[Diagnostic performance of κ/λ serum free light chain ratio (Freelite® assay) and IgGκ/IgGλ ratio (Hevylite® assay) as prognostic biomarkers of the evolution of immune thrombocytopenia into a chronic disease in adult patients]. / Performance diagnostique des rapports κ/λ des chaines légères libres sériques (test Freelite®) et IgGκ/IgGλ (test Hevylite®) comme marqueurs pronostiques de chronicisation du purpura thrombopénique immunologique de l'adulte.

INTRODUCTION: Immune thrombocytopenia (ITP) is an acquired hemorrhagic disease due to antiplatelet antibodies, that will become a chronic disease in 70% of adults. Most of chronic ITP patients display clonal restriction of antiplatelet antibodies. To date, there is no biomarker able to predict the evolution of the disease. The objective of the study is to determine whether Hevylite® and/or Freelite® assays are prognostic factors for progression to chronic ITP. METHODS: This is a retrospective, monocentric, prognostic study of a biomarker, performed using frozen samples stored in a serum library. Freelite® and a Hevylite® assays were performed on the samples collected at diagnosis for adult patients with newly diagnosed ITP at the University Hospital of Poitiers between 2014/01/01 and 2017/05/01. To predict the evolution into a chronic disease, a ROC curve analysis was performed on four variables: IgGκ, IgGκ/IgGλ ratio, IgGκ - IgGλ, and κ/λ ratio. RESULTS: Thirty-two patients were included and analyzed. No patient had an abnormal κ/λ ratio. Three patients had an abnormal IgGκ/IgGλ ratio. The following variables IgGκ, IgGκ/IgGλ, IgGκ - IgGλ, and κ/λ ratio were not able to predict progression to chronic ITP in our study. CONCLUSION: This study did not reveal any prognostic value of the Freelite® and Hevylite® tests on the evolution of ITP into a chronic disease.

IL-33 receptor ST2 deficiency attenuates renal ischaemia-reperfusion injury in euglycaemic, but not streptozotocin-induced hyperglycaemic mice.

AIM: Kidney hypoxia can predispose to the development of acute and chronic renal failure in diabetes. Ischaemia-reperfusion injury (IRI) causes inflammation, and diabetes is known to exacerbate this inflammatory response in the kidney, whereas alarmin IL-33 could act as an innate immune mediator during kidney IRI. Thus, the present study examined the impact of genetic IL-33 receptor ST2 deficiency (ST2-/-) on renal IRI in euglycaemic and hyperglycaemic mice. METHODS: Hyperglycaemia was induced with streptozotocin (STZ) in adult male C57BL/6JRj wild-type (WT) mice and ST2-/- mice. Unilateral renal IRI was achieved 3months after STZ treatment by left kidney nephrectomy (non-ischaemic control kidney) and clamping of the right renal artery for 32min in STZ- and vehicle-treated animals. At 24h after reperfusion, renal function and injury were determined by levels of plasma creatinine, blood urea nitrogen (BUN) and histological tubule scores. Also, in a complementary pilot clinical study, soluble ST2 concentrations were compared in diabetics and non-diabetics. RESULTS: Urinary albumin was significantly increased in STZ-induced hyperglycaemic mice, regardless of genotypic background. At 24h post-ischaemia, plasma creatinine, BUN and tubular injury were significantly reduced in ST2-/- mice compared with vehicle-treated WT mice, but this protective effect was lost in the STZ-induced hyperglycaemic ST2-/- animals. Plasma concentrations of soluble ST2 were significantly greater in type 2 diabetes patients vs non-diabetics. CONCLUSION: Our data suggest that the IL-33/ST2 pathway exerts differential effects depending on the glucose environment, opening-up new avenues for future research on alarmins and diabetes in ischaemia-related diseases.

Identification of invariant natural killer T cells in porcine peripheral blood.

The pig is a relevant preclinical model for numerous pathologies used to validate therapeutic strategies for translation to human. Although invariant natural killer T (iNKT) lymphocytes are a component of innate immunity implicated in many pathological processes, little is known on their characterization in swine. By addressing this issue using mouse α-galactosylceramide-loaded CD1d tetramers (α-GC-CD1dTT), which are commonly used to track iNKT cells, we were able to unequivocally identify CD3(+)α-GC-CD1dTT(+) cells in porcine peripheral blood, hereafter referred to as swine iNKT cells. These lymphocytes are enriched in CD4(-)CD8(+) and CD4(-)CD8(-) cells, harbor an activated-memory phenotype (SLA-DR(+)CD45RA(-)), express the intracellular promyelocytic-leukemia-zinc-finger (PLZF) transcription factor and are significantly enriched in IFN-γ-producing cells after in vitro activation in comparison with conventional T cells. Importantly, in presence of IL-2 and IL-15, the iNKT cell ligand α-GC induces selective expansion of CD3(+)α-GC-CD1dTT(+) cells, confirming the reactivity of swine iNKT cells against α-GC. When associated with α-GC, IL-33, an alarmin of IL-1 family recently described to target iNKT cells, leads to a greater expansion of CD3(+)α-GC-CD1dTT(+) cells than IL-2 and IL-15. Altogether, our results provide the first phenotypic and functional description of swine iNKT cells allowing to further study the critical role of iNKT cells in porcine models of organ injury.

Freshly isolated Valpha24+ CD4+ invariant natural killer T cells activated by alpha-galactosylceramide-pulsed B cells promote both IgG and IgE production.

CD1d-restricted invariant natural killer T (iNK T) cells activated by their experimental ligand alpha-galactosylceramide (alpha-GC) can produce both T helper 1 (Th1) and Th2 cytokines and display regulatory functions. Recent studies identified CD4(+) and CD4(-) CD8(-) double-negative (DN) iNK T cells as the two major components of the human population and suggest that they display a Th2 and a Th1 profile, respectively. We compared the Th2-promoting activity of freshly isolated human CD4(+) and DN iNK T cells in terms of their capacity to induce Ig production by autologous B cells. Secretion of IgG and IgE but not IgM was enhanced by the CD4(+) T cell subset (including iNK T cells) but not by its DN counterpart. iNK T cells were directly responsible for this pro-Th2 effect, as demonstrated by the requirement for both alpha-GC stimulation and CD1d presentation, as well as by its disappearance upon iNK T cell depletion. Interaction with iNK T cells led to progressive accumulation of isotype-switched and activated B cells. Myeloid dendritic cells (DC) completely block the induction of Ig production in co-culture. This dominant inhibitory effect of myeloid DC was concomitant with a specific loss of interleukin (IL)-4 production by CD4(+) iNK T but not by conventional T cells. These data support the conclusion that, conversely to the interferon (IFN)-gamma-producing DN human iNK T cell population, interleukin (IL)-4-producing CD4(+) iNK T cells can activate and help B cells to produce both IgG and IgE through a CD1d-dependent mechanism, in keeping with a functional Th1/Th2 dichotomy between these subsets.

Antiphospholipid syndrome in patients with retinal venous occlusion.

INTRODUCTION: We conducted a prospective study to determine the prevalence and the prognosis of antiphospholipid syndrome (APS) in patients with retinal venous occlusion (RVO). PATIENTS: Consecutive patients presenting with retinal vein occlusion were screened for vascular risk factors (diabetes mellitus, hypertension, hyperlipidemia) and for antiphospholipid antibodies (aPL): anticardiolipin (aCL), anti-beta2-glycoprotein I, and lupus anticoagulant. Patients with a serum sample positive for aPL returned at least 6 weeks later for a new screening to determine the prevalence of antiphospholipid syndrome. All patients were followed to determine the outcome. RESULTS: Sixty-eight patients presented with RVO, 16 had vascular risk factors for RVO. After two screenings for aPL, nine cases of antiphospholipid syndrome associated with RVO were diagnosed (13.2%). Eight patients were over age 50 years and none had a previous thrombotic event before RVO. All patients were treated with aspirin (160 mg/day). With a mean follow-up of 26.1+/-8.2 months (range, 16-36 months), there were no recurrences. CONCLUSION: Retinal venous occlusion is multifactorial in origin. In patients aged 50 years and older, without previous thrombotic event, aPL might not be predictive of recurrences and treatment with aspirin might be sufficient. In such patients, the routine screening for aPL does not appear warranted, but a randomized study should be conducted to really ascertain the pathogenic role of aPL and the most appropriate treatment in RVO.

Activation of natural killer T cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune Type 1 diabetes.

Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6-9). Thus, given that NKT cells respond to the alpha-galactosylceramide (alpha-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by alpha-GalCer might prevent the onset and/or recurrence of T1D. Here we show that alpha-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, alpha-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to alpha-GalCer. Protection from T1D by alpha-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet beta cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility that alpha-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.

A subset of NKT cells that lacks the NK1.1 marker, expresses CD1d molecules, and autopresents the alpha-galactosylceramide antigen.

In the present report, we characterize a novel T cell subset that shares with the NKT cell lineage both CD1d-restriction and high reactivity in vivo and in vitro to the alpha-galactosylceramide (alpha-GalCer) glycolipid. These cells preferentially use the canonical Valpha14-Jalpha281 TCR-alpha-chain and Vbeta8 TCR-beta segments, and are stimulated by alpha-GalCer in a CD1d-dependent fashion. However, in contrast to classical NKT cells, they lack the NK1.1 marker and express high surface levels of CD1d molecules. In addition, this NK1.1(-) CD1d(high) T subset, further referred to as CD1d(high) NKT cells, can be distinguished by its unique functional features. Although NK1.1(+) NKT cells require exogenous CD1d-presenting cells to make them responsive to alpha-GalCer, CD1d(high) NKT cells can engage their own surface CD1d in an autocrine and/or paracrine manner. Furthermore, in response to alpha-GalCer, CD1d(high) NKT cells produce high amounts of IL-4 and moderate amounts of IFN-gamma, a cytokine profile more consistent with a Th2-like phenotype rather than the Th0-like phenotype typical of NK1.1(+) NKT cells. Our work reveals a far greater level of complexity within the NKT cell population than previously recognized and provides the first evidence for T cells that can be activated upon TCR ligation by CD1d-restricted recognition of their ligand in the absence of conventional APCs.

IL-7 up-regulates IL-4 production by splenic NK1.1+ and NK1.1- MHC class I-like/CD1-dependent CD4+ T cells.

NK T cells are an unusual subset of T lymphocytes. They express NK1. 1 Ag, are CD1 restricted, and highly skewed toward Vbeta8 for their TCR usage. They express the unique potential to produce large amounts of IL-4 and IFN-gamma immediately upon TCR cross-linking. We previously showed in the thymus that the NK T subset requires IL-7 for its functional maturation. In this study, we analyzed whether IL-7 was capable of regulating the production of IL-4 and IFN-gamma by the discrete NK T subset of CD4+ cells in the periphery. Two hours after injection of IL-7 into mice, or after a 4-h exposure to IL-7 in vitro, IL-4 production by CD4+ cells in response to anti-TCR-alphabeta is markedly increased. In contrast, IFN-gamma production remains essentially unchanged. In beta2-microglobulin- and CD1-deficient mice, which lack NK T cells, IL-7 treatment does not reestablish normal levels of IL-4 by CD4+ T cells. Moreover, we observe that in wild-type mice, the memory phenotype (CD62L-CD44+) CD4+ T cells responsible for IL-4 production are not only NK1.1+ cells, but also NK1.1- cells. This NK1.1-IL-4-producing subset shares three important characteristics with NK T cells: 1) Vbeta8 skewing; 2) CD1 restriction as demonstrated by their absence in CD1-deficient mice and relative overexpression in MHC II null mice; 3) sensitivity to IL-7 in terms of IL-4 production. In conclusion, the present study provides evidence that CD4+MHC class I-like-dependent T cell populations include not only NK1.1+ cells, but also NK1.1- cells, and that these two subsets are biased toward IL-4 production by IL-7.

Mature mainstream TCR alpha beta+CD4+ thymocytes expressing L-selectin mediate "active tolerance" in the nonobese diabetic mouse.

Pathogenic autoreactive T lymphocytes are mediators of spontaneous insulin-dependent diabetes in nonobese diabetic (NOD) mice. This is demonstrated by their capacity to transfer diabetes into syngeneic immunoincompetent recipients. In addition, especially in prediabetic NOD mice, peripheral CD4+ T lymphocytes were identified that are highly effective, in conventional mixing cotransfer experiments, at preventing disease transfer. The present data demonstrate that mature heat-stable Ag-TCR alpha beta+CD8-thymocytes from prediabetic NOD mice also express this inhibitory capacity. Selection using an L-selectin (CD62L)-specific Ab showed that TCR alpha/beta+CD4+CD62L+ thymocytes, emerging from the mainstream differentiation pathway, concentrate this ability to regulate autoreactive effectors. Compared with mature TCR alpha beta+CD8- thymocytes, significantly lower numbers of TCR alpha beta+CD4+CD62L+ were sufficient to achieve an efficient inhibition of disease transfer into NOD-scid recipients. This protective ability was potentiated following in vitro culture in the presence of IL-7. In contrast, TCR alpha beta+CD62L- thymocytes, highly enriched in class I-restricted NK T cells, were unable to influence diabetes transfer. Identical results were obtained using thymocytes that have been cultured in vitro for 4 days in the presence of IL-7. These results support the active role in NOD mice of a thymus-derived CD4+ subset that controls peripheral pathogenic autoimmune effectors.

Requirement of IL-7 for IL-4-producing potential of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes.

IL-7 plays an important role in the growth and differentiation of T cells. We have previously reported that IL-7 induces preferential expansion of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes which express a skewed V beta repertoire and are potent IL-4 producers. In this report, we provide evidence that IL-1 in combination with granulocyte macrophage colony stimulating factor can also expand this population. Yet, these cells do not share the functional characteristics of those obtained in the presence of IL-7, i.e. cytotoxic activity and high IL-4 production. These functional capacities can be acquired by adding IL-7. In conclusion, our findings demonstrate that the capacity of MHC class I-selected CD4-CD8-TCR alpha beta+ thymocytes to produce IL-4 as well as to kill target cells is IL-7 dependent.

Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse.

An immunoregulatory role has recently been attributed to the discrete subset of major histocompatibility complex class I-restricted NK1+ mature heat-stable antigen- (HSA-) thymocytes expressing an unusual Vbeta8-biased T cell receptor repertoire. NK1+ T cells are the main interleukin (IL)-4 producers upon priming. We have studied the size and the function of this subset in the nonobese diabetic (NOD) mouse, a model of spontaneous T cell-mediated autoimmune insulin-dependent diabetes. This study was complicated by the absence in this strain of the NK1.1 allele, the only one for which an antibody is available. To circumvent this difficulty, the cells, hereafter designated the NK1+-like T subset, were characterized by the use of monoclonal antibodies which showed the Vbeta8 bias in the CD44+ Ly-49+ MEL-14- 3G11- thymocyte subset of non-autoimmune strains and of its absence in class I-deficient (beta2-microglobulin-/-) mice. A clear deficit in the number of NK1+-like cells was evidenced at 3 weeks of age in NOD mice. It was still present at 8 weeks of age in the double-negative CD4-CD8- population. The functional anomaly was even more striking: NOD mouse NK1+-like thymocytes virtually lacked the ability to produce IL-4 at 3 weeks and still showed a very reduced capacity at 8 weeks. NK1+ T cell deficiency was also suggested in the periphery by the reduction of Ly-49A+ cells in the spleen of 3- and 8-week-old NOD mice and the absence of short-term production of IL-4 in vitro by NOD mouse spleen cells 90 min after the administration of anti-CD3 antibody, a response attributed to NK1+ T cells. Taken together, these data demonstrate a very early defect in NK1+-like T cells which could be involved in the genesis of autoimmunity in NOD mice through a deficiency in Th2 cell function.

IL-7 reverses NK1+ T cell-defective IL-4 production in the non-obese diabetic mouse.

Converging data suggest an important role for IL-7 in T lymphocyte maturation as illustrated by the severe T lymphopenia observed in IL-7-deficient mice. We recently reported that IL-7 preferentially promotes the in vitro expansion of a discrete MHC class I-dependent lymphocyte subset comprising both CD4+ and CD4-CD8- TCR alpha beta + cells bearing several NK cells markers such NK1.1 and Ly-49. These T cells, designated as NK1+ T cells, have the unique property among thymocytes of producing large amounts of IL-4 upon primary stimulation via the TCR. We have further demonstrated that thymic NK1+ T cells of non-obese diabetic (NOD) mice, a spontaneous model of autoimmune type I diabetes, are markedly deficient in maturation both quantitatively and functionally (IL-4 production). In the present experiments, the addition of exogenous IL-7 completely restored IL-4 production by anti-TCR alpha beta-stimulated mature (HSA-CD8-) thymocytes in NOD mice. A short 2 h preincubation with IL-7 was sufficient to restore both the expression of IL-4 mRNA and IL-4 production capacity. This was related to a direct effect on NK1+ thymocytes since: (i) the effect of IL-7 was restricted to the non-mainstream MEL-14- 3G11- TCR alpha beta + subset which mostly concentrates the IL-4-producing capacity and (ii) IL-7 did not restore IL-4 production in class I-deficient mice which lack the NK1+ T cell subset. Importantly, this activity of IL-7 on NK1+ T cells was also demonstrated in non-autoimmune strains of mice. These results were extended in vivo by showing that the IL-7 treatment significantly increased the anti-CD3 triggered IL-4 production by NK1+ T spleen cells. These findings confirm the role of IL-7 in NK1+ T cell maturation and suggest that the NK1+ T cell defect in NOD mice could be related to insufficient intrathymic IL-7 bioavailability.

Early defect of immunoregulatory T cells in autoimmune diabetes.

A potential immunoregulatory function has recently been attributed to the discrete subset of major histocompatibility complex (MHC) class I-restricted TCR-alpha beta mature thymocytes expressing an unusual V beta 8-biased T cell receptor repertoire. This T cell subset which also selectively express the CD44 marker is the main IL-4 producer in the thymus. Nonobese diabetic (NOD) mice were found to have a marked deficit in the number and functional capacity of CD44+ TCR-alpha beta+ thymocytes from as early as 3 weeks of age. The deficiency in IL-4 production was completely corrected after incubation with interleukin-7 (IL-7), a selective growth factor for CD44+ TCR-alpha beta+ mature thymocytes. This abnormality in T cell differentiation could explain the Th2 functional deficiency that may be a key element in the emergence of Th1-driven autoimmune disease in NOD mice.

MHC class I-selected CD4-CD8-TCR-alpha beta+ T cells are a potential source of IL-4 during primary immune response.

Differentiation of naive CD4+ lymphocytes into either Th1 or Th2 cells is influenced by the cytokine present during initial Ag priming. IL-4 is the critical element in the induction of Th2 response; however, its origin during a primary immune response is not well defined. In the present study, we characterized a novel potential source of IL-4, the class I-selected CD4-CD8-TCR-alpha beta+ T cells. In a first set of experiments, we demonstrated that CD4-CD8-TCR-alpha beta+ thymocytes produce a large amount of IL-4 after in vitro anti-CD3 stimulation. This phenomenon was not observed in class I-deficient mice, demonstrating that among these cells, the class I-selected subset was predominantly responsible for IL-4 production. Further studies focused on the in vivo IL-4-producing capacity of peripheral CD4-CD8-TCR-alpha beta+ T cells. To this end, a single injection of anti-CD3 mAb, which promptly induces IL-4 mRNA expression, was used. Peripheral CD4-CD8-TCR-alpha beta+ T cells express high levels of IL-4 mRNA in response to in vivo anti-CD3 challenge. Furthermore, analysis performed in mice lacking MHC class I or class II molecules demonstrates that both the class I-selected subset of CD4-CD8-TCR+ and CD4+ peripheral T lymphocytes are the major IL-4 producers after in vivo anti-CD3 stimulation. These findings suggest that class I-selected CD4-CD8-TCR-alpha beta+ and CD4+ T cell populations are important sources of IL-4 probably implicated in the development of specific Th2 immune responses.

Interleukin 7 induces preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4+8- murine thymocytes positively selected by class I molecules.

We analyzed the phenotype and V beta-T cell receptor (TCR) repertoire, together with interleukin 7 receptor (IL-7R) expression in unfractionated thymocytes stimulated in vitro with IL-7. This culture system results in a specific proliferation of mature thymocytes belonging to the CD3+CD4-, CD4+8-, and CD4-8+ subsets. IL-7 induced a preferential expansion of V beta 8.2+CD4-8- and V beta 8.2+CD4-8- thymocytes. This phenomenon is not observed in beta 2-microglobulin-deficient mice, showing that a fraction of CD4+8- thymocytes, enriched in V beta 8.2+ cells, is selected by class I molecules in normal mice, as are a large proportion of CD4-8- alpha beta TCR+ thymocytes. Our findings also establish that IL-7 plays a major role in the expansion of rare thymocyte subsets, which could exert important functions in inflammatory and immune responses.

Anchored polymerase chain reaction based analysis of the V beta repertoire in the non-obese diabetic (NOD) mouse.

We have performed extensive analyses of T cell receptor V beta usage in the thymus, the spleen and the infiltrated islets of preclinical non-obese diabetic (NOD) mice. A semiquantitative anchored polymerase chain reaction (An-PCR) protocol has been developed for this purpose. The validity of the method has been first assessed by antibody staining with a panel of anti-V beta monoclonal antibodies (mAb). The results obtained by An-PCR are accurate, reproducible, and in good agreement with cell surface protein staining. A strict comparison between thymus and spleen repertoires reveals no major V beta-specific deletion except the already reported V beta 3 deletion due to Mtv-3. Certain V beta such as V beta 15, 18, 20 are found with a low frequency in the spleen, but the fact that they are also scarce in the thymus probably reflects a poor availability of these genetic elements during beta chain rearrangement rather than negative selection. Other V beta, such as V beta 2, V beta 12 and V beta 14 are significantly more abundant in the spleen than in the thymus. This finding was confirmed by mAb staining for V beta 2 and V beta 14. The expansion asymmetrically affects the CD4+ subset and can be traced back to the mature, single-positive thymocyte subset, suggesting an intrathymic positive selection event. V beta repertoires in infiltrated islets of 13- and 18-week-old, non-diabetic mice are polymorphic. Practically all the V beta found in the peripheral lymphoid tissues are present in the islets, in similar proportions. The major exception is V beta 12, one of the V beta which is subject to expansion during intrathymic differentiation and which is further augmented in the islets, both at 13 and 18 weeks. This increase probably reflects further peripheral amplification of the V beta 12-bearing subset due to encounter with the same ligand as in the thymus or with a cross-reactive motif. Finally, the nucleotide sequencing of all the V beta segments in usage in the NOD strain confirms the absence of allelic polymorphism of V beta-coding regions.