Interleukin 17 acts in synergy with B cell-activating factor to influence B cell biology and the pathophysiology of systemic lupus erythematosus.

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases, although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology, we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus.

Assessment of chimerism and immunomodulation to prevent post-transplantation relapse in childhood acute myeloblastic leukemia: is it the right approach?

Relapse of acute myeloblastic leukemia (AML) after first allogenic hematopoietic stem-cell transplantation (allo-HSCT) is a fatal complication. Sixty-five children transplanted for AML were included in a prospective national study from June 2005 to July 2008 to explore the feasibility of preemptive immune modulation based on the monitoring of blood chimerism. Relapse occurred in 23 patients (35%). The median time between the last complete chimerism and relapse was 13.5 days (2-138). Prompt discontinuation of cyclosporin and the administration of donor lymphocyte infusions (DLIs) based on chimerism monitoring failed as a preemptive tool, either for detecting relapse or certifying long-term remission.

Increased degradation of ATP is driven by memory regulatory T cells in kidney transplantation tolerance.

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function.

5' and 3' untranslated regions contribute to the differential expression of specific HLA-A alleles.

In hematopoietic stem cell transplantation (HSCT), when no HLA full-matched donor is available, alternative donors could include one HLA-mismatched donor. Recently, the low expressed HLA-C alleles have been identified as permissive mismatches for the best donor choice. Concerning HLA-A, the degree of variability of expression is poorly understood. Here, we evaluated HLA-A expression in healthy individuals carrying HLA-A*02 allele in different genotypes using flow cytometry and allele-specific quantitative RT-PCR. While an interindividual variability of HLA-A*02 cell surface expression, not due to the allele associated, was observed, no difference of the mRNA expression level was shown, suggesting the involvement of the posttranscriptional regulation. The results of qRT-PCR analyses exhibit a differential expression of HLA-A alleles with HLA-A*02 as the strongest expressed allele independently of the second allele. The associated non-HLA-A*02 alleles were differentially expressed, particularly the HLA-A*31 and HLA-A*33 alleles (strong expression) and the HLA-A*29 (low expression). The presence of specific polymorphisms in the 5' and 3' untranslated regions of the HLA-A*31 and HLA-A*33 alleles could contribute to this high level of expression. As previously described for HLA-C, low-expressed HLA-A alleles, such as HLA-A*29, could be considered as a permissive mismatch, although this needs to be confirmed by clinical studies.

Follow-up of post-transplant minimal residual disease and chimerism in childhood lymphoblastic leukaemia: 90 d to react.

Relapse after transplantation is a major cause of treatment failure in paediatric acute lymphoblastic leukaemia (ALL). Here, we report the findings of a prospective national study designed to investigate the feasibility of immune intervention in children in first or subsequent remission following myeloablative conditioning. This study included 133 children who received a transplant for ALL between 2005 and 2008. Minimal Residual Disease (MRD) based on T cell receptor/immunoglobulin gene rearrangements was measured on days -30, 30, 90 and 150 post-transplantation. Ciclosporin treatment was rapidly discontinued and donor lymphocyte infusions (DLI) were programmed for patients with a pre- or post-transplant MRD status ≥10(-3) . Only nine patients received DLI. Pre- and post-transplant MRD status, and the duration of ciclosporin were independently associated with 5-year overall survival (OS), which was 62·07% for the whole cohort. OS was substantially higher in patients cleared of MRD than in those with persistent MRD (52·3% vs. 14·3%, respectively). Only pre-transplant MRD status (Hazard Ratio 2·57, P = 0·04) and duration of ciclosporin treatment (P < 0·001) were independently associated with relapse. The kinetics of chimerism were not useful for predicting relapse, whereas MRD monitoring up to 90 d post-transplantation was a valuable prognostic tool to guide therapeutic intervention.

Comprehensive characterization of immunoglobulin gene rearrangements in patients with chronic lymphocytic leukaemia.

Previous studies have suggested a geographical pattern of immunoglobulin rearrangement in chronic lymphocytic leukaemia (CLL), which could be as a result of a genetic background or an environmental antigen. However, the characteristics of Ig rearrangements in the population from the South of France have not yet been established. Here, we studied CLL B-cell repertoire and mutational pattern in a Southern French cohort of patients using an in-house protocol for whole sequencing of the rearranged immunoglobulin heavy-chain genes. Described biased usage of variable, diversity and joining genes between the mutated and unmutated groups was found in our population. However, variable gene frequencies are more in accordance with those observed in the Mediterranean patients. We found that the third complementary-determining region (CDR) length was higher in unmutated sequences, because of bias in the diversity and joining genes usage and not due to the N diversity. Mutations found in CLL followed the features of canonical somatic hypermutation mechanism: preference of targeting for activation-induced cytidine deaminase and polymerase motifs, base change bias for transitions and more replacement mutations occurring in CDRs than in framework regions. Surprisingly, localization of activation-induced cytidine deaminase motifs onto the variable gene showed a preference for framework regions. The study of the characteristics at the age of diagnosis showed no difference in clinical outcome, but suggested a tendency of increased replacement and transition-over-transversion mutations and a longer third CDR length in older patients.

Lack of confirmation of anti-inward rectifying potassium channel 4.1 antibodies as reliable markers of multiple sclerosis.

BACKGROUND: auto-antibodies against the potassium channel inward rectifying potassium channel 4.1 (Kir4.1) have previously been identified in 46% of patients with multiple sclerosis (MS). OBJECTIVES: to confirm these findings. METHODS: we evaluated the presence of anti-Kir4.1 antibodies by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence in 268 MS patients, 46 patients with other neurological diseases (OND) and 45 healthy controls. RESULTS: anti-Kir4.1 antibodies were found in 7.5% of MS patients, 4.3% of OND patients and 4.4% of healthy controls. Immunofluorescence analysis did not identify any specific staining. CONCLUSIONS: we confirmed the presence of anti-Kir4.1 antibodies in MS patients, but at a much lower prevalence than previously reported.

Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE. METHODS: We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. RESULTS: We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. CONCLUSION: Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.

Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-matched related and unrelated hematopoietic stem cell transplantation.

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P = .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR] = 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P = .078), higher relapse-free survival (P = .029), and higher overall survival (P = .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner.

The intensity of immune activation is linked to the level of CCR5 expression in human immunodeficiency virus type 1-infected persons.

Immune activation is a main driver of AIDS- and non-AIDS-linked morbidities in the course of HIV-1 infection. As CCR5, the main HIV-1 co-receptor, is not only a chemokine receptor but also a co-activation molecule expressed at the surface of T cells, it could be directly involved in this immune activation. To test this hypothesis, we measured by flow cytometry the mean number of CCR5 molecules at the surface of non-activated CD4(+) T cells (CCR5 density), which determines the intensity of CCR5 signalling, and the percentage of CD8(+) T cells over-expressing CD38 (CD38 expression), a major marker of immune activation, in the blood of 67 HIV-1-infected, non-treated individuals. CCR5 density was correlated with CD38 expression independently of viral load (P=0.016). CCR5 density remained unchanged after highly active anti-retroviral therapy (HAART) introduction or cessation, whereas CD38 expression decreased and increased, respectively. Moreover, pre-therapeutic CCR5 density was highly predictive (r=0.736, P<10(-4) ) of residual CD38 over-expression after 9 months of HAART. Hence, CCR5 might play an immunological role in HIV-1 infection as a driver of immune activation. This could explain why CCR5 antagonists may have an inhibitory effect on immune activation.

Phenotype frequencies of autosomal minor histocompatibility antigens display significant differences among populations.

Minor histocompatibility (H) antigens are allogeneic target molecules having significant roles in alloimmune responses after human leukocyte antigen-matched solid organ and stem cell transplantation (SCT). Minor H antigens are instrumental in the processes of transplant rejection, graft-versus-host disease, and in the curative graft-versus-tumor effect of SCT. The latter characteristic enabled the current application of selected minor H antigens in clinical immunotherapeutic SCT protocols. No information exists on the global phenotypic distribution of the currently identified minor H antigens. Therefore, an estimation of their overall impact in human leukocyte antigen-matched solid organ and SCT in the major ethnic populations is still lacking. For the first time, a worldwide phenotype frequency analysis of ten autosomal minor H antigens was executed by 31 laboratories and comprised 2,685 randomly selected individuals from six major ethnic populations. Significant differences in minor H antigen frequencies were observed between the ethnic populations, some of which appeared to be geographically correlated.

Molecular identity of skin and blood T-cell clones in cutaneous T-cell lymphoma patients as determined from the migration pattern of the T-cell receptor-gamma gene by capillary electrophoresis.

The presence of a dominant clonal T-cell population in skin lesions is an important clue in the diagnosis of cutaneous T-cell lymphoma (CTCL). However, it has never been determined whether dominant T-cell receptor (TCR) rearrangements identified in skin lesions and blood from CTCL patients, displaying strictly identical migration patterns by capillary electrophoresis, actually correspond to identical clones. As this information has potential clinical relevance, TCR-gamma (TCRG) gene-derived amplified fragments from dominant blood and skin T-cell clones featuring either identical or slightly different capillary electrophoresis migration patterns were analyzed in eleven CTCL patients. In nine patients with identical electrophoretic migration patterns, sequence analyses revealed the dominant skin and blood T-cell clones to be identical. In contrast, in two patients displaying slight migration differences between skin and blood samples, the TCRG sequences were distinct. Additionally, capillary electrophoresis appears more sensitive and accurate than heteroduplex analysis and in silico analysis of samples of different origins is possible a posteriori. These results demonstrate the efficacy of capillary electrophoresis in assessing molecular identity and discrepancy of dominant T-cell populations obtained from different tissues or at different times, facilitating diagnosis and follow-up.

Blocking Antibodies Targeting the CD39/CD73 Immunosuppressive Pathway Unleash Immune Responses in Combination Cancer Therapies.

Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer.

Predictive factors and impact of full donor T-cell chimerism after reduced intensity conditioning allogeneic stem cell transplantation.

This study investigated the kinetics of CD3+-T cell chimerism (TCC) in 102 patients receiving reduced intensity conditioning allogeneic stem cell transplantation (RIC-allo-SCT) from an HLA-identical sibling. Patients with full donor TCC at day 30 had a higher incidence of grade 2-4 acute GVHD compared to patients in mixed TCC (cumulative-incidence, 61% vs. 35%; p=0.01). The delayed establishment of full donor TCC in myeloid malignancies was associated with a higher incidence of relapse (40% vs. 0; p=0.002), suggesting that monitoring of the kinetics of TCC is mandatory after RIC-allo-SCT.

PD-1 blockade at the time of tumor escape potentiates the immune-mediated antitumor effects of a melanoma-targeting monoclonal antibody.

Tumor antigen-targeting monoclonal antibodies (TA-targeting mAbs) are used as therapeutics in many malignancies and their capacity to mobilize the host immunity puts them at the forefront of anti-cancer immunotherapies. Both innate and adaptive immune cells have been associated with the therapeutic activity of such antibodies, but tumor escape from mAb-induced tumor immune surveillance remains one of the main clinical issues. In this preclinical study, we grafted immunocompetent and immunocompromised mice with the B16F10 mouse melanoma cell line and treated them with the TA99 TA-targeting mAb to analyze the immune mechanisms associated with the tumor response and resistance to TA99 monotherapy. In immunocompetent mice TA99 treatment strongly increased the fraction of CD8 and CD4 effector T cells in the tumor compared with isotype control, highlighting the specific immune modulation of the tumor microenvironment by TA99. However, in most mice, TA99 immunotherapy could not prevent immune effector exhaustion and the recruitment of regulatory CD4 T cells and consequently tumor escape from immune surveillance. Remarkably, anti-PD-1 treatment at the time of tumor emergence restored the Th1 effector functions of CD4 and CD8 T cells as well as of natural killer and γδT cells, which translated into a significant slow-down of tumor progression and extended survival. Our findings provide the first evidence that PD-1 blockade at the time of tumor emergence can efficiently boost the host anti-tumor immune response initiated several weeks before by the TA-targeting mAb. These results are promising for the design of combined therapies to sensitize non-responder or resistant patients.

IL-21 promotes the development of a CD73-positive Vγ9Vδ2 T cell regulatory population.

Vγ9Vδ2 T cells contribute to the immune response against many tumor types through their direct cytotoxic activity and capacity to regulate the biological functions of other immune cells, such as dendritic cells and IFN-γ-producing CD8+ T cells. However, their presence in the tumor microenvironment has also been associated with poor prognosis in breast, colon and pancreatic cancers. Additionally, recent studies demonstrated that cytokines can confer some plasticity to Vγ9Vδ2 T cells and promote their differentiation into cells with regulatory functions. Here, we demonstrated that activation of Vγ9Vδ2 T cells isolated from healthy donors and cultured in the presence of IL-21 favors the emergence of a subpopulation of Vγ9Vδ2 T cells that express the ectonucleotidase CD73 and inhibits T cell proliferation in a CD73/adenosine-dependent manner. This subpopulation produces IL-10 and IL-8 and displays lower effector functions and cytotoxic activity than CD73-negative Vγ9Vδ2 T cells. We also showed, in a syngeneic mouse tumor model, the existence of a tumor-infiltrating γδ T cell subpopulation that produces IL-10 and strongly expresses CD73. Moreover, maturation, IL-12 production and induction of antigen-specific T cell proliferation are impaired in DC co-cultured with IL-21-amplified Vγ9Vδ2 T cells. Altogether, these data indicate that IL-21 promotes Vγ9Vδ2 T cell regulatory functions by favoring the development of an immunosuppressive CD73+ subpopulation. Thus, when present in the tumor microenvironment, IL-21 might negatively impact γδ T cell anti-tumor functions.

The IL-17B-IL-17 receptor B pathway promotes resistance to paclitaxel in breast tumors through activation of the ERK1/2 pathway.

Interleukin 17B (IL-17B) is a pro-inflammatory cytokine that belongs to the IL-17 cytokines family and binds to IL-17 receptor B (IL-17RB). Here we found that high expression of IL-17B and IL-17RB is associated with poor prognosis in patients with breast cancer and that IL-17B expression upregulation is specifically associated with poorer survival in patients with basal-like breast cancer. We thus focused on IL-17B role in breast cancer by using luminal and triple negative (TN)/basal-like tumor cell lines. We found that IL-17B induces resistance to conventional chemotherapeutic agents. In vivo, IL-17B induced resistance to paclitaxel and treatment with an anti-IL-17RB neutralizing antibody completely restored breast tumor chemosensitivity, leading to tumor shrinkage. We next focused on the signaling pathways activated in human breast cancer cell lines upon incubation with IL-17B. We observed that IL-17B induces ERK1/2 pathway activation, leading to upregulation of anti-apoptotic proteins of the BCL-2 family. IL-17B-induced chemoresistance was completely abolished by incubation with PD98059, an inhibitor of the MAPK/ERK pathway, indicating that the ERK pathway plays a crucial role. Altogether our results emphasize the role of the IL-17B/IL-17RB signaling pathway in breast tumors and identify IL-17B and its receptor as attractive therapeutic targets for potentiating breast cancer chemotherapy.

Effects of alcohol withdrawal on monocyte subset defects in chronic alcohol users.

Excessive alcohol consumption has a modulating effect on immune functions that may contribute to decreased immunity and host defense. It is associated with increased intestinal permeability to endotoxins that is normalized after 14 d of abstinence. Whether and how blood monocyte subsets are impaired in patients with an AUD and what their evolution is after alcohol withdrawal are the paper's objectives. With the use of flow cytometry, blood monocyte subsets were quantified in AUDs before (n = 40) and 2 wk after (n = 33) alcohol withdrawal and compared with HC donors (n = 20). Expression of TLR2 and TLR4 on monocyte subsets was also quantified. Cytokine response of monocytes was monitored following PGN and LPS stimulation. The CD14+CD16- subset was decreased, whereas the CD14dimCD16+ subset was expanded (P < 0.001) in AUD compared with HC. The frequencies of TLR2- and TLR4-expressing monocytes were reduced in AUD compared with HC. Although the basal production of IL-1, IL-6, and TNF by monocytes in AUD was compared with HC, the PGN- and LPS-mediated IL-6 and TNF production was increased in AUD. Frequencies of IL-6-expressing monocytes were higher in AUD than HC. Alcohol withdrawal partially restored the distribution of monocyte subsets and the frequency of IL-6-producing monocytes and increased the frequency of TNF-producing cells in response to LPS and PGN stimulation to levels compared with those in HC. Our findings indicate that chronic alcohol use alters the distribution as well as the phenotypic and functional characteristics of blood monocyte subsets, which are partially restored following 2 wk of alcohol withdrawal.

Association of HLA-A and Non-Classical HLA Class I Alleles.

The HLA-A locus is surrounded by HLA class Ib genes: HLA-E, HLA-H, HLA-G and HLA-F. HLA class Ib molecules are involved in immuno-modulation with a central role for HLA-G and HLA-E, an emerging role for HLA-F and a yet unknown function for HLA-H. Thus, the principal objective of this study was to describe the main allelic associations between HLA-A and HLA-H, -G, -F and -E. Therefore, HLA-A, -E, -G, -H and -F coding polymorphisms, as well as HLA-G UnTranslated Region haplotypes (referred to as HLA-G UTRs), were explored in 191 voluntary blood donors. Allelic frequencies, Global Linkage Disequilibrium (GLD), Linkage Disequilibrium (LD) for specific pairs of alleles and two-loci haplotype frequencies were estimated. We showed that HLA-A, HLA-H, HLA-F, HLA-G and HLA-G UTRs were all in highly significant pairwise GLD, in contrast to HLA-E. Moreover, HLA-A displayed restricted associations with HLA-G UTR and HLA-H. We also confirmed several associations that were previously found to have a negative impact on transplantation outcome. In summary, our results suggest complex functional and clinical implications of the HLA-A genetic region.