Fleur Samantha Benghiat

Critical influence of natural regulatory CD25+ T cells on the fate of allografts in the absence of immunosuppression.

Background. Allografts are occasionally accepted in the absence of immunosuppression. Because naturally occurring CD4+CD25+ regulatory T cells (natural CD25+ Treg cells) have been shown to inhibit allograft rejection, we investigated their influence on the outcome of allografts in nonimmunosuppressed mouse recipients. Methods. We compared survival times of male CBA/Ca skin grafts in female CBA/Ca recipients expressing a transgenic anti-HY T-cell receptor on a RAG-1+/+ (A1[M]RAG+) or a RAG-1−/− (A1[M]RAG−) background. Depletion of natural CD25+ Treg cells in A1[M]RAG+ mice was achieved by in vivo administration of the PC61 monoclonal antibody. The influence of natural CD25+ Treg cells on the fate of major histocompatibility complex class II-mismatched (C57BL/6× bm12)F1 skin or bm12 heart transplants in C57BL/6 recipients was also assessed. Finally, we investigated the impact of natural CD25+ Treg cells on the production of T-helper (Th)1 and Th2 cytokines in mixed lymphocyte cultures between C57BL/6 CD4+ CD25− T cells as responders and bm12 or (C57BL/6× bm12)F1 antigen-presenting cells as stimulators. Results. Male allografts were spontaneously accepted by female A1(M)RAG+ mice but readily rejected by female A1(M)RAG+ mice depleted of natural CD25+ Treg cells by pretreatment with the PC61 monoclonal antibody. Depletion of CD25+ Treg cells also enhanced eosinophil-determined rejection of (C57BL/6× bm12)F1 skin grafts or bm12 cardiac grafts in C57BL/6 recipients. Finally, natural CD25+ Treg cells inhibited the production of interleukin (IL)-2, interferon-γ, IL-5, and IL-13 in mixed lymphocyte culture in a dose-dependent manner. Conclusion. Natural CD25+ Treg cells control Th1- and Th2-type allohelper T-cell responses and thereby influence the fate of allografts in nonimmunosuppressed recipients.

Critical Role of Regulatory T Cells in Th17-Mediated Minor Antigen-Disparate Rejection

Th17-mediated immune responses have been recently identified as novel pathogenic mechanisms in a variety of conditions; however, their importance in allograft rejection processes is still debated. In this paper, we searched for MHC or minor Ag disparate models of skin graft rejection in which Th17 immune responses might be involved. We found that T cell-derived IL-17 is critical for spontaneous rejection of minor but not major Ag-mismatched skin grafts. IL-17 neutralization was associated with a lack of neutrophil infiltration and neutrophil depletion delayed rejection, suggesting neutrophils as an effector mechanism downstream of Th17 cells. Regulatory T cells (Tregs) appeared to be involved in Th17 reactivity. We found that in vivo Treg depletion prevented IL-17 production by recipient T cells. An adoptive cotransfer of Tregs with naive monospecific antidonor T cells in lymphopenic hosts biased the immune response toward Th17. Finally, we observed that IL-6 was central for balancing Tregs and Th17 cells as demonstrated by the prevention of Th17 differentiation, the enhanced Treg/Th17 ratio, and a net impact of rejection blockade in the absence of IL-6. In conclusion, the ability of Tregs to promote the Th17/neutrophil-mediated pathway of rejection that we have described should be considered as a potential drawback of Treg-based cell therapy.

Interleukin 17–producing T helper cells in alloimmunity

Interleukin (IL) 17 is a proinflammatory cytokine already known to play a defense role against microbes and a pathogenic role in a number of autoimmune diseases. Although IL-17 can be produced by a variety of cells including neutrophils, CD8+, NK, and gamma-delta T cells, the concept of IL-17-secreting CD4+ T helper cells (Th17), distinct from Th1 and Th2, recently emerged. Herein, we discuss arguments in favor of a Th17-mediated alternative pathway of allograft rejection based on clinical and experimental observations drawn from the literature. We also discuss the complex interplays among regulatory T cells and Th17 cells in the allogeneic context.

IL-17 production elicited by allo-major histocompatibility complex class II recognition depends on CD25posCD4pos T cells.

Background. Interleukin (IL)-17 is involved in autoimmune inflammatory disorders and naturally occurring CD25pos regulatory T cells were shown to promote IL-17 synthesis. Because IL-17 is overproduced in certain types of allograft rejection, it is important to characterize the cells responsible for IL-17 synthesis and to define how IL-17 is regulated during alloimmune responses. Methods. Splenic CD4pos T cells were isolated from C57BL/6 mice and fractionated according to CD25 expression. T cells were stimulated by major histocompatibility complex class II-mismatched bone marrow-derived dendritic cells from bm12 mice, either immature or made mature by exposure to lipopolysaccharide. To track T cell populations, CD25negCD4pos and CD25posCD4pos were isolated from Thy1.1 and congenic Thy1.2 mice, respectively. Cell proliferation was quantified by CFSE dilution. IL-17-producing cells and FOXP3pos cells were enumerated by intracytoplasmic staining and cytokine levels in culture supernatants were measured by ELISA. Results. Addition of CD25posCD4pos T cells to CD25negCD4pos T cells inhibited IL-2, interferon-γ, and IL-13 production but promoted IL-17 synthesis on stimulation by allogenic immature DC. In this setting, IL-17 originated from CD25intCD4posFOXP3neg memory T cells, which depend on IL-2 to produce IL-17. Alloreactive CD25negCD4pos T cells were also induced to produce IL-17 when stimulated by mature DC in the presence of CD25highCD4posFOXP3pos T cells. Conclusions. We conclude that (1) the cellular source of IL-17 during an antiallo major histocompatibility complex class II response depends on the maturation status of allogenic DC, (2) whereas suppressing Th1 and Th2 cytokine synthesis, naturally occurring regulatory T cells, allow IL-17 production by alloreactive CD4pos T cells.

CD8+ T-Cell depletion and rapamycin synergize with combined coreceptor/stimulation blockade to induce robust limb allograft tolerance in mice.

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8+ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8+ T‐cell depletion and costimulation/coreceptor blockade.

Pertussis toxin activates adult and neonatal naive human CD4+ T lymphocytes

Pertussis toxin (PTX) is known to be mitogenic for T lymphocytes, but its direct action on naive human T cells has not been specified. Herein, we show that PTX induces the proliferation of purified adult CD45RA+CD4+ T cells independently of its ADP‐ribosyltransferase activity. PTX directly induces TNF‐α and IL‐2 mRNA expression, modulates the level of several cell surface receptors and induces Forkhead box p3 (Foxp3) protein accumulation in naive CD4+ T cells. Addition of autologous dendritic cells was found to be required for the production of high levels of IFN‐γ by PTX‐stimulated naive T cells. These effects of PTX occurred in conjunction with activation of NF‐κB and NFAT transcription factors. Overall, responses of neonatal CD4+ T cells to PTX were similar to those of adult CD45RA+CD4+ naive T cells except for their blunted CD40 ligand up‐regulation. We suggest that the adjuvant properties of PTX during primary cell‐mediated immune responses involve a direct action on naive T lymphocytes in addition to activation of antigen‐presenting cells.

Severe auto-immune hemolytic anemia in a fingolimod-treated multiple sclerosis patient

A 19-year-old multiple sclerosis (MS) patient was admitted to the emergency department with acute symptoms of fever, jaundice, nausea and fatigue. The patient did not have any history of hematological or systemic disease and was taking no medication but fingolimod for the last 10 months. Laboratory assessments confirmed the diagnosis of immune-mediated acute hemolytic anemia, as suggested by hematological and biochemical findings (hemoglobin 6.0 g/dl (11.8–15.5), serum bilirubin 2.3 mg/dl (< 1.2), serum unconjugated bilirubin 0.84 mg/dl (< 0.5), serum lactate dehydrogenase (LDH) 537 UI/l (< 214), serum haptoglobin < 5 mg/dl (30–200)). Liver enzymes and reticulocytes were in the normal range. Direct antiglobulin test (DAT) was positive (immunoglobulin (Ig)G-positive, C3d-negative) and cold agglutinins test was negative. Glucose-6-phosphate dehydrogenase test, antinuclear antibodies and lymphocyte immunophenotype analysis, serological tests (hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), parvovirus B19, Epstein-Barr virus (EBV), human herpes virus 6 (HHV6)) and abdominal ultrasound did not reveal any abnormality. The bone marrow analysis showed no sign of hematological malignancy, but a lack of mature red blood cell precursors, probably reflecting concomitant accelerated immune-mediated destruction of red blood precursors within the marrow as previously described.1 Two weeks later and despite an initial whole blood transfusion and methylprednisolone treatment (2 mg/kg/day) and an LDH and bilirubin improvement, hemoglobin was still at admission levels. Only after fingolimod cessation did biological parameters start to improve. Corticosteroids were tapered over a two-month period and laboratory findings remain normal several months later. Fingolimod (FTY720), a sphingosine-1-phosphatereceptor modulator, is the first oral treatment for relapsing– remitting MS. It is the first time that an autoimmune hemolytic anemia is reported in a patient treated with fingolimod. In our opinion, the causal link is quite probable, considering the absence of any other comorbidity and the improvement of biological parameters only after fingolimod cessation. No additional laboratory experiment could clarify the origin of this IgG hemolytic anemia, since in this kind of autoimmune phenomenon, antibodies are drug independent (drug need not be present to detect antibodies in vitro) and so it presents clinically and serologically as an autoimmune hemolytic anemia (AIHA) with red cell (RBC) autoantibodies in patients’ sera and in eluates from their RBCs.2 The causality in this particular patient could be proven only if the same adverse event appeared once again after fingolimod re-introduction, which could not be an option from the ethical point of view. Fingolimod has been shown to effect in several ways cell survival, from triggering apoptosis to even protecting against cell death.3 An in vitro apoptotic effect of fingolimod in erythrocytes partially due to an increase in cytosolic Ca2+ concentration has been reported. It is of interest that although fingolimod is considered to be effective as an antiinflammatory agent following a phosphorylation and subsequent stimulation of sphingosine phosphate receptor S1P1,4 erythrocytes are not able to phosphorylate FTY7205 and thus its apoptotic effect may be mediated by its nonphosphorylated form. In our opinion, particular attention should be paid to patients receiving drugs or suffering from disorders simulating eryptosis, since sensitivity to fingolimod could be enhanced.

Endotoxin hyperresponsiveness upon CD4+ T cell reconstitution in lymphopenic mice: control by natural regulatory T cells

Natural CD4+CD25+ regulatory T cells (nTreg) have been shown to control graft‐versus‐host disease after hematopoietic stem cell transplantation (HSCT). Herein, we considered the possibility that the beneficial action of nTreg upon immune reconstitution in lymphopenic hosts involves dampening of the inflammatory response induced by bacterial products. We first observed that transfer of syngeneic CD4+CD25– T cells in RAG‐deficient mice dramatically enhanced release of inflammatory cytokines and associated pathology upon endotoxin injection. Interferon (IFN)‐γ produced by T cells undergoing homeostatic proliferation was shown to be involved in the endotoxin hyperresponsiveness induced by CD4+ T cell reconstitution. Co‐transfer of CD4+CD25+ nTreg with CD4+CD25– T cells inhibited the expansion of IFN‐γ‐producing T cells and reduced endotoxin responses in RAG–/– mice. We conclude that (1) CD4+ T cell reconstitution sensitizes lymphopenic hosts to endotoxin‐induced pathology and (2) nTreg prevent this process by limiting the emergence of IFN‐γ‐producing cells.

Effect of N-acetylcysteine on pain in daily life in patients with sickle cell disease: a randomised clinical trial

N.J.S. collected and analysed the data and wrote the manuscript; H.K. designed the study and treated patients; E.J., J.E.C, D.A.T, M.E.R., N.D., Y.A., M.K., P.K., W.G.W. and C.D.D. treated patients; C.B., D.M. and M.A.R. collected and analysed the data; F.R. designed the study, collected and analysed the data, treated patients and wrote the manuscript. All authors approved the final version of the manuscript.

Adaptation of Trypanosoma rhodesiense to hypohaptoglobinaemic serum requires transcription of the APOL1 resistance gene in a RNA polymerase I locus

Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin‐related protein–haemoglobin complex, which binds to the parasite surface receptor for haptoglobin–haemoglobin. As haptoglobin–haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin‐poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol‐I)‐mediated expression of the serum resistance‐associated (SRA) protein, T. gambiense resists by pol‐II‐mediated expression of the T. gambiense‐specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin–haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin–haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor‐mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol‐I‐, but not with pol‐II‐mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol‐I locus. Thus, transcription by pol‐I favours adaptive gene regulation, explaining the presence of SRA in a pol‐I locus.