-) mice whereas adoptive transfer of LNH cells enabled Rag2(-/-)Il2rg(-/-) mice to respond to papain. Treatment of lung explants with papain induced IL-33 and TSLP production by stroma cells and IL-5 and IL-13 production by LNH cells. Thus, LNH cells are critical for protease allergen-induced airway inflammation.

IL-9 and Th9 Cells in Tumor Immunity.

T cells can be categorized into functionally diverse subpopulations, which include Th1, Th2, Th9, Th17, Th22, and Tfh cells and Foxp3+ Tregs, based on their role in maintaining normal immune homeostasis and affecting pathological immune-associated diseases. Among these subpopulations, Th9 cells are relatively new, and less is known about their signaling and effects on tumor immunity. Recently, some studies have focused on regulation of the IL-9/IL-9R signaling pathway and Th9 cell differentiation and their roles in tumor environments. Herein, we summarize recent progress in understanding the regulatory signaling of IL-9 and Th9 cells and their critical roles and mechanisms in antitumor immunity.

Formalizing and enriching phenotype signatures using Boolean networks.

In order to predict the behavior of a biological system, one common approach is to perform a simulation on a dynamic model. Boolean networks allow to analyze the qualitative aspects of the model by identifying its steady states and attractors. Each of them, when possible, is associated with a phenotype which conveys a biological interpretation. Phenotypes are characterized by their signatures, provided by domain experts. The number of steady states tends to increase with the network size and the number of simulation conditions, which makes the biological interpretation difficult. As a first step, we explore the use of Formal Concept Analysis as a symbolic bi-clustering technics to classify and sort the steady states of a Boolean network according to biological signatures based on the hierarchy of the roles the network components play in the phenotypes. FCA generates a lattice structure describing the dependencies between proteins in the signature and steady-states of the Boolean network. We use this lattice (i) to enrich the biological signatures according to the dependencies carried by the network dynamics, (ii) to identify variants to the phenotypes and (iii) to characterize hybrid phenotypes. We applied our approach on a T helper lymphocyte (Th) differentiation network with a set of signatures corresponding to the sub-types of Th. Our method generated the same classification as a manual analysis performed by experts in the field, and was also able to work under extended simulation conditions. This led to the identification and prediction of a new hybrid sub-type later confirmed by the literature.

Distribution of distinct subsets of circulating T follicular helper cells in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is an acute febrile vasculitis that primarily affects children. Previous studies have shown that both innate and adapt immune systems are involved in the immunopathogenesis of KD. The following study analyzes the distribution of the subsets of Circulating T follicular helper cells (cTfh cells) in KD patients with and without coronary artery lesions (CALs). METHODS: Twenty KD patients and fifteen healthy sex- and age- matched children were enrolled. Patients were divided into two groups depending on CALs. Blood samples were collected respectively before and after intravenous immunoglobulin (IVIG) administration. Circulating Tfh cells were categorized into three subsets by flow cytometry including cTfh1 (CXCR3 + CCR6-), cTfh2 (CXCR3-CCX6-) and cTfh17 (CXCR3-CCR6+) cells in circulating CD3 + CD4 + CXCR5 + CD45RA- T cells. Cytometric bead arrays were used to analyze the level of IFN-γ, IL-4 and IL-17A. RESULTS: We found that frequency of cTfh2 cells was significantly elevated in KD patients before IVIG administration with low expression of cTfh1 cells, where the ratio of cTfh2 + cTfh17/cTfh1 significantly increased. Levels of IFN-γ, IL-4 and IL-17A in KD were significantly higher compared to controls. Further analysis showed that cTfh1 cells were negatively correlated with serum CRP, whereas cTfh2 cells were positively correlated with serum CRP and ESR. Comparison of different groups showed that frequency of cTfh1 cells in CALs+ group were significantly lower compared to CALs- group. In contrast, cTfh2 cells in CALs+ group significantly increased. After IVIG administration, frequency of cTfh2 cells and the ratio significantly decreased while the frequency of cTfh1 cells significantly increased. Meanwhile, all levels of cytokines decreased. CONCLUSIONS: Our data demonstrated that cTfh1 and cTfh2 cells participate in the pathogenesis of KD, and that the two subsets might be associated with CALs.

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation.

Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that ß-glucosylceramide 22:0 (ßGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human ßGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, ßGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human ßGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders.

Th9 cells, new players in adaptive immunity.

Upon antigen-specific stimulation, naïve CD4⁺ T cells have the potential to differentiate into various T helper (Th) cell subsets. Earlier models of Th cell differentiation focused on IFN-γ-producing Th1 cells and IL-4-secreting Th2 cells. The discovery of additional CD4⁺ Th cell subsets has extended our understanding of Th cell differentiation beyond this dichotomy. Among these is the recently described Th9 cell subset, which preferentially produces interleukin (IL)-9. Here, we review the latest developments in Th9 cell development and differentiation, focusing on contributing environmental signals, and discuss potential physiological and pathophysiological functions of these cells. We describe the challenges inherent to unambiguously defining roles for Th9 cells using the available experimental animal models, and suggest new experimental models to address these concerns.

The predominance of a naive T helper cell subset in the immune response of experimental acute pancreatitis.

INTRODUCTION: In necrotizing acute pancreatitis (NAP), systemic inflammatory response syndrome (SIRS) and the compensatory anti-inflammatory response syndrome (CARS) decide overall outcome and mortality. In patients, low lymphocyte counts were found, but T-helper cells seemed to conversely increase. Our aim was to further categorize T-helper cells within the context of NAP induced SIRS and CARS. METHODS: NAP was induced by injection of sodium-taurocholate into the common bile duct of male BALB/c mice; sham treated animals received saline infusion. The animals were sacrificed at 6, 12, 24 and 48 h later. Lymphocytes from blood, liver and spleen were isolated and examined by flow cytometry. Staining was performed for CD4, CD8, CD19, CD45RB, CD25, CD69, and CD152. CD4+ cells were sorted for their CD45RB expression and sought for gene regulation associated to TH1/TH2 cells by quantitative RT-PCR. RESULTS: In NAP, CD4+ was solely increased in all compartments. CD8+ remained without substantial alterations. CD45RB showed significant expression in RBhigh in T-helper cells, confirmed by the CD45RBhigh/low ratio (Liver, 24 h: NAP 2.2, SHAM 0.6; p < 0.001). CD45RBhigh and -low cells were not associated to patterns of TH1/TH2 expression. In NAP, CCR4 expression was significantly decreased within RBhigh cells (fold change: 0.04, p < 0.05), while TLR6 showed significant overexpression (fold change: 2.36, p < 0.05). CONCLUSION: T-helper cells increase in NAP, leaning towards CD45RBhigh expression. They resemble naive T-cells, in which NAP leads to expression profiles associated with an innate immune response. This suggests new findings in immunological pathomechanisms of NAP.

An increased expression profile of Th9/IL-9 correlated with Th17/IL-17 in patients with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease, characterized by dysregulation of cellular immunity. Th9 cells were recently identified as a new subtype of Th cells, characterized by preferential production of IL-9. Given the pleiotropic function of IL-9, Th9 cells are demonstrated to be involved in various autoimmune diseases. However, whether Th9 cells are involved in the pathogenesis of ITP remains unclear. In this study, 49 active ITP patients, 39 ITP with remission and 20 healthy controls were included. Peripheral blood mononuclear cells (PBMCs) were isolated from ITP and controls for measuring Th9 and Th17 cells by flow cytometry. Meanwhile, RNA was isolated from PBMCs for the measurement of the mRNA level of PU.1, IRF4, BATF, and RORγt by quantitative real-time PCR. Plasma levels of IL-9 and IL-17 were detected by ELISA. Our results showed that higher expressions of Th9, IL-9, and associated transcription factors (PU.1, IRF4, and BATF) were found in active ITP patients and restored to the normal level (except IL-9) in patients in remission. Meanwhile, Th9 cells and the IL-9 plasma level were positively correlated with Th17 cells and the IL-17 level in ITP patients, respectively. Moreover, a positive correlation of IRF4 or BATF with RORγt was found. In conclusion, an aberrant expression profile of Th9/IL-9 was associated with pathogenesis of ITP possibly through cooperatively working with Th17/IL-17 and therapeutically targeting Th9/IL-9 might be a novel approach in the treatment of ITP.

Alteration of circulating type 2 follicular helper T cells and regulatory B cells underlies the comorbid association of allergic rhinitis with bronchial asthma.

Allergic rhinitis (AR), the most common allergic disorder of the airway, is often accompanied by bronchial asthma. However, little is known about the mechanism by which AR advances to AR comorbid with bronchial asthma (AR+Asthma). To determine the pathophysiologic features of AR and AR+Asthma, we examined subsets of follicular helper T (Tfh) cells and regulatory B (Breg) cells in peripheral blood from AR and AR+Asthma patients. The results showed polarization of Tfh2 cells within Tfh cell subsets in both AR and AR+Asthma cases. Interestingly, the %Breg cells in total B cells were decreased in AR cases and, more extensively, in AR+Asthma cases. Moreover, we found significant correlations of fractional exhaled nitric oxide and blood eosinophil levels with the index %Tfh2 cells per %Breg cells. Our findings indicate that relative decrease in Breg cells under the condition of Tfh2 cell skewing is a putative exaggerating factor of AR to bronchial asthma.

Defining Th-cell subsets in a classical and tissue-specific manner: Examples from the skin.

Th cells are important mediators of adaptive immunity and involved in various diseases. During the past decade, the Th family has expanded from including Th1 and Th2 cells to also encompass Th9, Th17, Th22, and Treg cells; the original classification using the expression of signature cytokines is still the gold standard for definition of subset affiliation. However, the identification of Th cells that do not fit into these tight conceptual boundaries has tumbled the field into an identity crisis. This review gives an overview on different Th-cell classification approaches, their advantages and drawbacks. In addition, this review highlights the functional properties of distinct Th subsets and their effector cytokines in tissues and disease-specific settings with a special focus on inflammatory skin diseases.

T helper 9 cells induced by plasmacytoid dendritic cells regulate interleukin-17 in multiple sclerosis.

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by persistent inflammation orchestrated by cluster of differentiation (CD) 4 T helper (Th) cells. In particular, Th1 and Th17 cells amplify, whereas T regulatory (Treg) cells moderate inflammation. The role of other Th subsets in MS is not clear. In the present study, we investigated the generation of different Th responses by human dendritic cells (DCs) in MS. We compared the production of several Th cytokines by naive CD4+ T-cells polarized with myeloid and plasmacytoid DCs (mDCs and pDCs) in healthy donors (HD) and relapsing-remitting (RR)-MS patients. We found that resiquimod-stimulated mDCs were able to activate Th17 differentiation, whereas pDCs induced interleukin (IL)-10-producing Th cells. Surprisingly, resiquimod-stimulated pDCs from MS patients also significantly induced the differentiation of Th9 cells, which produce IL-9 and are known to be involved in allergic diseases. We investigated the potential role of IL-9 in MS. We found that IL-9 activated signal transducer and activator of transcription (STAT) 1 and STAT5 phosphorylation and interfered with IL-17 and interferon (IFN) regulatory transcription factor (IRF)-4 expression in Th17-polarized cells. Moreover, in the cerebrospinal fluid (CSF) of 107 RR-MS patients, IL-9 inversely correlated with indexes of inflammatory activity, neurodegeneration and disability progression of MS. High levels of IL-9 were associated with the absence of IL-17 in the CSF of RR-MS patients. Our results demonstrate a Th9-inducing potential of pDCs in MS, suggesting an immunoregulatory role leading to attenuation of the exaggerated Th17 inflammatory response.

Review: Lymphocytes, cytokines, chemokines and the T-helper 1-T-helper 2 balance in canine atopic dermatitis.

BACKGROUND: The development of atopic dermatitis (AD) and other cutaneous hypersensitivities involves the activation and differentiation of allergen-specific lymphocytes. Although hypersensitivity is often considered to be a 'T-helper 2-polarized' lymphocyte response, recent evidence suggests that clinical disease is associated with the development of multiple lymphocyte phenotypes. OBJECTIVES: The purpose of this paper is to review recent advances in the understanding of the roles of lymphocytes, cytokines and noncytokine factors in the pathogenesis of canine AD. METHODS: Citation databases, abstracts and proceedings from international meetings published between 2001 and 2013 were reviewed in this update. Where necessary, older articles were included for background information. RESULTS: The development of canine AD is associated with changes in both cutaneous and circulating lymphocyte populations. These lymphocyte responses are characterized by the production of a complex variety of cytokines, including not only T-helper 2 but also T-helper 1, T-helper 17 and regulatory T-cell responses. In addition, microarray gene expression analysis has enabled the identification of a number of noncytokine factors that appear to be associated with atopic inflammation. These include the calcium-binding protein S100A8, serum amyloid A and a number of protease inhibitors, as well as genes involved in epidermal barrier formation, innate immunity receptors, cell cycle proteins and apoptosis. CONCLUSIONS: The development of AD in dogs is characterized by the development of a delicate balance between a variety of T-cell phenotypes and inflammatory mediators, including cytokines, chemokines and noncytokine factors.

Plasmacytoid dendritic cells engagement by influenza vaccine as a surrogate strategy for driving T-helper type 1 responses in human neonatal settings.

BACKGROUND: The elicitation of T-helper type 1 (Th1) cellular immunity to eradicate intracellular pathogens is a challenging task because of the interleukin 12 (IL-12) deficit observed in early infancy. METHODS: Screening cord blood responses to various pediatric vaccines and Toll-like receptor (TLR) agonists for innate responses and CD4(+) T-cell differentiation. RESULTS: We identified that nonadjuvanted inactivated trivalent influenza vaccine (TIV) was able to cosignal T cells for the production of interferon γ (IFN-γ) in a neonatal setting. This process includes the mobilization of neonatal plasmacytoid dendritic cells (pDCs) as antigen-presenting cells (APCs) that efficiently engage Th1 cells in an IL-12-independent but type I IFN-dependent manner. In addition, cord blood pDCs efficiently cross-presented antigen to CD8(+) T cells. Importantly, activation by TIV mainly requires TLR7; however, R848/TLR7- and CpGB/TLR9-activated pDCs, which poorly produced IFN-α, induce neonatal Th2 responses. CONCLUSIONS: TLR pathway engagement in pDCs is necessary but not sufficient for a successful neonatal Th1 outcome. We provide evidence of a mature and functional neonatal immune system at the level of APCs and T cells and propose to implement the IFN-α/IFN-γ axis in pediatric vaccination as a surrogate for the defective IL-12/IFN-γ axis.

CD45RA-Foxp3(low) non-regulatory T cells in the CCR7-CD45RA-CD27+CD28+ effector memory subset are increased in synovial fluid from patients with rheumatoid arthritis.

Increased numbers of regulatory T (Treg) cells are found in synovial fluid from patients with rheumatoid arthritis (RASF) compared with peripheral blood. However, Treg cells in RASF have been shown to have a decreased capacity to suppress T cells. Here we phenotypically classified CD4+ T cells in RASF into six subsets based on the expression of CD45RA, CCR7, CD27 and CD28, and demonstrated that the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in synovial fluid compared with peripheral blood. In addition, the proportion of Foxp3+ Treg cells in the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in RASF. Furthermore, most of the Foxp3+ Treg cells in RASF were non-suppressive CD45RA-Foxp3(low) non-Treg cells, and the frequency of the non-Treg cells in the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in RASF. Our findings suggest that the pro-inflammatory environment in RA joints may induce the increase of CD45RA-Foxp3(low) non-Treg cells in synovial fluid.

Induction and effector functions of T(H)17 cells.

T helper (T(H)) cells constitute an important arm of the adaptive immune system because they coordinate defence against specific pathogens, and their unique cytokines and effector functions mediate different types of tissue inflammation. The recently discovered T(H)17 cells, the third subset of effector T helper cells, have been the subject of intense research aimed at understanding their role in immunity and disease. Here we review emerging data suggesting that T(H)17 cells have an important role in host defence against specific pathogens and are potent inducers of autoimmunity and tissue inflammation. In addition, the differentiation factors responsible for their generation have revealed an interesting reciprocal relationship with regulatory T (T(reg)) cells, which prevent tissue inflammation and mediate self-tolerance.

Recent thymic emigrants are biased against the T-helper type 1 and toward the T-helper type 2 effector lineage.

After intrathymic development, T cells exit the thymus and join the peripheral T-cell pool. Such recent thymic emigrants (RTEs) undergo both phenotypic and functional maturation during the first 3 weeks they reside in the periphery. Using a well-controlled in vitro polarization scheme, we now show that CD4(+) RTEs are defective in T-helper (Th) type 0 (Th0), Th1, Th17, and regulatory T-cell lineage commitment, with dampened cytokine production and transcription factor expression. In contrast, CD4(+) RTES are biased toward the Th2 lineage both in vitro and in vivo, with more robust interleukin-4, interleukin-5, and interleukin-13 production than their mature naive counterparts. Coculture experiments demonstrate that mature naive T cells influence neighboring RTEs in their Th responses. In adoptive hosts, CD4(+) RTEs drive production of the Th2-associated antibody isotype immunoglobulin G1 and mediate airway inflammatory disease. This bias in RTEs likely results from dampened negative regulation of the Th2 lineage by diminished levels of T-bet, a key Th1 transcription factor. CD4(+) RTEs thus represent a transitional population with a distinct interpretation of, and response to, immunologic cues. These characteristics may be beneficial during the postthymic maturation period by leading to the avoidance of inappropriate immune responses, particularly in lymphopenic neonates and adults.

The important role of T cells and receptor expression in Sjögren's syndrome.

Sjögren's syndrome (SjS), an autoimmune disease characterized by exocrine gland dysfunction leading to dry mouth and dry eye diseases, is typified by progressive leucocyte infiltrations of the salivary and lacrimal glands. Histologically, these leucocyte infiltrations generally establish periductal aggregates, referred to as lymphocytic foci (LF), which occasionally appear as germinal centre (GC)-like structures. The formation and organization of these LF suggest an important and dynamic role for helper T cells (TH), specifically TH1, TH2 and the recently discovered TH17, in development and onset of clinical SjS, considered a B cell-mediated hypersensitivity type 2 disease. Despite an ever-increasing focus on identifying the underlying aetiology of SjS, defining factors that initiate this autoimmune disease remain a mystery. Thus, determining interactions between infiltrating TH cells and exocrine gland tissue (auto-)antigens represents a fertile research endeavour. This review discusses pathological functions of TH cells in SjS, the current status of TH cell receptor gene rearrangements associated with human and mouse models of SjS and potential future prospects for identifying receptor-autoantigen interactions.

Cutting edge: dendritic cell-restricted antigen presentation initiates the follicular helper T cell program but cannot complete ultimate effector differentiation.

Follicular helper T (T(FH)) cells are critical for germinal center (GC) formation. The processes that drive their generation and effector potential remain unclear. In this study, we define requirements for MHC class II APCs in murine T(FH) cell formation by either transiently ablating or restricting Ag presentation to dendritic cells (DCs). We find that cognate interactions with DCs are necessary and sufficient to prime CD4(+) T cells toward a CXCR5(+)ICOS(+)Bcl6(+) T(FH) cell intermediate. However, in the absence of additional APCs, these T(FH) cells fail to produce IL-21. Furthermore, in vitro priming of naive T cells by B cells engenders optimal production of IL-21, which induces a GC B cell transcriptional profile. These results support a multistep model for effector T(FH) cell priming and GC initiation, in which DCs are necessary and sufficient to induce a T(FH) cell intermediate that requires additional interactions with distinct APCs for full effector function.