Regulatory T-cell activation among patients who displayed operational tolerance following intra-portal administration of donor-specific antigens in living donor liver transplantation

Immunologic tolerance is the goal for all transplant surgeons. We have reported that repeated donor-specific antigen transfusion (DST) via the portal vein allowed rapid reduction of immunosuppressants with decreased acute cellular rejection episodes among living donor liver transplantations (LDLT). Moreover, we demonstrated that intraportal DST induced macrochimerism of donor type CD56⁺ T cells in the liver graft. We examined the impact of FoxP3⁺CD4⁺CD25⁺ T cells in recipients who acquired almost tolerance after LDLT with intraportal DST. We defined the amount of immunosuppressants administered less than one time per week as “almost tolerance” after LDLT, which occurred among 14% of DST patients after adult-to-adult LDLT. Two patients (4%) have gotten been we used from immunosuppressants more than 2 years after LDLT 4 years prior. We examined the impact of FoxP3⁺CD4⁺CD25⁺ T cells both in recipients with almost daily immunosuppressants and those who acquired almost tolerance. The proportion of FoxP3⁺/CD4⁺CD25⁺ T cells in the almost tolerance group was significantly higher than that in the almost daily immunosuppressant group (P < .05). The increased proportion of FoxP3⁺/CD4⁺CD25⁺ T cells significantly correlated with time after LRLT (y = 0.0964x + 42.02, R² = 0.8854). Repeated intraportal DST may be a goot tool to induce immunologic tolerance after LDLT. Both donor type CD56⁺ T cells and FoxP3⁺/CD4⁺CD25⁺ T cells may act as important regulatory cells for tolerance. The period after LDLT is important for acquiring immunologic tolerance.     

Influence of Immunosuppressive Drugs on the Development of CD4CD25 Foxp3 T Cells in Liver Transplant Recipients

Introduction

Many studies suggest that CD4⁺CD25^high T regulatory cells (Tregs) have a crucial role in downregulating the immune response to alloantigens. In this study, we investigated the possible influence of immunosuppressive therapy, including rapamycin and calcineurin inhibitors (CNIs; tacrolimus), on level of Tregs in liver allograft recipients.

Materials and Methods

We assessed 47 liver transplant recipients with stable liver function for ≥2 years, dividing them into 2 groups: Patients receiving rapamycin (n = 15), and those receiving tacrolimus (n=32). Thirty-eight, age-matched healthy subjects were used as normal controls. We examined the expression of CD4, CD25, and Foxp3 in peripheral blood T cells. Flow cytometry was performed with a FACSCalibur instrument with data analysis using Cell Quest software.

Results

Rapamycin significantly increased the prevalence of Tregs, including the percentage of CD4⁺CD25^high T cells in total lymphocytes and among total CD4⁺ T cells, compared with the healthy subjects and the CNI group. The prevalence of Tregs in the CNIs group was significantly lower than that of controls. Foxp3 was expressed in >95% of CD4⁺CD25^highT cells, whereas it was in <20% of CD4⁺CD25^low T cells and not expressed among CD4⁺CD25⁻ T cells.

Conclusions

Immunosuppressive therapy (rapamycin or CNIs) may have a different roles in tolerance induction among liver transplant recipients. Namely, rapamycin promoted the induction of a profile consistent with alloantigen tolerance; CNIs hampered this progression.     

Decreased circulating CD4+CD25highFoxp3+ T cells during acute rejection in liver transplant patients

Background

CD4⁺CD25^highFoxP3⁺ regulatory T (Treg) cells have been implicated to maintain immunologic tolerance. They have been investigated in acute renal allografts rejection episodes (ARE). This study was performed to examine the frequency of peripheral blood (PB) CD4⁺CD25^highFoxP3⁺ Treg cells among liver transplantation patients with prior benign end-stage liver diseases in relation to ARE.

Methods

This prospective analysis of 55 patients who underwent liver transplantation from 2004 to 2009 did not include prisoners either as donors or recipients. PB was obtained from liver transplant patients longitudinally: pretransplantation, posttransplantation within 1 year, and at the time of an episode of ARE to measure by flow cytometry circulating CD4⁺CD25^highFoxP3⁺ T cells. Blood samples were drawn during ARE with concomitant liver biopsies. The rejector group was defined in the 14/55 cases who suffered an ARE; in the other patients with stable liver function were classified as the nonrejector group. We compared the number of circulating CD4⁺CD25^highFoxP3⁺ T cells between the 2 groups.

Results

There was no difference in the levels of circulating CD4⁺CD25^highFoxP3⁺ T cells pretransplantation. Interestingly, circulating CD4⁺CD25^highFoxP3⁺ T cells were significantly lower among the rejector compared with the nonrejector (2.23 ± 0.54% vs 2.99 ± 0.86%; P < .01). Longitudinal analysis revealed circulating CD4⁺CD25^highFoxP3⁺ T cells of patients in the rejector group to be significantly lower during rejection than during quiescence (2.23 ± 0.54% vs 3.68 ± 0.70%; P < .0001). The frequency of circulating CD4⁺CD25^highFoxP3⁺ T cells negatively correlated with a Rejection Activity Index (r = −0.80; P < .01).

Conclusion

Monitoring peripheral CD4⁺CD25^highFoxP3⁺ T cell levels may be useful to evaluate the immune state, potentially acting as a sensitive marker for ARE diagnosis among liver transplantation patients. Moreover, they may contribute to the mechanisms of Treg-mediated acceptance of liver transplantations.     

Monitoring of cytomegalovirus-specific CD8+ T-cell response with major histocompatibility complex pentamers in kidney transplant recipients

Introduction

Cytomegalovirus (CMV) can reactivate causing serious clinical problems during immunosuppression. CMV-specific CD8⁺ T cells play an important role in the control of CMV reactivation. Using pentameric major histocompatibility complex (MHC) peptide complexes, we investigated cellular immune responses to CMV among healthy individuals and kidney transplantation recipients in Korea, which is an endemic area of CMV infection.

Materials and methods

Analysis of CMV-specific T cells was performed on 28 healthy individuals and 40 recipients who bore human leukocyte antigen (HLA)-A2 or -A24. CMV pp65 pentamer-binding cells incubated with various monoclonal antibodies were measured by four-color flow cytometry.

Results

Detectable levels of pentamer⁺ CD8⁺ T cells were present in 109/139 samples (78.4%) that stained with the A*02NLV-pentamer, while 15/67 samples (22.4%) stained with the A*24QYD-pentamer (P < .01). Among patients with HLA-A2, 22/24 (91.7%) samples showing positive CMV antigenemia revealed detectable pentamer⁺ CD8⁺ T cells, while 87/115 (75.7%) displaying negative CMV antigenemia had detectable pentamer⁺ CD8⁺ T cells (P = .04). There was no significant difference in percentages of pentamer⁺ CD8⁺ T cells between patients who did versus who did not experience episodes of CMV infection. The subpopulation of CMV-specific CD8⁺ T cells in transplantation recipients was evaluated using phenotypic markers; memory cells comprised the majority of the CMV-specific CD8⁺ T-cell population.

Conclusion

The A*02NLV-pentamer complex was useful to monitor CMV-specific T cells. However, MHC pentamer-based techniques did not provide a clear distinction between patients who are or are not at risk for CMV infection.     

Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4+ memory T cells in mice

Donor-reactive memory T (Tm) cells undermine transplanted organs more readily than naive T cells. Rapamycin (RAPA) and tacrolimus (FK-506) are current mainstay immunosuppressants used for preventing acute allograft rejection. Although their efficacy in suppressing naive T cell is established, their suppressing effect on memory T cells is undefined. This study was conducted to investigate the inhibiting capability of RAPA or FK-506 against transferred alloreactive CD4⁺ Tm cells in a mouse cardiac transplant model. We found that these drugs alone prolonged the median survival time (MST) of allograft from 5 days to 9 days in recipient mice with CD4⁺ Tm infusion (P < 0.01), which however was not significantly longer than that (8 days) in untreated recipient mice without CD4⁺ Tm infusion (naive control). Mean histologic rank of rejection activity in section of cardiac allograft on day 5 postgrafting was Grade 4 in the Tm control recipients versus Grade 3A in both of the immunosuppressant treatment recipients with CD4⁺ Tm infusion. RAPA or FK-506 alone failed to completely suppress proliferation and differentiation of the alloreactive CD4⁺ Tm, which was confirmed by in vitro mixed lymphocyte reaction (MLR) and by flow cytometry (FCM) of the splenocytes for detecting CD44^highCD62L⁻ effector/memory as well as CD69⁺/CD25⁺ activation phenotype cells from the respective recipients. Furthermore, the agent alone didn't completely inhibit the activation of CD4⁺ Tm, for serum level of IFN-γ and its gene expression at the cardiac allograft from the immunosuppressant-treated recipients were as still high as the untreated naive control. Thus, RAPA or FK-506 alone couldn't completely suppress the proliferation and activation of the alloantigen-primed CD4⁺ Tm cells responding to the alloantigen, indicating that alloreactive CD4⁺ Tm was insensitive to these immunosuppressants. The characteristics of alloreactive CD4⁺ Tm to resist immunosuppressants and its potency to initiate quick and vigorous rejection despite treatment with the immunosuppressant make it to be a critical barrier to prolongation of allograft survival and induction of transplant tolerance.     

Lymphocyte subtypes CD3+, CD19+, CD16+CD56+, CD4+, CD8+, and CD3+HLA-DR+ in peripheral blood obtained from patients after thoracic organ transplantation

Introduction

The aim of this study was to assess peripheral blood lymphocyte subtypes (CD3⁺, CD19⁺, CD16⁺CD56⁺, CD4⁺, CD8⁺, and CD3⁺HLA-DR⁺) obtained from thoracic organ recipients at various periods after transplantation.

Material and Methods

Seventeen patients after lung transplantation (LT) and 5 patients after heart transplantation (HT) included 13 males (76.5%) and 4 females (23.5%) of overall mean age at the time of transplantation of 46.7 ± 11.55 years and mean body mass index of 21.1 ± 4. Lymphocyte phenotypes were estimated using Simultest IMK Plus.

Results

A significant decrease in lymphocytes of the majority of subtypes was observed at 1 year posttransplantation compared with normal ranges: CD19⁺ B lymphocytes in 56% of patients, CD8⁺ T cells among 48% and CD16⁺CD56⁺ natural killer elements, 56%. In contrast, there were increased numbers of activated lymphocytes (CD3⁺HLA-DR⁺). Beyond the 1-year observation, we observed a trend to normalize parameters among the majority of subjects.

Conclusion

A clear tendency to a decrease number of peripheral blood lymphocytes of various subtypes was observed among thoracic organ recipients in the first year posttransplantation with the exception of activated HLA-DR⁺ cells. After the first year, there was slow restoration of lymphocytes.     

Paradoxical Functions of B7: CD28 Costimulation in a MHC Class II‐Mismatched Cardiac Transplant Model

Blockade of the B7: CD28 costimulatory pathway has emerged as a promising therapy to prevent allograft rejection. However, this pathway has also been demonstrated to be important for the generation and maintenance of regulatory T cells. In this study, we investigated the role of the B7: CD28 pathway in the ‘bm12 into B6’ MHC class II‐mismatched vascularized cardiac transplant model of chronic rejection. Allograft rejection was remarkably accelerated in B6 background B7DKO and CD28KO recipients compared with B6 wild‐type (WT) recipients. Allograft rejection was associated with a significantly enhanced Th1/Th2 alloreactivity and marked reduction in the ratio of regulatory T cells to CD4⁺ effector/memory cells. We noted that administration of anti‐B7‐1 and anti‐B7‐2 mAb prior to transplantation also accelerated allograft rejection. Furthermore, depleting CD25⁺ cells in B6 WT recipients of bm12 hearts prior to transplant also precipitated rejection at a similar rate. Neither B7/CD28 deficiency nor CD25 depletion affected graft survival in single MHC class I‐mismatched (bm1 into B6) recipients. This study highlights the paradoxical functions of B7: CD28 costimulation in a MHC class II‐mismatched model, in which the B7: CD28 pathway is demonstrated to be important in preventing rejection through the generation and maintenance of Tregs.     

Long‐term survival of pig‐to‐rhesus macaque renal xenografts is dependent on CD4 T cell depletion

The shortage of available organs remains the greatest barrier to expanding access to transplant. Despite advances in genetic editing and immunosuppression, survival in experimental models of kidney xenotransplant has generally been limited to <100 days. We found that pretransplant selection of recipients with low titers of anti‐pig antibodies significantly improved survival in a pig‐to–rhesus macaque kidney transplant model (6 days vs median survival time 235 days). Immunosuppression included transient pan–T cell depletion and an anti‐CD154–based maintenance regimen. Selective depletion of CD4⁺ T cells but not CD8⁺ T cells resulted in long‐term survival (median survival time >400 days vs 6 days). These studies suggested that CD4⁺ T cells may have a more prominent role in xenograft rejection compared with CD8⁺ T cells. Although animals that received selective depletion of CD8⁺ T cells showed signs of early cellular rejection (marked CD4⁺ infiltrates), animals receiving selective CD4⁺ depletion exhibited normal biopsy results until late, when signs of chronic antibody rejection were present. In vitro study results suggested that rhesus CD4⁺ T cells required the presence of SLA class II to mount an effective proliferative response. The combination of low pretransplant anti‐pig antibody and CD4 depletion resulted in consistent, long‐term xenograft survival.     

Elevated CD4/CD25 T-cell Frequency and Function During Hepatitis C Virus Recurrence After Liver Transplantation

Background/Aim

Factors involved in hepatitis C virus (HCV) recurrence versus acute cellular rejection are not fully understood. The aim of the present study was to investigate whether patients with recurrence after liver transplantation (OLT) showed similar CD4⁺/CD25⁺ cell frequency and function as those who became chronically infected.

Patients and Methods

After written informed consent, we enrolled 20 patients (group A) who underwent OLT with HCV recurrence within 6 months. HCV-RNA and hypertransaminasemia were used to assess the reactivation of viral hepatitis. CD4⁺/CD25⁺ T cells were enumerated using a flow cytometry assay, gated on CD3 cells, stained for FoxP3. After immunomagnetic sorting (Dynal, Oslo, NW), Treg suppressor activity was measured, as the ability to inhibit proliferation of autologous CD4⁺/CD25⁻ T cells (anti-CD3/CD28 stimulation—1:2, 1:20 ratio). Eight patients with acute hepatitis C who evolved to a chronic infection after 6 months (group B) were used as positive controls, while 10 healthy individuals were negative controls (group C).

Results

We did not observe any difference in CD4⁺/CD25⁺ frequency or function among group A compared with group B (CD4⁺/CD25⁺ = 14% ± 2% versus CD4⁺/CD25⁺ = 16% ± 3%), although both groups were significantly increased with respect to group A (CD4⁺/CD25⁺ = 6% ± 3%; Mann-Whitney U test, P < .01).

Conclusion

Patients developing HCV recurrence after OLT have the same immunoregulatory network as patients with acute hepatitis C evolving to persistent infection, likely suggesting that CD4⁺/CD25⁺ numbers may be a marker to predict recurrence of HCV after OLT.     

Donor major histocompatibility complex class I expression determines the outcome of prenatal transplantation

Purpose

The failure of in utero transplantation in immune-competent recipients suggests the existence of a fetal immune barrier. The importance of donor major histocompatibility complex (MHC) class I expression in the induction of prenatal tolerance remains undefined. We hypothesized that donor cell MHC class I expression facilitates engraftment in prenatal allogeneic recipients rather than promoting immune rejection.

Methods

B6.Ly5.2 (class I⁺) or B6.TAP^−/− (class I⁻) murine fetal liver cells were transplanted into age-matched allogeneic fetal recipients. Survival to weaning and subsequent growth was assessed. Engraftment rates and peripheral blood chimerism levels were measured serially.

Results

The presence or absence of class I expression did not affect survival or growth of recipients and no graft-vs-host disease developed. Allogeneic recipients of B6.Ly5.2 cells exhibited significantly higher levels of donor hematopoietic chimerism when compared to recipients of B6.TAP^−/− cells (27% + 10% vs 11% + 8%; P = .004) that deteriorated further over time.

Conclusions

Donor class I MHC antigen expression is essential for stable long-term engraftment and maintenance of donor-specific tolerance. Further studies are needed to better characterize the role of the fetal innate immune system in prenatal allotransplantation.     

Immune profile of pediatric renal transplant recipients following alemtuzumab induction

The incidence of developing circulating anti-human leukocyte antigen antibodies and the kinetics of T cell depletion and recovery among pediatric renal transplant recipients who receive alemtuzumab induction therapy are unknown. In a collaborative endeavor to minimize maintenance immunosuppression in pediatric renal transplant recipients, we enrolled 35 participants from four centers and treated them with alemtuzumab induction therapy and a steroid-free, calcineurin-inhibitor–withdrawal maintenance regimen. At 3 months after transplant, there was greater depletion of CD4⁺ than CD8⁺ T cells within the total, naive, memory, and effector memory subsets, although depletion of the central memory subset was similar for CD4⁺ and CD8⁺ cells. Although CD8⁺ T cells recovered faster than CD4⁺ subsets overall, they failed to return to pretransplant levels by 24 months after transplant. There was no evidence for greater recovery of either CD4⁺ or CD8⁺ memory cells than naïve cells. Alemtuzumab relatively spared CD4⁺CD25⁺FoxP3⁺ regulatory T cells, resulting in a rise in their numbers relative to total CD4⁺ cells and a ratio that remained at least at pretransplant levels throughout the study period. Seven participants (20%) developed anti-human leukocyte antigen antibodies without adversely affecting allograft function or histology on 2-year biopsies. Long-term follow-up is underway to assess the potential benefits of this regimen in children.The effects of alemtuzumab on T cell subsets have been extensively studied in adults since its introduction in the 1990s. It has been associated with profound depletion of total T cells and differential recovery among T cell subsets, with early and near-complete recovery of CD8⁺ T cells, but late, partial recovery of CD4⁺ T cells.^1–3 CD4⁺ memory T cells were relatively spared compared with other CD4⁺ subsets; some investigators reported preferential sparing of central memory (T_CM) cells, whereas others observed preferential sparing of the effector memory (T_EM) subset. Emergence of the T_EM subset, whether identified peripherally or in the allograft, has been associated with acute rejection, raising concerns about the tolerogenic potential of alemtuzumab.^1–4 Although the use of alemtuzumab was not associated with an increase in either FoxP3 expression or regulatory T cell counts in vitro, both transient and sustained expansion of regulatory T cells were observed when alemtuzumab was used in association with sirolimus in vivo.^2,5,6 Notwithstanding this, however, the combination of alemtuzumab and sirolimus in protocols free of calcineurin inhibitor (CNI) was associated with rates of acute rejection exceeding 20%, with a humoral rejection rate as high as 62.5%.^7,8 In the absence of long-term CNI treatment, alemtuzumab-treated adults may therefore have a propensity to develop alloantibodies and antibody-mediated rejection.^9,10In contrast, there is a paucity of data regarding the use of alemtuzumab in pediatric solid organ transplantation.¹¹ From a clinical perspective, two small series investigating the outcomes of selected high-risk recipients of renal, liver, and intestinal transplants treated with alemtuzumab yielded conflicting results, whereas a recent series of 42 renal transplant recipients of living donor grafts reported few cases of acute rejection and excellent graft function up to 4 years after transplant.^12–14 From a mechanistic perspective, only one study reported T cell counts in a single pediatric patient, demonstrating profound and prolonged depletion of CD3⁺, CD4⁺, CD8⁺, and CD20⁺ cells, with counts only reaching 50% of their baseline levels 12 months after transplant.¹²In this study, we investigated the longitudinal immune profiles of pediatric renal transplant recipients treated with alemtuzumab induction therapy, followed by a CNI-withdrawal regimen. Specific aims were to characterize the depletion and recovery patterns of various T cell subsets and to screen for anti-human leukocyte antigen (anti-HLA) antibody development.     

CD154 Deficiency Uncouples Allograft CD8+ T‐Cell Effector Function from Proliferation and Inhibits Murine Airway Obliteration

Obliterative bronchiolitis (OB) limits the long‐term success of lung transplantation, while T‐cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8⁺ T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8⁺ T cells, as airway rejection has been reported to be CD154‐dependent. Airway allografts from CD154^−/− recipients had significantly lower day 28 OAD scores compared to wild‐type (WT) recipients, and adoptive transfer of CD8⁺ T cells from WT recipients, but not CD154^−/− recipients, were capable of airway rejection in fresh CD154^−/− allograft recipients. Intragraft CD8⁺ T cells from CD154^−/− mice showed similar expression of the surface markers CD69, CD62L^low CD44^high and PD‐1, but markedly impaired IFN‐γ and TNF‐α secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8⁺ T cells from CD154^−/− recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8⁺ T cells required for murine OAD.     

Increased Pretransplant Frequency of CD28+ CD4+ TEM Predicts Belatacept‐Resistant Rejection in Human Renal Transplant Recipients

While most human T cells express the CD28 costimulatory molecule constitutively, it is well known that age, inflammation, and viral infection can drive the generation of CD28^null T cells. In vitro studies have demonstrated that CD28^null cell effector function is not impacted by the presence of the CD28 costimulation blocker belatacept. As such, a prevailing hypothesis suggests that CD28^null cells may precipitate costimulation blockade‐resistant rejection. However, CD28⁺ cells possess more proliferative and multifunctional capacity, factors that may increase their ability to successfully mediate rejection. Here, we performed a retrospective immunophenotypic analysis of adult renal transplant recipients who experienced acute rejection on belatacept treatment as compared to those who did not. Intriguingly, our findings suggest that patients possessing higher frequency of CD28⁺ CD4⁺ T_EM prior to transplant were more likely to experience acute rejection following treatment with a belatacept‐based immunosuppressive regimen. Mechanistically, CD28⁺ CD4⁺ T_EM contained significantly more IL‐2 producers. In contrast, CD28^null CD4⁺ T_EM isolated from stable belatacept‐treated patients exhibited higher expression of the 2B4 coinhibitory molecule as compared to those isolated from patients who rejected. These data raise the possibility that pretransplant frequencies of CD28⁺ CD4⁺ T_EM could be used as a biomarker to predict risk of rejection following treatment with belatacept.     

Bile CXC Motif Chemokine 10 Levels Correlate With Anti-donor Cytotoxic T Cell Responses After Liver Transplantation

Background

CXC motif chemokine 10 (CXCL10), known as interferon-γ−induced protein 10, is an inflammatory cytokine secreted by various cells in response to interferon-γ. CXCR3, the receptor of CXCL10, is predominantly expressed on activated T, B, natural killer, and dendritic cells, as well as macrophages. CXCR3 promotes chemotaxis upon binding CXCL10. Serum CXCL10 levels have recently attracted attention as a post-transplantation biomarker for graft rejection. However, the correlation between the degree of T cell response to allostimulation and CXCL10 levels remains unclear. In this study, we investigated the serum and bile CXCL10 levels of patients who underwent living donor liver transplantation (LDLT) and compared them with the T cell responses to allostimulation.

Patients and Methods

Between February 2009 and August 2012, 41 patients underwent LDLT at Hiroshima University Hospital. Serum and bile CXCL10 levels were measured weekly for 4 weeks after surgery, while the T cell responses to allostimulation were evaluated using a mixed lymphocyte reaction with an intracellular carboxyfluorescein diacetate succinimidyl ester−labeling technique that we regularly use to monitor the immune response to anti-donor and anti−third-party stimulation after liver transplantation. The stimulation index (SI) and CD25 expression of the CD4+ and CD8+ T cell subsets in response to allostimulation were then analyzed using flow cytometry.

Results

Serum CXCL10 levels were significantly correlated with the SI values for CD8+ T cells in response to both types of allostimulation. Bile CXCL10 levels were significantly correlated with CD25 expression of CD8+ T cell subsets, especially in response to anti-donor stimulation. Patients with higher bile CXCL10 levels suffered from severe acute cellular rejection that was refractory to steroid pulse.

Conclusion

Measurements of bile CXCL10 levels could predict anti-donor cytotoxic T cell responses in liver transplant recipients.     

Belatacept‐Resistant Rejection Is Associated With CD28+ Memory CD8 T Cells

Recently, newer therapies have been designed to more specifically target rejection in an effort to improve efficacy and limit unwanted toxicity. Belatacept, a CD28‐CD80/86 specific reagent, is associated with superior patient survival and graft function compared with traditional therapy, but its adoption as a mainstay immunosuppressive therapy has been tempered by increased rejection rates. It is essential that the underlying mechanisms associated with this rejection be elucidated before belatacept is more widely used. To that end, we designed a study in a nonhuman primate kidney transplant model where animals were treated with either a belatacept‐ or a tacrolimus‐based immunosuppressive regimen. Interestingly, we found that elevated pretransplant frequencies of CD28⁺CD8⁺T_EMRA cells are associated with rejection on belatacept but not tacrolimus treatment. Further analysis showed that the CD28⁺CD8⁺T_EMRA cells rapidly lose CD28 expression after transplant in those animals that go on to reject with the allograft infiltrate being predominantly CD28^?. These data suggest that CD28⁺ memory T cells may be resistant to belatacept, capable of further differentiation including loss of CD28 expression while maintaining effector function. The unique signaling requirements of CD28⁺ memory T cells provide opportunities for the development of targeted therapies, which may synergize with belatacept to prevent costimulation‐independent rejection.     

Correlation Between Survival Interval and CD4+ T-Cell Intracellular ATP Levels in Liver Transplant Recipients

Objective

The objectives of this study were to analyze the potential correlation between post–liver transplantation survival interval and CD4⁺ T-cell intracellular ATP (iATP) levels, and to describe the distribution of CD4⁺ T-cell iATP levels in liver transplant recipients.

Immune-mediated liver injury: prognostic value of CD4+, CD8+, and CD68+ in infants with extrahepatic biliary atresia

Background

Hepatic fibrosis and cirrhosis develop progressively in extrahepatic biliary atresia (EHBA) despite timely surgical intervention.

Purpose

The aim of the study was to define CD4⁺ helper T lymphocytes, cytotoxic CD8⁺ T lymphocytes, and CD68⁺ (macrophages) infiltration of portal tracts and lobules and hepatic fibrosis as possible predictive measures of outcome of infants having EHBA.

Methods

The outcome of 32 infants with EHBA was correlated to their percutaneous biopsy and postportoenterostomy core liver tissue infiltration by CD4⁺, CD68⁺, and CD8⁺ cells and to the degree of detected fibrosis.

Results

Portoenterostomy cores were heavily infiltrated by CD4⁺, CD8⁺, and CD68⁺, compared with the preoperative liver biopsy (P = .008, .004, and .017, respectively). Infants having favorable outcome had more macrophage infiltration in portoenterostomy core compared with those having an unfavorable outcome (25.66 ± 29.77 per HPF compared with 11.62 ± 4.58, P = .000). Mean CD4⁺/CD8⁺ ratio was 1.54 ± 1.37 in those who died within 18 months postoperatively and 0.733 ± 0.48 in others (P = .021).

Conclusion

Immune-mediated destruction of portal tracts is an integral part of pathogenesis of EHBA.     

CD28 biomarker quantification and expression level profiles in CD4+ T-lymphocytes in solid organ transplantation

The introduction of anti-calcineurin-based therapies has led to an increase in the one-year survival as well as graft function rates in patients undergoing solid organ transplantation (SOT). Nonetheless, early cellular acute rejection (EAR) incidence still remains a major challenge that irrevocably heads to poor outcomes. The mechanisms underlying CD4 T cell activation in SOT are still under research. In this sense, CD28 co-stimulatory molecule plays a pivotal role triggering CD4 T cell activation as well as survival maintenance. Previous own studies stated the role that CD4⁺ CD28⁺ circulating T lymphocytes plays before and during EAR episodes. We assessed the percentage as well as the absolute number of CD28 molecules on CD4⁺ T cells as predictive surrogate biomarker of EAR in a prospective cohort of liver and kidney transplant recipients. Quantitative analysis of CD28 was carried out on whole peripheral blood samples by flow cytometry. Decreased pre-transplant expression of CD28 was associated with EAR in both study groups. Furthermore, the expression of CD28 within the rejected group, experimented an up-regulation upon transplantation. These preliminary results suggest that patients undergoing liver or kidney transplant can be stratified at high risk of EAR according to their CD28 molecule expression on peripheral CD4⁺ T lymphocytes.