A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells

In this study we show the inheritance of a CD4⁺CD8⁺ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co-express CD4 and CD8α, but not CD8β. CD4⁺ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co-receptor. These cells induce in vivo a graft versus host-reaction, providing further evidence for their function as CD4⁺ T cells. The CD4⁺CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co-expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross-breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4⁺ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.     

Transforming growth factor-beta 1 (TGF-beta 1)- and beta 2-like activities in malignant pleural effusions caused by malignant mesothelioma or primary lung cancer.

We investigated the levels of TGF-beta in malignant pleural effusions (MPE) caused by malignant mesothelioma (MESO) or primary lung cancer. TGF-beta levels in MPE caused by MESO were 283.9 +/- 219.2 pm (mean +/- s.d.) and were three to six times higher than those due to primary lung cancers (P < 0.01 or P < 0.05). We also evaluated TGF-beta 1- and beta 2-like activities in MPE using specific polyclonal antibodies. Although TGF-beta 1-like activity could be detected in all cases, TGF-beta 2-like activities were detected in five of seven in MESO and in a few cases with primary lung cancer. These results demonstrate that the levels of total TGF-beta and TGF-beta 2-like activity may be clinically useful to differentiate MESO from primary lung cancer. Our data also suggest that TGF-beta may help further characterize the clinical features of MESO.     

Secreted IL‐1α promotes T‐cell activation and expansion of CD11b+Gr1+ cells in carbon tetrachloride‐induced liver injury in mice

Interleukin‐1α is mainly expressed on the cell membrane, but can also be secreted during inflammation. The roles of secreted and membrane IL‐1α in acute liver inflammation are still not known. Here, we examined the functions of secreted and membrane IL‐1α in a mouse model of carbon tetrachloride‐induced acute liver injury. We show that secreted IL‐1α aggravates liver damage and membrane IL‐1α slightly protects mice from liver injury. Further studies showed that secreted IL‐1α promotes T‐cell activation. It also increased the expansion of CD11b⁺Gr1⁺ myeloid cells, which may serve as a negative regulator of acute liver inflammation. Moreover, secreted IL‐1α induced IL‐6 production from hepatocytes. IL‐6 neutralization reduced the proliferation of CD11b⁺Gr1⁺ myeloid cells in vivo. CCL2 and CXCL5 expression was increased by secreted IL‐1α in vitro and in vivo. Antagonists of the chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of CD11b⁺Gr1⁺ myeloid cells. These results demonstrate that secreted and membrane IL‐1α play different roles in acute liver injury. Secreted IL‐1α could promote T‐cell activation and the recruitment and expansion of CD11b⁺Gr1⁺ myeloid cells through induction of CCL2, CXCL5, and IL‐6. The controlled release of IL‐1α could be a critical regulator during acute liver inflammation.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

Role of CD4 T cell helper subsets in immune response and deviation of CD8 T cells in mice*

The ability of different CD4⁺ T cell subsets to help CD8⁺ T‐cell response is not fully understood. Here, we found using the murine system that Th17 cells induced by IL‐1β, unlike Th1, were not effective helpers for antiviral CD8 responses as measured by IFNγ‐producing cells or protection against virus infection. However, they skewed CD8 responses to a Tc17 phenotype. Thus, the apparent lack of help was actually immune deviation. This skewing depended on both IL‐21 and IL‐23. To overcome this effect, we inhibited Th17 induction by blocking TGF‐β. Anti‐TGF‐β allowed the IL‐1β adjuvant to enhance CD8⁺ T‐cell responses without skewing the phenotype to Tc17, thereby providing an approach to harness the benefit of common IL‐1‐inducing adjuvants like alum without immune deviation.     

Interleukin‐17A participates in podocyte injury by inducing IL‐1β secretion through ROS‐NLRP3 inflammasome‐caspase‐1 pathway

Studies show that the Th17/IL ‐17A axis plays an important role in the pathogenesis of kidney diseases. Previously, we also showed that IL ‐17A may play a role in the pathogenesis of primary nephrotic syndrome; however, the underlying mechanism(s) is unclear. The aim of this study was to explore the molecular mechanism of IL ‐17A‐inducing podocyte injury in vitro. In this study, the NLRP 3 inflammasome activation and the morphology of podocytes were detected by Western blot and immunofluorescence. The results showed that podocytes persistently expressed IL ‐17A receptor and that NLRP 3 inflammasome in these cells was activated upon exposure to IL ‐17A. Also, activity of caspase‐1 and secretion of IL ‐1β increased in the presence of IL ‐17A. In addition, IL ‐17A disrupted podocyte morphology by decreasing expression of podocin and increasing expression of desmin. Blockade of intracellular ROS or inhibition of caspase‐1 prevented activation of the NLRP 3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL ‐17A induces podocyte injury by activating the NLRP 3 inflammasome and IL ‐1β secretion and contributes to disruption of the kidney's filtration system.     

T regulatory cells are critical for the maintenance,anamnestic expansion and protection elicited by vaccine‐induced CD8 T cells

T regulatory (Treg) cells are critical for preventing autoimmunity and suppressing immune responses during cancer and chronic infection. However, the role of Treg cells in the generation of vaccine‐induced immune memory remains ill‐defined. Using the mouse model of lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate that transient absence of Treg cells during effector to memory CD8 T‐cell transition results in a permanent impairment in the maintenance, function and recall capacity of CD8 T cells. Memory CD8 T cells in mice that were transiently depleted of Treg cells exhibited defective up‐regulation of memory markers with a significant decrease in polyfunctionality. However, Treg‐depleted mice showed no significant change in CD4 T‐cell responses, and antibody levels relative to control. Altogether, this study evaluates the role of Treg cells in the formation of immune memory and demonstrates an important role for Treg cells in promoting memory CD8 T‐cell differentiation and vaccine‐induced immune protection against intracellular pathogens.     

The siRNA cocktail targeting interleukin 10 receptor and transforming growth factor‐β receptor on dendritic cells potentiates tumour antigen‐specific CD8+ T cell immunity

Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune‐priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)‐10 and transforming growth factor (TGF)‐β that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL‐10 receptor alpha (siIL‐10RA) initiated the strongest antigen‐specific CD8⁺ T cell immune responses. The potency of siIL‐10RA was enhanced further by combining it with siRNA targeting TGF‐β receptor (siTGF‐βR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl‐2‐like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL‐10RA and siTGF‐βR generated the strongest antigen‐specific CD8⁺ T cell immunity. Concordantly, the knock‐down of both IL‐10RA and TGF‐βR in DCs induced the strongest anti‐tumour effects in the TC‐1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)‐16 E7 antigen, and even in the immune‐resistant TC‐1 (P3) tumour model that secretes more IL‐10 and TGF‐β than the parental tumour cells (TC‐1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail‐mediated strategy by co‐targeting immunosuppressive molecules to enhance the potency of DC‐based vaccines.     

The regulatory role of interferon‐γ producing gamma delta T cells via the suppression of T helper 17 cell activity in bleomycin‐induced pulmonary fibrosis

Interstitial pneumonia (IP) is a chronic progressive interstitial lung disease associated with poor prognosis and high mortality. However, the pathogenesis of IP remains to be elucidated. The aim of this study was to clarify the role of pulmonary γδT cells in IP. In wild‐type (WT) mice exposed to bleomycin, pulmonary γδT cells were expanded and produced large amounts of interferon (IFN)‐γ and interleukin (IL)‐17A. Histological and biochemical analyses showed that bleomycin‐induced IP was more severe in T cell receptor (TCR‐δ‐deficient (TCRδ^–/–) mice than WT mice. In TCRδ^–/– mice, pulmonary IL‐17A⁺CD4⁺ Τ cells expanded at days 7 and 14 after bleomycin exposure. In TCRδ^–/– mice infused with γδT cells from WT mice, the number of pulmonary IL‐17A⁺ CD4⁺ T cells was lower than in TCRδ^–/– mice. The examination of IL‐17A^–/– TCRδ^–/– mice indicated that γδT cells suppressed pulmonary fibrosis through the suppression of IL‐17A⁺CD4⁺ T cells. The differentiation of T helper (Th)17 cells was determined in vitro, and CD4⁺ cells isolated from TCRδ^–/– mice showed normal differentiation of Th17 cells compared with WT mice. Th17 cell differentiation was suppressed in the presence of IFN‐γ producing γδT cells in vitro. Pulmonary fibrosis was attenuated by IFN‐γ‐producing γδT cells through the suppression of pulmonary IL‐17A⁺CD4⁺ T cells. These results suggested that pulmonary γδT cells seem to play a regulatory role in the development of bleomycin‐induced IP mouse model via the suppression of IL‐17A production.     

TGF‐β downregulates KLRG1 expression in mouse and human CD8+ T cells

The inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) and the integrin α_E (CD103) are expressed by CD8⁺ T cells and both are specific for E‐cadherin. However, KLRG1 ligation by E‐cadherin inhibits effector T‐cell function, whereas binding of CD103 to E‐cadherin enhances cell–cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8⁺ T cells from untreated and virus‐infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8⁺ T cells‐infiltrating hepatocellular carcinomas. As TGF‐β is known to induce CD103 expression in CD8⁺ T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF‐β signaling in mouse as well as in human CD8⁺ T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8⁺ T‐cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8⁺ T cells if lymphocytes from tissues expressing high levels of TGF‐β are analyzed.     

Cytomegalovirus vector expressing RAE‐1γ induces enhanced anti‐tumor capacity of murine CD8+ T cells

Designing CD8⁺ T‐cell vaccines, which would provide protection against tumors is still considered a great challenge in immunotherapy. Here we show the robust potential of cytomegalovirus (CMV) vector expressing the NKG2D ligand RAE‐1γ as CD8⁺ T cell‐based vaccine against malignant tumors. Immunization with the CMV vector expressing RAE‐1γ, delayed tumor growth or even provided complete protection against tumor challenge in both prophylactic and therapeutic settings. Moreover, a potent tumor control in mice vaccinated with this vector can be further enhanced by blocking the immune checkpoints TIGIT and PD‐1. CMV vector expressing RAE‐1γ potentiated expansion of KLRG1⁺ CD8⁺ T cells with enhanced effector properties. This vaccination was even more efficient in neonatal mice, resulting in the expansion and long‐term maintenance of epitope‐specific CD8⁺ T cells conferring robust resistance against tumor challenge. Our data show that immunomodulation of CD8⁺ T‐cell responses promoted by herpesvirus expressing a ligand for NKG2D receptor can provide a powerful platform for the prevention and treatment of CD8⁺ T‐cell sensitive tumors.     

Functional exhaustion of CD4+ T cells induced by co‐stimulatory signals from myeloid leukaemia cells

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T‐cell exhaustion. T‐cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co‐stimulatory molecules to T‐cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia‐derived co‐stimulatory signals on CD4⁺ T helper (Th) cell exhaustion, which may limit anti‐tumour immunity. Here, CD86 and inducible T‐cell co‐stimulator ligand (ICOS‐LG) co‐stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co‐cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro‐generated exhausted Th cells were defined by up‐regulation of programmed cell death 1 (PD‐1), cytotoxic T‐lymphocyte antigen 4 (CTLA‐4), lymphocyte activation gene 3 (LAG3) and T‐cell immunoglobulin and mucin domain‐containing protein 3 (TIM‐3) inhibitory receptors. They were reluctant to proliferate upon re‐stimulation and produced reduced amounts of interleukin‐2 (IL‐2), tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ). Nonetheless, IL‐2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co‐stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD‐1, TIM‐3 and/or LAG3 was identified together with CD86⁺ and/or ICOS‐LG⁺ myeloid blasts. Collectively, co‐stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.     

c‐Jun NH2‐terminal kinase is a critical node in the death of CD4+CD8+ thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy, due to the depletion of CD4⁺CD8⁺ thymocytes, is observed during infections with numerous pathogens. Several mechanisms, such as glucocorticoids and inflammatory cytokines, are known to be involved in this process; however, the roles of intracellular signaling molecules have not been investigated. In this study, the functional role of c‐Jun NH₂‐terminal kinase (JNK) during infection‐induced thymic atrophy was addressed. The levels of phosphorylated JNK in immature CD4⁺CD8⁺ thymocytes from C57BL/6 (Nramp‐deficient) and 129/SvJ (Nramp‐sufficient) mice were increased upon oral infection of mice with Salmonella enterica serovar Typhimurium (S. typhimurium). Furthermore, inhibition of JNK signaling, but not ERK or p38 MAPK, prevented the in vitro death of infected thymocytes. Importantly, the in vivo inhibition of JNK signaling with SP600125 protected C57BL/6 CD4⁺CD8⁺ thymocytes from depletion via multiple mechanisms as follows: lower intracellular ROS, inflammatory cytokines, Bax and caspase 3 activity, increase in Bcl‐xL amounts, and prevention of the loss in mitochondrial membrane potential. Notably, thymic architecture was preserved in infected mice treated with SP600125. Overall, this study identifies a novel role for JNK as a crucial regulator of the death of CD4⁺CD8⁺ thymocytes during S. typhimurium infection.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

Bronchiolitis obliterans syndrome is associated with increased p‐glycoprotein expression and loss of glucocorticoid receptor from steroid‐resistant proinflammatory CD8+ T cells

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8⁺ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8⁺ cells, and we hypothesized that the drug membrane efflux pump, p‐glycoprotein‐1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)‐γ/tumour necrosis factor (TNF)‐α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged‐matched controls (± prednisolone ± Pgp inhibitor, cyclosporin A ± GCR activator, Compound A) using flow cytometry. Both Pgp⁺ and Pgp^– lymphocyte subsets from all subjects produced IFN‐γ/TNF‐α proinflammatory cytokines. Pgp expression was increased in CD8⁺Pgp⁺ T cells and correlated with IFN‐γ/TNF‐α expression and BOS grade. Reduced GCR was observed in CD8⁺Pgp^– T, natural killer (NK) T‐like and NK cells from stable patients compared with controls, and reduced further in CD8⁺Pgp^– T cells in BOS. The addition of 2·5 ng/ml cyclosporin A and 1 µM prednisolone inhibit IFN‐γ/TNF‐α production significantly by CD8⁺Pgp⁺ T cells from BOS patients. The addition of 10 µM Compound A and 1 µM prednisolone inhibit IFN‐γ/TNF‐α production significantly by CD8⁺Pgp^– T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid‐resistant proinflammatory CD8⁺ T cells. Treatments that inhibit Pgp and up‐regulate GCR in CD8⁺ T cells may improve graft survival.     

Avidity of influenza‐specific memory CD8+ T‐cell populations decays over time compromising antiviral immunity

Decline of cell‐mediated immunity is often attributed to decaying T‐cell numbers and their distribution in peripheral organs. This study examined the hypothesis that qualitative as well as quantitative changes contribute to the declining efficacy of CD8⁺ T‐cell memory. Using a model of influenza virus infection, where loss of protective CD8⁺ T‐cell immunity was observed 6 months postinfection, we found no decline in antigen‐specific T‐cell numbers or migration to the site of secondary infection. There was, however, a large reduction in antigen‐specific CD8⁺ T‐cell degranulation, cytokine secretion, and polyfunctionality. A profound loss of high‐avidity T cells over time indicated that failure to confer protective immunity resulted from the inferior functional capacity of remaining low avidity cells. These data imply that high‐avidity central memory T cells wane with declining antigen levels, leaving lower avidity T cells with reduced functional capabilities.