Esther Mancebo

Longitudinal analysis of immune function in the first 3 years of life in thymectomized neonates during cardiac surgery

The purpose of this study is to evaluate the effects of neonatal thymectomy in the functional capacity of the immune system. We selected a group of 23 subjects, who had undergone thymectomy in their first 30 days of life, during an intervention for congenital heart disease. Several parameters of the immune system were evaluated during their first 3 years of life. Lymphocyte populations and subpopulations (including naive, memory and effector subpopulations), T cell receptor (TCR) Vβ repertoire, response of T cells following in vitro stimulation by mitogen, quantification of immunoglobulins, TCR excision circles (TRECS) and interleukin (IL)‐7 were measured. We found that neonatal thymectomy produces long‐term diminution in total lymphocyte counts, especially in naive CD4+ and CD8+ T cells. Additionally, TRECS were decreased, and plasma IL‐7 levels increased. A statistically significant negative correlation was found between absolute CD4+ T cells and IL‐7 (r = −0·470, P = 0·02). The patients did not suffer more infectious events than healthy control children, but thymectomy in neonates resulted in a significant decrease in T lymphocyte levels and TRECS, consistent with cessation of thymopoiesis. This could produce a compromise in immune function later in life, especially if the patients suffer T cell depletion and need a reconstitution of immune function.

Phenotypic and functional evaluation of CD3+CD4−CD8− T cells in human CD8 immunodeficiency

Background Human CD8 immunodeficiency is characterized by undetectable CD8+ lymphocytes and an increased population of CD4−CD8− (double negative) T lymphocytes. Design and Methods We hypothesized that the double negative subset corresponds to the cellular population that should express CD8 and is committed to the cytotoxic T lymphocyte lineage. To assess this, we determined the phenotype and function of peripheral blood mononuclear cells and/or magnetically isolated double negative T lymphocytes from two CD8-deficient patients. To analyze the expression and co-localization with different organelles, 293T cells were transfected with plasmids bearing wild-type or mutated CD8α. Results CD8α mutated protein was retained in the cytoplasm of transfected cells. The percentages of double negative cells in patients were lower than the percentages of CD8+ T cells in healthy controls. Double negative cells mostly had an effector or effector memory phenotype whereas naïve T cells were under-represented. A low concentration of T-cell receptor excision circles together with a skewed T-cell receptor-V repertoire were observed in the double negative population. These data suggest that, in the absence of CD8 co-receptor, the thymic positive selection functions suboptimally and a limited number of mature T-cell clones would emerge from the thymus. In vitro, the double negative cells showed a mild defect in cytotoxic function and decreased proliferative capacity. Conclusions It is possible that the double negative cells are major histocompatibility complex class-I restricted T cells with cytolytic function. These results show for the first time in humans that the presence of the CD8 co-receptor is dispensable for cytotoxic ability, but that it affects the generation of thymic precursors committed to the cytotoxic T lymphocyte lineage and the proliferation of mature cytotoxic T cells.

Peripheral blood lymphocyte populations in end-stage liver diseases.

Goals/Background The aim of this study was to decipher whether end-stage liver failure modifies peripheral blood lymphocytes (PBL) in a homogeneous manner, independently of the base pathology, or, if on the contrary, PBL subsets show a different profile in each hepatic disease. Methods We studied PBL subsets in 71 patients with end-stage liver disease, before liver transplant, and 74 healthy controls by flow cytometry. The results were statistically compared between patients and controls, and cohorts of patients classified according to their base pathology. Results We observed lower absolute numbers in all lymphocyte populations in patients compared with controls. We found an increment of CD3+ activated cells (P<10−5) and CD45RO+CD4+ (P<10−5) in chronic hepatitis C virus versus controls; hepatitis B virus showed high TCRγδ+ and CD8+ T cells with respect to controls (P=0.008 and P=0.029, respectively); alcoholic cirrhotic patients showed low CD8+, mainly CD45RA+CD8+ (P=0.007) and high CD45RO+CD4+ (P<10−5) compared with the normal population; autoimmune diseases showed lower CD3+ and TCRαβ+ (P=0.002 and P=0.0001) than controls. Conclusions Regardless of the base pathology, patients with end-stage liver disease show a low absolute number of lymphocyte populations compared with controls. However, PBL profiles are different, characteristic, and specific of every disease causing chronic liver failure.

Early renal graft function deterioration in recipients with preformed anti-MICA antibodies: partial contribution of complement-dependent cytotoxicity

BACKGROUND We previously reported that preformed anti-MHC class I-related chain A (MICA) antibodies increase the risk for renal graft rejection and enhance the deleterious effect of PRA(+) status early after transplantation. METHODS We studied 727 kidney recipients. Days to reach optimal serum creatinine level, estimated glomerular filtration rate (eGFR) at Month 3 and chronic kidney disease (CKD) stages were recorded. Anti-MICA specificities and C1q binding were tested by solid-phase assay. Complement-dependent cytotoxicity (CDC) and flow cytometry (FC) cross-matches with HeLa and PMA/CD28-T-blasts were performed. RESULTS PRA(+)MICA(+) recipients exhibited longer time to reach optimal serum creatinine level after transplantation (P = 0.005) and had the lowest eGFR at Month 3 (P = 0.006). PRA(+)MICA(+) status independently increased the risk for CKDT stage 5 at Month 3 [hazard ratio (HR) 4.92, P = 0.030]. Pre-transplant anti-MICA antibodies were polyspecific and showed stronger reactions when coexisting with anti-HLA antibodies (mean standard fluorescent intensity 112 157 ± 44 426 in HLA(+)MICA(+) sera versus 49 680 ± 33 116 in HLA(-)MICA(+) sera, P = 0.0006). Anti-AYVE supereplet reactivity was significantly higher in HLA(+)MICA(+) versus HLA(-)MICA(+) patients (P < 0.001) and significantly superior than anti-CMGWS supereplet within HLA(+)MICA(+) patients (P = 0.001). Three of 13 anti-MICA(+) pre-transplant sera were positive for the C1q binding assay; one of them (serum 3) exclusively recognized AYVE supereplet with a strong reactivity against MICA*027 antigen (same as MICA*008). Anti-MICA antibodies in anti-HLA-absorbed serum 3 bound native MICA molecules in MICA*008(+) HeLa and PMA/CD28-T-blasts and mediated cell death by activating complement. CONCLUSION Preformed anti-MICA antibodies may occasionally be cytotoxic by fixing and activating complement. This way they might contribute to worse early kidney graft function.

High frequency of central memory regulatory T cells allows detection of liver recipients at risk of early acute rejection within the first month after transplantation

Several studies have analyzed the potential of T regulatory cells (Treg cells) as biomarkers of acute rejection (AR). The aim of the present multicenter study was to correlate the percentage of peripheral Treg cells in liver graft recipients drawn at baseline up to 12 months after transplantation with the presence of AR. The percentage of central memory (cm) Treg cells (CD4(+)CD25(high)CD45RO(+)CD62L(+)) was monitored at pre-transplant and at 1 and 2 weeks, and 1, 2, 3 and 6 months and 1 year post-transplantation. The same validation standard operating procedures were used in all participating centers. Fifteen patients developed AR (23.4%). Hepatitis C virus recurrence was observed in 16 recipients, who displayed low peripheral blood cmTreg levels compared with patients who did not. A steady increase of cmTregs was observed during the first month after transplantation with statistically significant differences between AR and non-AR patients. The high frequency of memory Treg cells allowed us to monitor rejection episodes during the first month post-transplantation. On the basis of these data, we developed a prediction model for assessing risk of AR that can provide clinicians with useful information for managing patients individually and customizing immunosuppressive therapies.

Activated Regulatory T Cells Expressing CD4+CD25highCD45RO+CD62L+ Biomarkers Could Be a Risk Factor in Liver Allograft Rejection

Activated regulatory T cells (aTregs) are nowadays a hot topic in organ transplantation to establish their role during acute rejection (AR) episodes. The aim of this multi-center study was to monitor the frequency of aTregs within the first year after transplantation in a cohort of first-time liver transplant recipients enrolled from 2010 to 2012. aTregs frequency was analyzed by means of flow cytometry. Patients who had AR showed higher levels of aTregs during first year after transplantation in comparison with patients who did not have higher levels. High levels of aTregs in liver recipients might be used as a biomarker of AR; however, further studies must be done to address the potential role of aTregs as biomarkers of AR in liver transplantation.

High proportion of CD95+ and CD38+ in cultured CD8+ T cells predicts acute rejection and infection, respectively, in kidney recipients

The aim of this study was to find noninvasive T-cell markers able to predict rejection or infection risk after kidney transplantation. We prospectively examined T-lymphocyte subsets after cell culture stimulation (according to CD38, CD69, CD95, CD40L, and CD25 expression) in 79 first graft recipients from four centers, before and after transplantation. Patients were followed up for one year. Patients who rejected within month-1 (n=10) showed high pre-transplantation and week-1 post-transplantation percentages of CD95(+), in CD4(+) and CD8(+) T-cells (P<0.001 for all comparisons). These biomarkers conferred independent risk for early rejection (HR:5.05, P=0.061 and HR:75.31, P=0.004; respectively). The cut-off values were able to accurately discriminate between rejectors and non-rejectors and Kaplan-Meier curves showed significantly different free-of-rejection time rates (P<0.005). Patients who rejected after the month-1 (n=4) had a higher percentage of post-transplantation CD69(+) in CD8(+) T-cells than non-rejectors (P=0.002). Finally, patients with infection (n=41) previously showed higher percentage of CD38(+) in CD8(+) T-cells at all post-transplantation times evaluated, being this increase more marked in viral infections. A cut-off of 59% CD38(+) in CD8(+) T-cells at week-1, week-2 and month-2 reached 100% sensitivity for the detection of subsequent viral infections. In conclusion, predictive biomarkers of rejection and infection risk after transplantation were detected that could be useful for the personalized care of kidney recipients.

High expression of CD38, CD69, CD95 and CD154 biomarkers in cultured peripheral T lymphocytes correlates with an increased risk of acute rejection in liver allograft recipients.

The mayor goal still outstanding into the solid organ transplantation field involves the search of surrogate biomarkers able to predict several clinical events, such as acute rejection (AR) or opportunistic infection. In the present multicenter study, a series of interesting surface antigens with important activator or inhibitory immune functions on cultured peripheral T cells were monitored in liver transplant recipients drawn at baseline and up to one year after transplantation. Sixty-four patients were included in the multicenter study during 3 years. Pre- and post-transplantation surface antigens levels displayed significant differences between AR and non acute rejection (NAR) groups, and also this differential expression was used to construct a risk predictive model based on a composite panel of outcome biomarkers (CD38, CD69, CD95 and CD154). The model was able to stratify these patients at high risk of AR. These preliminary results could provide basic information to improve the immunosuppressive treatment and it might better help to predict AR episodes.

Isolated De Novo Antiendothelial Cell Antibodies and Kidney Transplant Rejection.

BACKGROUND Studies analyzing the role of antiendothelial cell antibodies (AECAs) in large series of kidney transplant recipients are scarce, and HLA, MHC (major histocompatibility complex) class I-related chain A (MICA), and angiotensin II type 1 receptor have not been formally excluded as targets. STUDY DESIGN Retrospective study of a cohort of kidney transplant recipients. SETTING & PARTICIPANTS 324 kidney transplant recipients who were negative for anti-HLA, anti-MICA, and anti-angiotensin II type 1 receptor antibodies were tested for AECAs in pre- and posttransplantation serum samples. PREDICTORS AECA-positive (preformed [pre+/post+] vs de novo [pre-/post+]) versus AECA-negative (pre-/post-) before or after transplantation. OUTCOMES Patient mortality, transplant loss, and acute rejection events. RESULTS 66 (20%) patients were AECA positive (39 [12%] preformed, 27 [8%] de novo) and 258 (80%) were AECA negative. During a follow-up of 10 years, 7 (18%) AECA pre+/post+ patients had rejections compared with 14 (52%) AECA pre-/post+ and 57 (22%) AECA pre-/post- recipients (OR, 3.80; P=0.001). AECA pre-/post+ status emerged as an independent risk factor for transplant rejection compared to the AECA pre-/post- group (OR, 5.17; P<0.001). However, AECA pre+/post+ and AECA pre-/post+ patients did not show higher risk for either patient death (ORs of 1.49 [P=0.7] and 1.06 [P=0.9], respectively) or transplant loss (ORs of 1.22 and 0.86, respectively; P for both = 0.8) compared to the AECA pre-/post- population. LIMITATIONS Retrospective study. Posttransplantation sera were collected before or after rejection, entailing a nearly cross-sectional relationship between the exposure and outcome. Lack of identification of precise antigens for AECAs. CONCLUSIONS De novo AECAs may be associated with rejection. These antibodies might serve as biomarkers of endothelium damage in kidney transplant recipients.

Helper Innate Lymphoid Cells (hILC) resist Immunosuppressive Therapy: an Observation from Kidney and Liver Transplantation

Introduction We have previously observed that CD3+ CD8+ lymphocytes are depleted in the graft epithelium of intestinal transplant recipients who receive immunosuppressive therapy, while innate lymphoid cells type 1 and 3 (ILC1 and ILC3) persist in high proportions. In this study we compare the ILC representation and its subsets (ILC1, including NK and non-cytotoxic helper ILC1 cells (hILC1), ILC2 and ILC3) in peripheral blood of kidney and liver transplanted recipients versus control subjects (CS) in order to identify possible frequency variations in the context of immune alloresponse and immunosuppressive therapy (IT). Materials and Methods Peripheral blood mononuclear cells (PBMCs) were obtained at day +14, from 88 kidney recipients (KTR14): 11 received only triple therapy (No-I), 46 received induction therapy with thymoglobulin (I-TMG) and 31 with Basiliximab (I-Bas), from 19 liver recipients (LTR14): 16 were No-I and 3 I-Bas) and from 48 CS. Total ILC were identified by flow cytometry as CD45+ Lin- (CD3, CD19, CD14) and its subsets were defined as: ILC1 (CD117-CRTH2-), ILC2 (CD117-CRTH2+) and ILC3 (CD117+ CRTH2-). ILC1 were subdivided in NK and hILC1 according to CD127 and CD94. Results and Discussion • ILC2 and ILC3 are higher in KRT14 vs CS (p<0.0001 and p=0.0002, respectively) whereas ILC1 (NK and hILC1) are lower (p<0.0001) (Figure 1A). These differences in ILC subsets frequencies are due to decreasing of ILC1 absolute number (Figure 1B) whereas ILC2 and ILC3 numbers are not statistically different between KRT14 and CS. Similar results were obtained from the liver cohort (Figure 1C (p<0.0001 for ILC1 and ILC2, p=0.0005 for ILC3)) and 1D (p<0.0001)). • hILC1 frequency is higher (p=0.0001) whereas NK frequency is lower (p=0.0001) in KRT14 vs CS and LTR14 vs CS (p=0.0002 and p=0.008, respectively). These differences in ILC1 subsets frequencies are due to decreasing of NK absolute number (p<0.0001) (Figure 2A) whereas hILC1 number (Figure 2B) is not statistically different between KRT14 vs CS and LTR14 vs CS (Figure 2C (p<0.0001) and Figure 2D). • Decreasing of NK in KRT14 was mostly observed in patients who received TMG at pretransplant as induction therapy (Figure 3). • The obtained results from comparing LTR14 vs CS are comparable to those observed between KTR14 vs CS. Due to the fact that most of LTR14 only received triple therapy it seems that it leads to NK but no ILC depletion. Figure. No caption available. Figure. No caption available. Figure. No caption available. Conclusion hILC are unaffected by TMG and Basiliximab used as induction therapy for kidney and liver transplantation. hILC are also unaffected by tacrolimus-based triple therapy used as maintenance treatment.