Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8+ memory T cells

Protection against malaria can be achieved by induction of a strong CD8⁺ T‐cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long‐lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8⁺ T‐cell response and sterile protection, which is short‐lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite‐specific antibodies that showed protective capacity. The numbers of CSP‐specific CD8⁺ T cells were not substantially enhanced by sporozoite infections; however, CSP‐specific memory CD8⁺ T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized‐only mice. CD4⁺ T cells contributed to protection as well; but CD8⁺ memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8⁺ effector‐memory T cells with increased cytotoxic activity as well as CD4⁺ memory T cells and neutralizing antibodies.     

Sublingual tolerance induction with antigen conjugated to cholera toxin B subunit induces Foxp3+CD25+CD4+ regulatory T cells and suppresses delayed-type hypersensitivity reactions

Although sublingual (s.l.) immunotherapy with selected allergens is safe and often effective for treating patients with allergies, knowledge of the immunological mechanisms involved remains limited. Can s.l. administration of antigen (Ag) induce peripheral immunological tolerance and also suppress delayed‐type hypersensitivity (DTH) responses? To what extent can s.l.‐induced tolerance be explained by the generation of Foxp3⁺CD25⁺CD4⁺ regulatory T cells (T_reg)? This study addressed these questions in mice and compared the relative efficacy of administering ovalbumin (OVA) conjugated to cholera toxin B (CTB) subunit with administration of the same Ag alone. We found that s.l. administration of a single or even more efficiently three repeated 40‐μg doses of OVA/CTB conjugate suppressed T‐cell proliferative responses to OVA by cervical lymph node (CLN), mesenteric lymph node (MLN) and spleen cells and concurrently strongly increased the frequency of Ag‐specific T_reg in CLN, MLN and spleen and also transforming growth factor‐β (TGF‐β) levels in serum. The CLN and splenic cells from OVA/CTB‐treated BALB/c mice efficiently suppressed OVA‐specific T‐cell receptor (TCR) transgenic (DO11.10) CD25^?CD4⁺ effector T‐cell proliferation in vitro. Further, s.l. treatment with OVA/CTB completely suppressed OVA‐specific DTH responses in vivo and T‐cell proliferative responses in mice immunized subcutaneously with OVA in Freund's complete adjuvant. The intracellular expression of Foxp3 was strongly increased in OVA‐specific (KJ1‐26⁺) CD4⁺ T cells from OVA/CTB‐treated mice. Thus, s.l. administration of CTB‐conjugated Ag can efficiently induce peripheral T‐cell tolerance associated with strong increases in serum TGF‐β levels and in Ag‐specific Foxp3⁺CD25⁺CD4⁺ T_reg cells.     

FTY720-induced conversion of conventional Foxp3- CD4+ T cells to Foxp3+ regulatory T cells in NOD mice

Citation Sun Y, Wang W, Shan B, Di J, Chen L, Ren L, Li W, Li D‐J, Lin Y. FTY720‐induced conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in NOD mice. Am J Reprod Immunol 2011; 66: 349–362 Problem FTY720 is known as an agonist of sphingosine‐1‐phosphate (S1P) receptor, but little is known about the possibility that FTY720 induces the conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in non‐obese diabetic (NOD) mice. Method of study FTY720 treatment was performed using Foxp3^?CD4⁺ T cells purified from NOD mice. Results FTY720 caused an increase in Foxp3⁺ Treg cells in lymphoid organs in NOD mice. FTY720 effectively induced Foxp3 expression in Foxp3^?CD4⁺ T cells both in vitro and in vivo, an effect that was inhibited by a TGF‐β‐neutralizing antibody or the proinflammatory cytokine IL‐6. T‐cell‐mediated embryo rejection in NOD mice was prevented upon FTY720 treatment. Conclusions The use of FTY720 along with Ag administration may represent a useful therapeutic strategy to selectively expand Ag‐specific Foxp3⁺ Tregs to intervene autoimmune and infectious diseases.     

T‐cell tolerance induced by repeated antigen stimulation: Selective loss of Foxp3− conventional CD4 T cells and induction of CD4 T‐cell anergy

Repeated immunization of mice with bacterial superantigens induces extensive deletion and anergy of reactive CD4 T cells. Here we report that the in vitro proliferation anergy of CD4 T cells from TCR transgenic mice immunized three times with staphylococcal enterotoxin B (SEB) (3× SEB) is partially due to an increased frequency of Foxp3⁺ CD4 T cells. Importantly, reduced number of conventional CD25^? Foxp3^? cells, rather than conversion of such cells to Foxp3⁺ cells, was the cause of that increase and was also seen in mice repeatedly immunized with OVA (3× OVA) and OVA—peptide (OVAp) (3× OVAp). Cell‐transfer experiments revealed profound but transient anergy of CD4 T cells isolated from 3× OVAp and 3× SEB mice. However, the in vivo anergy was CD4 T‐cell autonomous and independent of Foxp3⁺ Treg. Finally, proliferation of transferred CD4 T cells was inhibited in repeatedly immunized mice but inhibition was lost when transfer was delayed, despite the maintenance of elevated frequency of Foxp3⁺ cells. These data provide important implications for Foxp3⁺ cell‐mediated tolerance in situations of repeated antigen exposure such as human persistent infections.     

Activation of CD4+ CD25+ regulatory T cell suppressor function by analogs of the selecting peptide

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells can undergo both thymic selection and peripheral expansion in response to self peptides that are agonists for their T cell receptors (TCR). However, the specificity by which these TCR must recognize peptide:MHC complexes to activate Treg cell function is not known. We show that CD4⁺CD25⁺Foxp3⁺ Treg cells can mediate suppression in response to peptides that are only weakly cross‐reactive with the self peptide that induced their formation in vivo. Moreover, suppression could be efficiently activated by peptide analogs that were inefficient at inducing CD69 up‐regulation, and that also induced little or no proliferation of naïve CD4⁺CD25^–Foxp3^– T cells expressing the same TCR. These findings provide evidence that self peptide‐specific CD4⁺CD25⁺Foxp3⁺ Treg cells can exert regulatory function in response to self‐ and/or pathogen‐derived peptides with which they are only weakly cross‐reactive.     

Heterologous vaccination against human tuberculosis modulates antigen‐specific CD4+ T‐cell function

Heterologous prime‐boost strategies hold promise for vaccination against tuberculosis. However, the T‐cell characteristics required for protection are not known. We proposed that boost vaccines should induce long‐lived functional and phenotypic changes to T cells primed by Bacille Calmette Guerin (BCG) and/or natural exposure to mycobacteria. We characterized changes among specific CD4⁺ T cells after vaccination with the MVA85A vaccine in adults, adolescents, and children. CD4⁺ T cells identified with Ag85A peptide‐bearing HLA class II tetramers were characterized by flow cytometry. We also measured proliferative potential and cytokine expression of Ag85A‐specific CD4⁺ T cells. During the effector phase, MVA85A‐induced specific CD4⁺ T cells coexpressed IFN‐γ and IL‐2, skin homing integrins, and the activation marker CD38. This was followed by contraction and a transition to predominantly IL‐2‐expressing, CD45RA^?CCR7⁺CD27⁺ or CD45RA⁺CCR7⁺CD27⁺ specific CD4⁺ T cells. These surface phenotypes were similar to Ag85A‐specific T cells prior to MVA85A. However, functional differences were observed postvaccination: specific proliferative capacity was markedly higher after 6–12 months than before vaccination. Our data suggest that MVA85A vaccination may modulate Ag85A‐specific CD4⁺ T‐cell function, resulting in greater recall potential. Importantly, surface phenotypes commonly used as proxies for memory T‐cell function did not associate with functional effects of vaccination.     

Immunogenicity and protective efficacy of a DNA vaccine encoding the fusion protein of mycobacterium heat shock protein 65(Hsp65) with human interleukin‐2 against Mycobacterium tuberculosis in BALB/c mice

Developing a new generation of vaccines is important for preventing tuberculosis (TB). DNA vaccine is one promising candidate. In this study we evaluated the immunogenicity and protective efficacy of the DNA vaccine encoding the fusion protein of Mycobacterium tuberculosis heat shock protein 65 (Hsp65) with human interleukin‐2 (hIL‐2) in BALB/c mice. We showed that the DNA vaccine pcDNA‐Hsp65‐hIL‐2 could induce high levels of antigen‐specific antibody, IFN‐γ, CD4⁺ and CD8⁺ T cell production. When the immunized mice were infected with M. tuberculosis H37Rv, the organ bacterial loads in the DNA immunized group were significantly reduced compared to those of the saline control group, but the ability to reduce bacteria was not better than for BCG. The histopathology in lungs of the DNA vaccine immunized mice was similar to that of BCG immunized mice, which was obviously ameliorated compared to that of the saline control group. Overall, the DNA vaccine could afford protection against M. tuberculosis infection, though the protection efficacy was not as great as that of conventional BCG.     

CD4+ Foxp3+ regulatory T‐cell number increases in the gastric tissue of C57BL/6 mice infected with Helicobacter pylori

Helicobacter pylori (H. pylori), one of the most common infections, is associated with various clinical outcomes. In addition to inducing inflammation, immunological clearance of the pathogen is often incomplete. Regulatory T cells (Treg cells) have been recently demonstrated to play an important role in H. pylori infection and the final clinical outcome. The aim of this study was to investigate the number and localization of CD4⁺Foxp3⁺ Treg cells in stomachs and spleens of H. pylori‐infected mice. The expression levels of Foxp3 as well as anti‐ and pro‐inflammatory cytokines before and after H. pylori triple eradication therapy were examined. We found that the percentages of CD4⁺Foxp3⁺ Treg cells out of the lamina propria lymphocytes (LPLs) and spleen lymphocytes in the infection group were higher than the PBS negative control group and the treatment group. H. pylori antigen stimulation was associated with an increased number of Treg cells in vitro. Furthermore, compared with the PBS and treatment groups, a higher mRNA expression level of Foxp3 in the gastric tissue was detected in the infection group. IL‐10 and TGF‐β1 contents were increased significantly in the culture supernatant of spleen lymphocyte stimulated with H. pylori antigen. A marked elevation in serum IFN‐γ level was observed in H. pylori‐infected mice. In addition, gastric tissues of the infection group contained more Foxp3⁺ cells. These results indicate that the percentage of CD4⁺Foxp3⁺ Treg cells are increased in H. pylori‐infected mice, suggesting a role of Treg cells in H. pylori‐induced pathologies, even at the early stages of chronic gastritis and gastric tumorigenesis.     

Early recruitment of natural CD4+Foxp3+ Treg cells by infective larvae determines the outcome of filarial infection

Human helminth infections are synonymous with impaired immune responsiveness indicating suppression of host immunity. Using a permissive murine model of filariasis, Litomosoides sigmodontis infection of inbred mice, we demonstrate rapid recruitment and increased in vivo proliferation of CD4⁺Foxp3⁺ Treg cells upon exposure to infective L3 larvae. Within 7 days post‐infection this resulted in an increased percentage of CD4⁺T cells at the infection site expressing Foxp3. Antibody‐mediated depletion of CD25⁺ cells prior to infection to remove pre‐existing ‘natural’ CD4⁺CD25⁺Foxp3⁺ Treg cells, while not affecting initial larval establishment, significantly reduced the number of adult parasites recovered 60 days post‐infection. Anti‐CD25 pre‐treatment also impaired the fecundity of the surviving female parasites, which had reduced numbers of healthy eggs and microfilaria within their uteri, translating to a reduced level of blood microfilaraemia. Enhanced parasite killing was associated with augmented in vitro production of antigen‐specific IL‐4, IL‐5, IL‐13 and IL‐10. Thus, upon infection filarial larvae rapidly provoke a CD4⁺Foxp3⁺ Treg‐cell response, biasing the initial CD4⁺ T‐cell response towards a regulatory phenotype. These CD4⁺Foxp3⁺ Treg cells are predominantly recruited from the ‘natural’ regulatory pool and act to inhibit protective immunity over the full course of infection.     

CTLA4-Ig modifies dendritic cells from mice with collagen-induced arthritis to increase the CD4+CD25+Foxp3+ regulatory T cell population

Cytotoxic T lymphocyte antigen-4 (CTLA4) and IgG fusion protein, CTLA4-Ig, is a therapeutic agent used for rheumatoid arthritis. It binds B7 molecules on dendritic cells (DCs) and thereby blocks B7/CD28 costimulatory interaction and inhibits effective T cell proliferation. However, the effect of CTLA4-Ig on the regulatory T cell (Treg) is still not known. In this study, we investigated the influence of CTLA4-Ig on the CD4+CD25+Foxp3+ Treg population in collagen-induced arthritis (CIA) mouse model. CTLA4-Ig suppressed CIA and increased the CD4+CD25+Foxp3+ Treg population in joint and spleen. When CD11c + DCs and CD4+T cells from CIA mice were cultured with anti-CD3, CTLA4-Ig increased the CD4+CD25 + Foxp3+ Treg population in a TGF-β-dependent manner. When CD11c + DCs from CIA mice were treated with CTLA4-Ig and adoptively transferred into CIA-induced mice, arthritis did not develop in association with the increase in CD4+CD25+Foxp3+ Treg population. However, in CTLA4-Ig-untreated DC-transferred CIA mice, arthritis developed and then rapidly progressed. Our study demonstrated that CTLA4-Ig suppressed CIA by modifying DCs from CIA mice into tolerogenic DCs to increase the CD4+CD25+Foxp3+ Treg population and this seems to be the new immune regulatory mechanism of CTLA4-Ig.     

Hsp65-producing Lactococcus lactis prevents experimental autoimmune encephalomyelitis in mice by inducing CD4+LAP+ regulatory T cells

Heat shock proteins (Hsps) participate in the cellular response to stress and they are hiperexpressed in inflammatory conditions. They are also known to play a major role in immune modulation, controlling, for instance, autoimmune responses. In this study, we showed that oral administration of a recombinant Lactococcus lactis strain that produces and releases LPS-free Hsp65 prevented the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. This was confirmed by the reduced inflammatory cell infiltrate and absence of injury signs in the spinal cord. The effect was associated with reduced IL-17 and increased IL-10 production in mesenteric lymph node and spleen cell cultures. Hsp65-producing-L. lactis-fed mice had a remarkable increase in the number of natural and inducible CD4+Foxp3+ regulatory T (Treg) cells and CD4+LAP+ (Latency-associated peptide) Tregs - which express the membrane-bound TGF-β - in spleen, inguinal and mesenteric lymph nodes as well as in spinal cord. Moreover, many Tregs co-expressed Foxp3 and LAP. In vivo depletion of LAP+ cells abrogated the effect of Hsp65-producing L. lactis in EAE prevention and worsened disease in medium-fed mice. Thus, Hsp65-L.lactis seems to boost this critical regulatory circuit involved in controlling EAE development in mice.     

MHC‐restricted Ag85B‐specific CD8+ T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice

Despite efforts to develop effective treatments and vaccines, Mycobacterium tuberculosis (Mtb), particularly pulmonary Mtb, continues to provide major health challenges worldwide. To improve immunization against the persistent health challenge of Mtb infection, we have studied the CD8⁺ T cell response to Bacillus Calmette‐Guérin (BCG) and recombinant BCG (rBCG) in mice. Here, we generated CD8⁺ T cells with an rBCG‐based vaccine encoding the Ag85B protein of M. kansasii, termed rBCG‐Mkan85B, followed by boosting with plasmid DNA expressing the Ag85B gene (DNA‐Mkan85B). We identified two MHC‐I (H2‐K^d)‐restricted epitopes that induce cross‐reactive responses to Mtb and other related mycobacteria in both BALB/c (H2^d) and CB6F1 (H2^b/d) mice. The H2‐K^d‐restricted peptide epitopes elicited polyfunctional CD8⁺ T cell responses that were also highly cross‐reactive with those of other proteins of the Ag85 complex. Tetramer staining indicated that the two H2‐K^d‐restricted epitopes elicit distinct CD8⁺ T cell populations, a result explained by the X‐ray structure of the two peptide/H2‐K^d complexes. These results suggest that rBCG‐Mkan85B vector‐based immunization and DNA‐Mkan85B boost may enhance CD8⁺ T cell response to Mtb, and might help to overcome the limited effectiveness of the current BCG in eliciting tuberculosis immunity.     

UV inhibits allergic airways disease in mice by reducing effector CD4+ T cells

Background In human asthma, and experimental allergic airways disease in mice, antigen‐presenting cells and CD4⁺ effector cells at the airway mucosa orchestrate, and CD4⁺CD25⁺ regulatory T cells attenuate, allergen immunity. UV irradiation of skin before sensitization with ovalbumin (OVA) causes significantly reduced asthma‐like responses in respiratory tissues. Objective To determine whether UV‐induced changes in CD11c⁺ cells, CD4⁺CD25⁺ effector cells or CD4⁺CD25⁺ regulatory cells in the trachea and airway draining lymph nodes (ADLNs) were responsible for reduced allergic airways disease. Methods The phenotype and function of CD11c⁺ cells and CD4⁺CD25⁺ cells in the trachea and ADLNs of UV‐ and non‐irradiated, OVA‐sensitized mice was examined 24 h after a single exposure to aerosolized OVA. Results No changes in the function of CD11c⁺ cells from UV‐irradiated mice were observed. CD4⁺CD25⁺ cells from UV‐irradiated, OVA‐sensitized mice harvested 24 h after OVA aerosol proliferated less in response to OVA in vitro and were unable to suppress the proliferation of OVA‐sensitized responder cells. This result suggested reduced activation of effector T cells in the airway mucosa of UV‐irradiated, OVA‐sensitized mice. To exclude regulatory cells of any type, there was similar proliferation in vivo to aerosolized OVA by CFSE‐loaded, OVA‐TCR‐specific CD4⁺ cells adoptively transferred into UV‐ and non‐irradiated, OVA‐sensitized mice. In addition, there was no difference in the expression of regulatory T cell markers (Foxp3, IL‐10, TGF‐β mRNA). To examine effector T cells, ADLN cells from UV‐irradiated, OVA‐sensitized and ‐challenged mice were cultured with OVA. There was reduced expression of the early activation marker CD69 by CD4⁺CD25⁺ cells, and reduced proliferation in the absence of the regulatory cytokine, IL‐10. Conclusion Reduced allergic airways disease in UV‐irradiated mice is due to fewer effector CD4⁺CD25⁺ cells in the trachea and ADLNs, and not due to UV‐induced regulatory cells. Cite this as: J. P. McGlade, D. H. Strickland, M. J. M. Lambert, S. Gorman, J. A. Thomas, M. A. Judge, J. T. Burchell, G. R. Zosky and P. H. Hart, Clinical & Experimental Allergy, 2010 (40) 772–785.     

Control of Schistosoma mansoni egg‐induced inflammation by IL‐4‐responsive CD4+CD25−CD103+Foxp3− cells is IL‐10‐dependent

Host protection to helminth infection requires IL‐4 receptor α chain (IL‐4Rα) signalling and the establishment of finely regulated Th2 responses. In the current study, the role of IL‐4Rα‐responsive T cells in Schistosoma mansoni egg‐induced inflammation was investigated. Egg‐induced inflammation in IL‐4Rα‐responsive BALB/c mice was accompanied with Th2‐biased responses, whereas T‐cell‐specific IL‐4Rα‐deficient BALB/c mice (iLck^creIl4ra^?/lox) developed Th1‐biased responses with heightened inflammation. The proportion of Foxp3⁺ Treg in the draining LN of control mice did not correlate with the control of inflammation and was reduced in comparison to T‐cell‐specific IL‐4Rα‐deficient mice. This was due to IL‐4‐mediated inhibition of CD4⁺Foxp3⁺ Treg conversion, demonstrated in adoptively transferred Rag2^?/? mice. Interestingly, reduced footpad swelling in Il4ra^?/lox mice was associated with the induction of IL‐4 and IL‐10‐secreting CD4⁺CD25^?CD103⁺Foxp3^? cells, confirmed in S. mansoni infection studies. Transfer of IL‐4Rα‐responsive CD4⁺CD25^?CD103⁺ cells, but not CD4⁺CD25^high or CD4⁺CD25^?CD103^? cells, controlled inflammation in iLck^creIl4ra^?/lox mice. The control of inflammation depended on IL‐10, as transferred CD4⁺CD25^?CD103⁺ cells from IL‐10‐deficient mice were not able to effectively downregulate inflammation. Together, these results demonstrate that IL‐4 signalling in T cells inhibits Foxp3⁺ Treg in vivo and promotes CD4⁺CD25^?CD103⁺Foxp3^? cells that control S. mansoni egg‐induced inflammation via IL‐10.     

Follicular regulatory T cells repress cytokine production by follicular helper T cells and optimize IgG responses in mice

Follicular helper T (Tfh) cells provide crucial help to germinal center B (GCB) cells for proper antibody production, and a specialized subset of regulatory T cells, follicular regulatory T (Tfr) cells, modulate this process. However, Tfr‐cell function in the GC is not well understood. Here, we define Tfr cells as a CD4⁺ Foxp3⁺ CXCR5^hi PD‐1^hi CD25^low TIGIT^high T‐cell population. Furthermore, we have used a novel mouse model (“Bcl6FC”) to delete the Bcl6 gene in Foxp3⁺ T cells and thus specifically deplete Tfr cells. Following immunization, Bcl6FC mice develop normal Tfh‐ and GCB‐cell populations. However, Bcl6FC mice produce altered antigen‐specific antibody responses, with reduced titers of IgG and significantly increased IgA. Bcl6FC mice also developed IgG antibodies with significantly decreased avidity to antigen in an HIV‐1 gp120 “prime‐boost” vaccine model. In an autoimmune lupus model, we observed strongly elevated anti‐DNA IgA titers in Bcl6FC mice. Additionally, Tfh cells from Bcl6FC mice consistently produce higher levels of Interferon‐γ, IL‐10 and IL‐21. Loss of Tfr cells therefore leads to highly abnormal Tfh‐cell and GCB‐cell responses. Overall, our study has uncovered unique regulatory roles for Tfr cells in the GC response.     

Mycobacterium chelonae sensitisation induces CD4+‐mediated cytotoxicity against BCG

Significant variability in efficacy of live Mycobacterium bovis BCG as a tuberculosis vaccine is observed globally. Effects of pre‐vaccination sensitisation to non‐tuberculous environmental mycobacteria (Env) are suspected to underlie this phenomenon, but the mechanisms remain unclear. We postulated that it could be due to Env‐specific T cells exerting cytotoxicity against BCG‐infected host cells. After murine sensitisation with heat‐killed antigens of different Env species, splenocytes from M. chelonae (CHE)‐sensitised mice exerted the strongest cytotoxicity against autologous BCG‐infected macrophages. This cytotoxicity was correlated with reduced BCG viability. The cytotoxicity was reduced by the depletion of CD4⁺, but not CD8⁺ or CD56⁺ cells, and CD4⁺ cells showed higher percentage of cytotoxicity than CD4^? cells, supporting a role for CD4⁺ cells in CHE‐induced, BCG‐specific cytotoxicity. Additionally, this cytotoxicity was IFN‐γ, perforin and FasL dependent. After CHE‐sensitisation and subsequent BCG intranasal infection, there was significant expansion of lung CD4⁺ cells, the main cell type producing IFN‐γ. This was associated with 2‐ and 6‐fold reductions in lung BCG counts 1 and 3 wk, respectively post‐ infection, relative to non‐sensitised mice. This is the first report describing cytotoxicity against BCG‐infected cells as a mechanism underlying the influence of Env sensitisation on subsequent BCG responses.     

The Ex Vivo Induction of Human CD103+ CD25hi Foxp3+ CD4+ and CD8+ Tregs is IL‐2 and TGF‐β1 Dependent

The expression of the integrin αE (CD103), may enhance the retention of regulatory T cells to peripheral inflammatory sites and possibly contribute to their suppressive potential. The aim of this study was to define the regulatory role of IL‐2 and TGF‐β1 on the CD103 expression and the optimal in vitro conditions for the induction/expansion of human CD4⁺ and CD8⁺ Tregs. Cord blood mononuclear cells (CBMC) were stimulated under various culture conditions, including anti‐CD3, anti‐CD28, IL‐2 and TGF‐β1. TGF‐β1 and IL‐2 were both required for optimal expression of CD103. In addition, TGF‐β1 and IL‐2 synergistically induced CD103 expression on CD8⁺ T cells, whereas, only additive induced expression was noted on CD4⁺ T cells. Surprisingly, CD103 expression was not dependent upon CD28 costimulation. IL‐2 also played a central role in CD103 expression by CD25^hi Foxp3+ Tregs. IL‐2, TGF‐β1 and anti‐CD3 defined the optimal stimulatory conditions favouring the induction/expansion of both CD4⁺ and CD8⁺ human Tregs from naive CBMC. Thus, this study provides new insights into the regulatory role of IL‐2 upon CD103 expression by human cord blood CD4⁺ and CD8⁺ T cells. Furthermore, it identifies the in vitro culture conditions driving the differentiation of the novel phenotype CD4⁺ and CD8⁺ CD103⁺ CD25^hi Foxp3+ Tregs from human CBMC.     

CD4+CD25+Foxp3+ Regulatory T Cells Contribute in Liver Fibrosis Improvement with Interferon Alpha

The aim of this study is to investigate the optimal dose, treatment time, and possible immunologic mechanisms of interferon alpha (IFN-α) in the treatment of liver fibrosis. Mice were injected intraperitoneally with 10 % carbon tetrachloride to induce liver fibrosis, except in the normal control group. The experimental mice were randomly divided into four groups: physiological saline group, 20 U/gb wt IFN-α group, 40 U/gb wt IFN-α group, and 60 U/gb wt IFN-α group. After 3 and 6 weeks, type I collagen was detected in liver by hematoxylin and eosin (HE) stain, Masson’s trichrome stain, and immunohistochemical staining. The number of CD8⁺ T cells, the number of CD4⁺CD25⁺Foxp3⁺ Tregs and the activation of CD4⁺ T cells were detected in liver and spleen. Beneficial effects were observed in the 40 U/gb wt IFN-α group by pathological analysis. The number of CD8⁺ T cells in the liver was significantly lower in mice receiving middle-dose IFN-α therapy as compared to mice receiving physiological saline (P?<?0.05), while CD4⁺CD25⁺Foxp3⁺ Tregs and activation of CD4⁺ T cells in the liver were significantly higher in the therapeutic group than in the physiological saline group (P?<?0.05). CD8⁺ T cells (r?=?0.3796) and activated CD4⁺ T cells (r?=?0.2437) were found to be positively correlated with the degree of liver fibrosis. CD4⁺CD25⁺Foxp3⁺ Tregs (r?=??0.7932) was found to be negatively correlated with the degree of liver fibrosis. IFN-α can inhibit liver fibrosis following 6 weeks of middle-dose IFN-α therapy by upregulating CD4⁺CD25⁺Foxp3⁺ Tregs and suppressing CD8⁺ T cells.