Mycobacterial antigen‐induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non‐diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non‐diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell‐mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non‐diabetic TB patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen‐induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐β], and Th2 (IL‐4, IL‐5, IL‐10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1_pool stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1_pool in healthy subjects. In response to complex mycobacterial antigens, both IFN‐γ and TNF‐β were secreted by PBMC of all groups whereas diabetic TB patients secreted IL‐10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN‐γ and IL‐10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.     

Reverse plasticity: TGF‐β and IL‐6 induce Th1‐to‐Th17‐cell transdifferentiation in the gut

Th17 cells are a heterogeneous population of pro‐inflammatory T cells that have been shown to mediate immune responses against intestinal bacteria. Th17 cells are highly plastic and can transdifferentiate to Th1/17 cells or unconventional Th1 cells, which are highly pathogenic in animal models of immune‐mediated diseases such as inflammatory bowel diseases. A recent European Journal of Immunology article by Liu et al. (Eur. J. Immunol. 2015. 45:1010–1018) showed, surprisingly, that Th1 cells have a similar plasticity, and could transdifferentiate to Th17 cells. Thus, IFN‐γ‐producing Th1 effector cells specific for an intestinal microbial antigen were shown to acquire IL‐17‐producing capacities in the gut in a mouse model of colitis, and in response to TGF‐β and IL‐6 in vitro. TGF‐β induced Runx1, and together with IL‐6 was shown to render the ROR‐γt and IL‐17 promoters in Th1 cells accessible for Runx1 binding. In this commentary, we discuss how this unexpected plasticity of Th1 cells challenges our view on the generation of Th1/17 cells with the capacity to co‐produce IL‐17 and IFN‐γ, and consider possible implications of this Th1‐to‐Th17‐cell conversion for therapies of inflammatory bowel diseases and protective immune responses against intracellular pathogens.     

The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

Dendritic cells (DCs) play a critical role in controlling T helper 2 (Th2) cell-dependent diseases, but the signaling mechanism that triggers this function is not fully understood. We showed that p38α activity in DCs was decreased upon HDM stimulation and dynamically regulated by both extrinsic signals and Th2-instructive cytokines. p38α-specific deletion in cDC1s but not in cDC2s or macrophages promoted Th2 responses under HDM stimulation. Further study showed that p38α in cDC1s regulated Th2-cell differentiation by modulating the MK2−c-FOS−IL-12 axis. Importantly, crosstalk between p38α-dependent DCs and Th2 cells occurred during the sensitization phase, not the effector phase, and was conserved between mice and humans. Our results identify p38α signaling as a central pathway in DCs that integrates allergic and parasitic instructive signals with Th2-instructive cytokines from the microenvironment to regulate Th2-cell differentiation and function, and this finding may offer a novel strategy for the treatment of allergic diseases and parasitic infection.     

TGF‐β indirectly favors the development of human Th17 cells by inhibiting Th1 cells

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti‐proliferative effect of TGF‐β than Th1 and Th2 clones or circulating Th1‐oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl‐2 expression and reduced apoptosis in the presence of TGF‐β, in comparison with Th1 cells. Umbilical cord blood naïve CD161⁺CD4⁺ T cells, which contain the precursors of human Th17 cells, differentiated into IL‐17A‐producing cells only in response to IL‐1β plus IL‐23, even in serum‐free cultures. TGF‐β had no effect on constitutive RORγt expression by umbilical cord blood CD161⁺ T cells but it increased the relative proportions of CD161⁺ T cells differentiating into Th17 cells in response to IL‐1β plus IL‐23, whereas under the same conditions it inhibited both T‐bet expression and Th1 development. These data suggest that TGF‐β is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.     

γδ+ and CD4+ αβ+ human T cell subset responses upon stimulation with various Mycobacterium tuberculosis soluble extracts

By using a flow cytometric technique which allows direct identification of proliferating cells within mixed cell populations, we have previously described that soluble extracts obtained from Mycobacterium tuberculosis or M. avium represent strong stimuli for human γδ⁺ T cells. In the present study, we demonstrate that the protocol used for the preparation of M. tuberculosis soluble extracts may have an impact on their γδ⁺ T cell stimulatory capacity. In agreement with our previous data, soluble extracts prepared from bacteria killed at 85°C and directly disrupted by prolonged sonication (TBe), elicited a strong proliferation of γδ⁺ T cells after 6–7 days of stimulation. In contrast, when soluble extracts were obtained from bacteria autoclaved (121°C, 25 min) and then washed by centrifugation, a predominant proportion of CD4⁺ αβ⁺ T cells was achieved in the responding population. The stimulatory activity for γδ⁺ T cells was recovered in the supernatant of the autoclaved bacteria, indicating that autoclaving of M. tuberculosis bacilli releases an antigen(s) into the supernatant which stimulates human γδ⁺ T cells. While protease digestion of TBe only partially reduced its stimulatory capacity on γδ⁺ T cells, the stimulatory component(s) released into the supernatant after autoclavation of bacilli was found to be sensitive to protease digestion. Interestingly, in contrast to the preponderant proportion of γδ⁺ T cells induced in the responding population by unfractionated TBe, when the extract was fractionated by fast performance liquid chromatography (FPLC), most of the fractions exhibited a strong stimulatory capacity on CD4⁺ αβ⁺ T cells only. The γδ⁺ T cell stimulatory activity was confined to the low molecular weight range FPLC fractions. Such results may suggest a possible regulatory role of γδ⁺ T cells on CD4⁺ αβ⁺ T cells.     

Coincident diabetes mellitus modulates Th1‐, Th2‐, and Th17‐cell responses in latent tuberculosis in an IL‐10‐ and TGF‐β‐dependent manner

Type 2 diabetes mellitus (DM) is a risk factor for the development of active tuberculosis (TB), although its role in the TB‐induced responses in latent TB (LTB) is not well understood. Since Th1, Th2, and Th17 responses are important in immunity to LTB, we postulated that coincident DM could alter the function of these CD4⁺ T‐cell subsets. To this end, we examined mycobacteria‐induced immune responses in the whole blood of individuals with LTB‐DM and compared them with responses of individuals without DM (LTB‐NDM). T‐cell responses from LTB‐DM are characterized by diminished frequencies of mono‐ and dual‐functional CD4⁺ Th1, Th2, and Th17 cells at baseline and following stimulation with mycobacterial antigens‐purified protein derivative, early secreted antigen‐6, and culture filtrate protein‐10. This modulation was at least partially dependent on IL‐10 and TGF‐β, since neutralization of either cytokine resulted in significantly increased frequencies of Th1 and Th2 cells but not Th17 cells in LTB‐DM but not LTB individuals. LTB‐DM is therefore characterized by diminished frequencies of Th1, Th2, and Th17 cells, indicating that DM alters the immune response in latent TB leading to a suboptimal induction of protective CD4⁺ T‐cell responses, thereby providing a potential mechanism for increased susceptibility to active disease.     

Comparative analysis of αβ and γδ T cell activation by Mycobacterium tuberculosis and isopentenyl pyrophosphate

Phosphorylated nonpeptide compounds have recently been identified as potent mycobacteria-derived ligands for human Vγ9/Vδ2-expressing γδ T cells. Crude mycobacterial extracts also contain protein antigens which stimulate CD4 αβ T cells to produce growth factors that are used by γδ T cells for clonal expansion. We have investigated the dynamics in vitro of expansion of CD4 T cells and Vγ9 cells in cultures of peripheral blood mononuclear cells stimulated with synthetic isopentenyl pyrophosphate (IPP) in the absence or presence of additional stimuli. The results indicated that following stimulation with IPP, γδ T cells express CD25 and CD69 antigens, but fail to proliferate unless growth factors are provided exogenously or endogenously through activation of CD4 T cells by additional stimuli such as tetanus toxoid, alloantigen, or superantigens. Furthermore, the presence of antigen presenting cells are required for expansion of γδ T cells. In response to IPP stimulation, purified CD4 T cells neither express CD25 or CD69, nor do they proliferate even in the presence of exogenous IL-2. Apart from IL-2, IL-15 and, less efficiently, IL-4, IL-7, and IL-12 can contribute to cellular expansion of IPP-reactive Vγ9 cells. Together, the results demonstrate that peripheral blood γδ T cells proliferate in response to IPP only if CD4 T cells are simultaneously activated by an additional stimulus. This mechanism provides a tight control of the reactivity of γδ T cells towards phosphorylated nonpeptide antigens.     

Mycobacterium tuberculosis RpfE promotes simultaneous Th1‐ and Th17‐type T‐cell immunity via TLR4‐dependent maturation of dendritic cells

Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation‐promoting factor (Rpf) E, a latency‐associated member of the Rpf family, in promoting naïve CD4⁺ T‐cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL‐6, IL‐1β, IL‐23p19, IL‐12p70, and TNF‐α but not IL‐10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF‐κB signaling. RpfE‐treated DCs effectively caused naïve CD4⁺ T cells to secrete IFN‐γ, IL‐2, and IL‐17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T‐bet and RORγt but not GATA‐3. Furthermore, lung and spleen cells from Mtb‐infected WT mice but not from TLR4^?/? mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1‐ and Th17‐polarized T‐cell expansion.     

Expression of HNF-1α and HNF-1β in various histological differentiations of hepatocellular carcinoma

Hepatic nuclear factor 1 (HNF-1) regulates genes in a hepatocyte-specific manner. It has been previously reported that the ratio of HNF-1α and HNF-1β mRNA is related to histological differentiation hepatocellular carcinoma (HCC). In this study, the expression levels of the HNF-1α and HNF-1β proteins were analysed relatively and quantitatively in various histologically differentiated HCC and surrounding non-cancerous tissues, and HNF-1α binding activity for the AT element of the B domain of the human α-fetoprotein enhancer was examined. Western blot analysis demonstrated that HNF-1α protein was expressed at a higher level in well-differentiated HCC tissues than in the surrounding non-HCC tissues; on the other hand, the HNF-1α protein was expressed at lower levels in moderately and poorly differentiated HCCs than in the surrounding non-HCC tissues. The levels of HNF-1β expression in well-differentiated and poorly differentiated HCCs were similar to and higher than those found in the respective surrounding non-cancerous portions. In binding assays, HNF-1 binding activity was high in well-differentiated HCC and lower in moderately and poorly differentiated HCCs. Most well-differentiated HCC cases showed immunohistochemical expression of HNF-1α. These findings show that poor histological differentiation of HCC correlates with decreases in the level and activity of HNF-1α proteins. © 1998 John Wiley & Sons, Ltd.     

Quantitative analysis of specific Th1/Th2 helper cell responses and IgG subtype antibodies in interferon‐α‐treated patients with chronic hepatitis C

This study aimed to characterise the immune mechanisms relevant to viral clearance in interferon (IFN)‐α‐treated chronic hepatitis C virus (HCV) infection. Proliferative responses of peripheral blood mononuclear cells from sustained complete IFN‐α therapy responders (n = 8), nonresponders (n = 13), untreated patients (n = 10), and healthy controls (n = 5) were measured retrospectively upon stimulation with recombinant HCV‐antigens (core, helicase, NS3, NS4, and NS5) and the secretion of IFN‐γ and interleukins (IL‐4, IL‐5, IL‐10, and IL‐12) were tested by ELISA. Furthermore, IFN‐γ as well as IL‐10 secreting CD4+ T cells were quantitated by intracellular cytokine staining. Anti‐HCV core and NS3‐specific IgG subclass antibodies were quantitated in the corresponding patient sera. Sustained therapy responders had more frequent and stronger NS3 and helicase‐specific cellular immune responses than nonresponders, untreated HCV patients and healthy controls. Independent from therapy outcome HCV‐stimulated T cells in IFN‐α treated patients secreted preferentially IFN‐γ The Th2 cytokines IL‐4 and IL‐10 were even decreased in nonresponders, while the IL‐12 secretion was not influenced. With respect to the humoral immune response sustained complete responders showed significantly reduced IFN‐γ independent anti‐HCV‐core and ‐NS3 IgG1 antibody synthesis. In conclusion, vigorous NS3‐specific T‐helper cell responses were associated with viral clearance in IFN‐α recipients; however, the cytokine and antibody analysis argues against a Th1/Th2 imbalance as a major factor that influence the therapy outcome. J. Med. Virol. 64:340–349, 2001. © 2001 Wiley‐Liss, Inc.     

γδ T cells are secondary participants in acute graft-versus-host reactions initiated by CD4+ αβT cells

To examine the role of T cell subpopulations in an acute graft-versus-host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4⁺ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell-initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.     

Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-γ production

The interaction of CD40 ligand (CD40L) on activated T cells with CD40 on B cells, monocytes and dendritic cells is essential for humoral immunity and for up-regulation of antigen-presenting cell (APC) functions, as a result of signaling through CD40. There are also some indications that after interaction with CD40, CD40L can directly signal T cells. In this study we demonstrate that upon stimulation of human peripheral blood T cells through the T cell receptor (TCR)/CD3 complex, CD40/CD40L interaction strongly enhances the production of Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-γ and Th2 cytokines such as IL-4, IL-5 and IL-10 by a direct effect on T cells. Furthermore, CD40/CD40L interaction synergizes with IL-12 in selectively enhancing IFN-γ production by purified anti-CD3-stimulated T cells. These effects were observed at both the protein and the mRNA level. Both CD4⁺ and CD8⁺ T cells were able to produce IFN-γ in the presence of helper signals from IL-12 and CD40, although CD8⁺ T cells were less active. Since CD40/CD40L interaction also up-regulates IL-12 production and B7 expression by APC, our results suggest that CD40/CD40L interaction is bidirectional, and promotes activation of both APC and T cells.     

Distribution of α4β7 and αEβ7 integrins on thymocytes,intestinal epithelial lymphocytes and peripheral lymphocytes

β₇ is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of β₇. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express α₄β₇ and α_Eβ₇ using mAb against α_Eβ₇ and mAb DATK32 which recognizes a combinatorial epitope on α₄β₇. β₇⁺ thymocytes have a mature phenotype: TcR⁺, CD11a^hi CD44^hi HSA^dull. Small subsets of double-negative CD4_?CD8_?, single-positive CD4⁺ and CD8⁺ thymocytes express α₇, while double-positive CD4⁺ CD8⁺ thymocytes are β₇. However, two integrins α_Eβ₇ and α₄β₇ recognized by anti-β₇ are not expressed on an identical subpopulation of thymocytes, as β_Eα₇⁺α₄β₇^?, α_Eβ₇⁺α₄β₇⁺ and α_Eβ₇^?α₄β₇⁺ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of α_Eβ₇ but little α₄β₇. In the spleen, Peyer's patches and lymph nodes, α₄β₇ is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4⁺ and CD8⁺ lymphocytes, express high levels of β₇ in the form of α₄β₇ and α_Eβ₇, although, as observed with lymphocytes, not all α₄β₇^hi CD4^? lymphocytes expressed α₄β₇. The population of α₄β₇^hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4⁺ lymphocyte population, which can be further subdivided on the basis of α_Eβ₇, L-selectin and α₄ expression. Therefore, memory CD4⁺ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.