[Show abstract][Hide abstract] ABSTRACT: Cord blood (CB) is a well-established source of stem cells for hematopoietic reconstitution that contains immune cells with a unique profile. This results in a balanced graft versus host alloreactivity allowing tolerance to human leukocyte antigen mismatch while maintaining a potent antitumor effect. CB immune cells are constitutively programmed to tolerate a wide range of nonself antigens presented both during pregnancy and early after birth during the intense period of bacterial colonization. Thus, there is a growing interest in characterizing better the properties of CB immune cells, in assessing the kinetics of immune reconstitution (IR) after CB transplantation (CBT), and in defining ex vivo strategies to induce fully competent cells for immunotherapy. Notably, this chapter aims to present up-to-date data on these topics. Published findings showed that CB cells are immature and produce less cytokines than their adult counterparts. The immaturity and naivety of CB cells could explain the lower Graft-versus-Host Disease (GvHD) risk after CBT but also raise some concerns about the quality of IR in CB transplanted patients. In this regard, recent data suggest that the use of appropriate conditioning regimes and GvHD prophylaxis, including the omission of T-cell depletion, can promote rapid expansion of naïve lymphocytes with a consequent restoration of the adaptive immunity early after transplantation. This evidence supports projects aiming to ex vivo generate fully competent immune cells for adaptive cellular immunotherapy. In conclusion, a better understanding of CB cell properties and of the environmental and epigenetic mechanisms involved during IR could help improving clinical outcomes and purposely designing cell therapy products derived from CB for use after CBT but also in broader clinical indications.
[Show abstract][Hide abstract] ABSTRACT: Chronic infection perturbs immune homeostasis. While prior studies have reported dysregulation of effector and memory cells, little is known about the effects on naïve T cell populations. We performed a cross-sectional study of chronic hepatitis C (cHCV) patients using tetramer-associated magnetic enrichment to study antigen-specific inexperienced CD8
T cells (i.e., tumor or unrelated virus-specific populations in tumor-free and sero-negative individuals). cHCV showed normal precursor frequencies, but increased proportions of memory-phenotype inexperienced cells, as compared to healthy donors or cured HCV patients. These observations could be explained by low surface expression of CD5, a negative regulator of TCR signaling. Accordingly, we demonstrated TCR hyperactivation and generation of potent CD8
T cell responses from the altered T cell repertoire of cHCV patients. In sum, we provide the first evidence that naïve CD8
T cells are dysregulated during cHCV infection, and establish a new mechanism of immune perturbation secondary to chronic infection.
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) is a heterogeneous group of malignancies that may be sensitive to the NK cell antitumor response. However, NK cells are frequently defective in AML. In this study, we found in an exploratory cohort (n = 46) that NK cell status at diagnosis of AML separated patients in two groups with a different clinical outcome. Patients with a deficient NK cell profile, including reduced expression of some activating NK receptors (e.g., DNAX accessory molecule-1, NKp46, and NKG2D) and decreased IFN-γ production, had a significantly higher risk of relapse (p = 0.03) independently of cytogenetic classification in multivariate analysis. Patients with defective NK cells showed a profound gene expression decrease in AML blasts for cytokine and chemokine signaling (e.g., IL15, IFNGR1, IFNGR2, and CXCR4), Ag processing (e.g., HLA-DRA, HLA-DRB1, and CD74) and adhesion molecule pathways (e.g., PVR and ICAM1). A set of 388 leukemic classifier genes defined in the exploratory cohort was independently validated in a multicentric cohort of 194 AML patients. In total, these data evidenced the interplay between NK cells and AML blasts at diagnosis allowing an immune-based stratification of AML patients independently of clinical classifications.
No preview · Article · Sep 2015 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Previous studies of non-histocompatibility leukocyte antigen (HLA) gene single-nucleotide polymorphisms (SNPs) on subgroups of patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) revealed an association with transplant outcome. This study further evaluated the association of non-HLA polymorphisms with overall survival in a cohort of 762 HSCT patients using data on 26 polymorphisms in 16 non-HLA genes. When viewed in addition to an already established clinical risk score (EBMT-score), three polymorphisms: rs8177374 in the gene for MyD88-adapter-like (MAL; P=0.026), rs9340799 in the oestrogen receptor gene (ESR; P=0.003) and rs1800795 in interleukin-6 (IL-6; P=0.007) were found to be associated with reduced overall survival, whereas the haplo-genotype (ACC/ACC) in IL-10 was protective (P=0.02). The addition of these non-HLA polymorphisms in a Cox regression model alongside the EBMT-score improved discrimination between risk groups and increased the level of prediction compared with the EBMT-score alone (gain in prediction capability for EBMT-genetic-score 10.8%). Results also demonstrated how changes in clinical practice through time have altered the effects of non-HLA analysis. The study illustrates the significance of non-HLA genotyping prior to HSCT and the importance of further investigation into non-HLA gene polymorphisms in risk prediction.Bone Marrow Transplantation advance online publication, 27 July 2015; doi:10.1038/bmt.2015.173.
No preview · Article · Jul 2015 · Bone marrow transplantation
[Show abstract][Hide abstract] ABSTRACT: Background: The incidence of cancer is increased after solid organ transplantation. Natural killer (NK) cells are key effectors of the tumor immune response. Methods: We conducted a cross sectional multicentre matched case-control study including 42 kidney transplant recipients (KTRs) on diagnosis of cancer and 41 KTRs without cancer. Extensive phenotyping of NK cells populations and functional tests of NK cells were performed. Results: Kidney transplant recipients with cancer had a higher incidence of acute rejection (P = 0.02) and cytomegalovirus (CMV) infection (P = 0.03) than controls. They had more lymphopenia than control KTRs (1020/mm3 +/- 32 vs 1218/mm3 +/- 34; P = 0.001) including a CD4+ lymphopenia (P = 0.01). Total CD3-/CD56+ NK cell counts were similar in both groups. However, KTRs with cancer had a lower frequency of the cytokine-enriched CD56bright NK cell subset (P = 0.001). The percentage of NK cells expressing NKp46 was decreased in KTRs with cancer (45% vs 53 %, P = 0.001). Furthermore, the ability of NK cells to degranulate CD107a+ cytolytic vesicles was reduced (11% vs 22%; P = 0.02), and the percentage of NK cells secreting IFN[gamma] was decreased (7.5% vs 28.8%; P = 0.01) in KTRs with cancer. Conclusions: These results reveal an imbalance between NK cell subpopulations and functional NK cell defects in KTRs at the diagnosis of malignancy, including a decreased expression of NKp46 and decreased numbers of NK cells producing INF[gamma]. This study highlights the role of NKp46, a major activating NK cell receptor, which could be considered as a potential marker during immunological follow-up of KTRs.
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancers (CRC) develop in the face of an important immune system associated with the intestinal mucosal tissue. The immune response against the tumor has been proposed to impact the prognostic of patients undergoing treatment for CRC. In this study T cells infiltrating the tumor were compared to cells populating the unaffected neighboring mucosal tissue and cells from the peripheral blood. We observed that T cells from the tumor harbor an activated phenotype, with engagement of the NKG2D pathway in CD8 T cells. We show that mucosal and tumor-infiltrating T cells are enriched in NKG2D CD4 T cells, which exhibit cytotoxic functions. Finally, T cell populations in the tumor were modified according to its oncogenetic status, with higher percentages of CD8 T cells isolated from MSI patients.
[Show abstract][Hide abstract] ABSTRACT: Over the past 15 years, SCT has emerged as a promising treatment option for patients with severe autoimmune diseases (ADs). Mechanistic studies recently provided the proof-of-concept that restoration of immunological tolerance can be achieved by haematopoietic SCT in chronic autoimmunity through eradication of the pathologic, immunologic memory and profound reconfiguration of the immune system, that is, immune 'resetting'. Nevertheless, a number of areas remain unresolved and warrant further investigation to refine our understanding of the underlying mechanisms of action and to optimize clinical SCT protocols. Due to the low number of patients transplanted in each centre, it is essential to adequately collect and analyse biological samples in a larger cohort of patients under standardized conditions. The European society for blood and marrow transplantation Autoimmune Diseases and Immunobiology Working Parties have, therefore, undertaken a joint initiative to develop and implement guidelines for 'good laboratory practice' in relation to procurement, processing, storage and analysis of biological specimens for immune reconstitution studies in AD patients before, during and after SCT. The aim of this document is to provide practical recommendations for biobanking of samples and laboratory immune monitoring in patients with ADs undergoing SCT, both for routine supportive care purposes and investigational studies.Bone Marrow Transplantation advance online publication, 10 November 2014; doi:10.1038/bmt.2014.251.
No preview · Article · Nov 2014 · Bone Marrow Transplantation
[Show abstract][Hide abstract] ABSTRACT: Background
Post-transplant non-Hodgkin lymphoma (NHL) is a well-recognized complication of solid organ transplantation, and pharmacologic suppression of adaptive immunity plays a major role in its development. However, the role of natural killer (NK) cells in post-lung transplant de novo NHL is unknown.
Extensive phenotypic analyses of NK cells from patients diagnosed with NHL after liver or lung transplantation were conducted with multicolor flow cytometry. Polyfunctionality assays simultaneously assessed NK-cell degranulation (CD107a) and intracellular cytokine production (IFN-γ and TNF-α) in the presence of NHL target cells.
The development of de novo NHL is linked to NK-cell maturation defects, including overexpression of NKG2A and CD62L and down-modulation of inhibitory killer immunoglobulin-like receptors and CD57 receptors. More Importantly, in patients who developed NHL after lung transplantation, we observed a specific down-modulation of the activating receptors (NKp30, NKp46, and NKG2D), and a sharp decrease in perforin expression and degranulation against NHL target cells.
Our results suggest that accumulation of abnormal NK cells could play a role in the outgrowth of NHL after lung transplantation, independently of the immune-suppressive regimen.
No preview · Article · Oct 2014 · The Journal of Heart and Lung Transplantation
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) cells are killed by allogeneic NK cells. However, autologous NK cells from AML patients express decreased levels of activating receptors, and show reduced cytotoxicity. Here, we investigated how interactions between NK and AML cells might cause loss of NK cell activity in patients. Our results show that AML cell lines and primary blasts alter the NK cell phenotype, reducing their cytotoxic potential upon prolonged contact. Down-regulation of NK-cell-activating receptors was contact-dependent and correlated with conjugate formation. Time-lapse imaging of HL60 AML cell line and NK-cell interactions showed a high proportion of non-cytolytic contacts. Studies of NK-cell immunological synapses revealed a defect in lytic synapse formation. Namely, despite correct F-actin and LFA-1 recruitment, polarization of lytic granules toward primary blasts or AML cell lines was reduced. The NK-AML cell line synapses showed impairment of CD3ζ recruitment. Attempts to correct these synapse defects by cytokine stimulation of NK cells improved conjugate formation, but not granule polarization. Pre-treatment of AML cell lines with the immuno-modulating molecule lenalidomide significantly enhanced granule polarization. We speculate that combining immunomodulatory drugs and cytokines could increase AML cell sensitivity to autologous NK cells and reinforce the activity of allogeneic NK cells in adoptive immunotherapy.This article is protected by copyright. All rights reserved
Full-text · Article · Oct 2014 · European Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Immune reconstitution after allogeneic stem cell transplantation is a dynamic and complex process depending on the recipient and donor characteristics, on the modalities of transplantation and on the occurrence of graft-versus-host disease. Multivariate methods widely used for gene expression profiling, can simultaneously analyze the patterns of a great number of biological variables on a heterogeneous set of patients. Here we use these methods on flow cytometry assessment of up to 25 lymphocyte populations to analyze the global pattern of long-term immune reconstitution after transplantation. Immune patterns were most distinct from healthy controls at 6 months, and had not yet fully recovered as long as 2 years, after transplant. The two principal determinants of variability were linked to the balance of B and CD8+ T cells and of natural killer and B cells, respectively. Recipient's cytomegalovirus serostatus, cytomegalovirus replication, and chronic graft-versus-host disease were the main factors shaping the immune pattern 1 year after transplant. We identified a complex signature of under- and over-representation of immune populations dictated by recipient's cytomegalovirus seropositivity. Finally, we identified dimensions of variance in immune patterns as significant predictors of long-term non-relapse mortality, independently of chronic graft-versus-host disease.
[Show abstract][Hide abstract] ABSTRACT: Using B-cell rearrangement excision circles measurements, we analyzed B-cell reconstitution in a cohort of 243 patients who underwent allogeneic stem cell transplantation. Acute and chronic graft vs. host disease (aGVHD and cGVHD) transiently increased B-cell replication but decreased overall B-cell neogenesis with a clear difference in term of kinetics. Moreover, impact of aGVHD in absence of cGVHD was transient, recovering at month 6 similar values than in patients who did not suffer from GVHD. Conversely, impact of cGVHD at month 12 in multivariate analysis was independent of previous aGVHD effect on B-cell output. Finally, we showed in patients affected with cGVHD a higher B-cell division rate that correlates with an elevated BAFF/CD19+ B cell ratio, supporting a B-cell hyperactivation state in vivo.
[Show abstract][Hide abstract] ABSTRACT: The diverse aspects of Cutaneous T-cell lymphomas may impede the diagnosis of Sézary syndrome (SS) and mycosis fungoides (MF), in particular at early stages of the disease. We defined the CD158k/KIR3DL2 molecule as a first positive cell surface marker for Sézary cells (SCs). Here, we designed an optimized flow-cytometry gating strategy, allowing the definition of lymphocytes of different sizes and defects of cell surface markers. Quantification by cytomorphology, flow-cytometry or clonal evaluation, gave similar results at initial time points and during the evolution in a prospective study involving 64 consecutive cutaneous T-cell lymphoma or erythrodermic patients. We found that CD158k+T-cells and circulating CD4+T-cells from MF patients exhibited unexpected patterns of cell surface expression with a marked heterogeneity of circulating lymphocytes even at initial diagnosis. Taken together, our results show that a multistep gating of CD158k+ cells is reliable to assess tumor burden in case of SS and suggest that both circulating MF CD4+T-cells and CD158k+T-cells are not homogeneous distinct memory populations. Further phenotypic and functional characterization of such subsets is needed in order to better understand the underlying molecular mechanisms leading to the development of these diseases.Journal of Investigative Dermatology accepted article preview online, 26 August 2014. doi:10.1038/jid.2014.356.
Full-text · Article · Aug 2014 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: Natural killer cells are the first lymphocyte subset to reconstitute, and play a major role in early immunity after allogeneic hematopoietic stem cell transplantation. Cells expressing the activating receptor NKG2C seem crucial in the resolution of cytomegalovirus episodes, even in the absence of T-cells. We prospectively investigated Natural Killer cells reconstitution in a cohort of 439 adult recipients who underwent non T-cell depleted allogeneic hematopoietic stem cell transplantation between 2005 and 2012. Freshly collected blood samples were analyzed 3, 6, 12 and 24 months after transplantation. Data were studied with respect to conditioning regimen, source of stem cells, underlying disease, occurrence of Graft-versus-Host Disease, and profiles of cytomegalovirus reactivation. In multivariate analysis, we show that the absolute numbers of CD56brightNatural killer cells at month 3 were significantly higher after myeloablative than after reduced intensity conditioning. Acute Graft-versus-Host Disease impaired reconstitution of total and CD56dimNatural killer cells at month 3. In contrast, high Natural killer cell count at month 3 was associated with a lower incidence of chronic Graft-versus-Host Disease, independently from a previous episode of acute Graft-versus-Host Disease and stem cell source. NKG2C+CD56dim and total Natural killer cell count at M3 was lower in patients reactivating CMV between month 0 and month 3, but expanded greatly afterwards. These cells were also less numerous in patients who experienced subsequent reactivation between month 3 and month 6. Our results advocate a direct role of NKG2C-expressing Natural killer cells in the early control of cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.
[Show abstract][Hide abstract] ABSTRACT: Background. Activating anti-angiotensin type 1 receptor antibodies (AT1R-AA) have been described in patients with systemic scleroderma, an auto-immune disorder with clinical fibrotic features. Chronic graft-versus-host disease (cGvHD) after hematopoietic stem cell transplantation may have clinical fibrotic features, whose pathogenesis may be similar with systemic sclerosis. Objective. To evaluate the presence of AT1R-AA and their association with clinical and biological symptoms in cGvHD patients. Material and Methods. Sera from 87 patients including 45 extensive cGvHD and 42 hematopoietic stem cell transplantation patients without cGvHD were retrospectively analyzed for the presence of AT1R-AA using an enzymatic immunoassay. Results. The frequency of AT1R-AA was significantly increased (odds ratio [OR]-3.4, P=0.04) in the cGvHD group (24.4%) compared with the non-cGvHD group (7.1%). In the cGvHD group the positivity of AT1R-AA was significantly associated with: i/ the presence of antinuclear antibodies (OR=5.9, P=0.04) ii/a more severe global and organ-specific cGvHD scoring (PG0.05), iii/ the presence of active skin or mucosal erosions (OR=19.2, P<0.01). There was no difference between the number and the types of organs involved by the cGvHD between the AT1R-AA-positive versus AT1R-AA-negative subgroups. Conclusion. This preliminary study suggests a potential role and prognostic value of AT1R-AA in cGvHD.
[Show abstract][Hide abstract] ABSTRACT: Previous studies on regulatory T-cell (Treg) reconstitution after allogeneic hematopoietic SCT (HSCT) have suggested that, within the GVHD process, imbalance between effector T cells and Tregs may be more important than the absolute numbers of circulating Tregs. No study has analyzed naive vs memory Treg reconstitution in a longitudinal cohort with large numbers of patients. The reconstitution of total and subsets of Treg was prospectively analyzed by flow cytometry in 185 consecutive recipients at 3, 6, 12 and 24 months after allogeneic HSCT. The levels of total, naive and memory Tregs increased, mainly within the memory subset, but remained lower than healthy controls up to 2 years after transplantation. Reduced-intensity conditioning and peripheral blood (PBSC) as the source of stem cells were associated with better 3-month reconstitution. In multivariate analysis, PBSC, recipient age ⩽25 and no anti-thymoglobulin in the conditioning regimen were associated with a better Treg reconstitution. Naive Treg long-term reconstitution was mainly influenced by recipient age. Whereas prior acute GVHD impaired Treg reconstitution, Treg subsets (absolute numbers and frequencies relative to CD4(+) T-cell subsets) at 3, 6 and 12 months after HSCT were not associated with the occurrence of a later episode of chronic GVHD.Bone Marrow Transplantation advance online publication, 19 May 2014; doi:10.1038/bmt.2014.105.
Full-text · Article · May 2014 · Bone Marrow Transplantation
[Show abstract][Hide abstract] ABSTRACT: Despite the effectiveness of immunosuppressive drugs, kidney transplant recipients still face late graft dysfunction. Thus, it is necessary to identify biomarkers to detect the first pathologic events and guide therapeutic target development. Previously, we identified differences in the T-cell receptor Vβ repertoire in patients with stable graft function. In this prospective study, we assessed the long-term effect of CD8(+) T-cell differentiation and function in 131 patients who had stable graft function. In 45 of 131 patients, a restriction of TCR Vβ diversity was detected and associated with the expansion of terminally differentiated effector memory (TEMRA; CD45RA(+)CCR7(-)CD27(-)CD28(-)) CD8(+) T cells expressing high levels of perforin, granzyme B, and T-bet. This phenotype positively correlated with the level of CD57 and the ability of CD8(+) T cells to secrete TNF-α and IFN-γ. Finally, 47 of 131 patients experienced kidney dysfunction during the median 15-year follow-up period. Using a Cox regression model, we found a 2-fold higher risk (P=0.06) of long-term graft dysfunction in patients who had increased levels of differentiated TEMRA CD8(+) T cells at inclusion. Collectively, these results suggest that monitoring the phenotype and function of circulating CD8(+) T cells may improve the early identification of at-risk patients.
No preview · Article · Mar 2014 · Journal of the American Society of Nephrology
[Show abstract][Hide abstract] ABSTRACT: Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin-CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24- progenitor subpopulations. CD34+lin-CD10+CD24- lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin-CD10+CD24- cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution.