T and B lymphocyte dynamics after genetically-modified pig-to-baboon kidney xenotransplantation with an anti-CD40mAb-based immunosuppressive regimen

BackgroundThe aim was to monitor recovery of T/B lymphocytes in baboons after depletion by anti-thymocyte globulin (ATG) and anti-CD20mAb (Rituximab), followed by pig kidney transplantation and maintenance therapy with an anti-CD40mAb-based regimen.MethodsIn baboons (n = 14), induction was with ATG and anti-CD20mAb, and maintenance with (i) anti-CD40mAb, (ii) rapamycin, and (iii) methylprednisolone. Follow-up was for 6 months, or until rejection or other complication developed. Baboon blood was collected at intervals to measure T/B cells and subsets by flow cytometry. In a separate study in baboons receiving the same immunosuppressive regimen (n = 10), the populations of T/B lymphocytes in PBMCs, lymph nodes, and spleen were examined.ResultsAfter induction therapy, the total lymphocyte count and the absolute numbers of CD3⁺, CD4⁺, and CD8⁺T cells fell by >80%, and no CD22⁺B cells remained (all p < 0.001). T cell numbers began to recover early, but no CD22⁺B cells were present in the blood for 2 months. Recovery of both T and B cells remained at <30% of baseline (p < 0.001), even if rejection developed. At 6 months, effector memory CD8⁺T cells had increased more than other T cell subsets, but a greater percentage of B cells were naïve. In contrast to blood and spleen, T and B cells were not depleted in lymph nodes.ConclusionsATG and anti-CD20mAb effectively decreased T and B lymphocytes in the blood and, in the presence of anti-CD40mAb maintenance therapy, recovery of these cells was inhibited. The recovery of effector memory CD8⁺T cells may be detrimental to long-term graft survival.     

Relationship between antithymocyte globulin,T cell phenotypes,and clinical outcomes in pediatric kidney transplantation

Depletional induction using antithymocyte globulin (ATG) reduces rates of acute rejection in adult kidney transplant recipients, yet little is known about its effects in children. Using a longitudinal cohort of 103 patients in the Immune Development in Pediatric Transplant (IMPACT) study, we compared T cell phenotypes after ATG or non-ATG induction. We examined the effects of ATG on the early clinical outcomes of alloimmune events (development of de novo donor specific antibody and/or biopsy proven rejection) and infection events (viremia/viral infections). Long-term patient and graft outcomes were examined using the Scientific Registry of Transplant Recipients. After ATG induction, although absolute counts of CD4 and CD8 T cells were lower, patients had higher percentages of CD4 and CD8 memory T cells with a concomitant decrease in frequency of naïve T cells compared to non-ATG induction. In adjusted and unadjusted models, ATG induction was associated with increased early event-free survival, with no difference in long-term patient or allograft survival. Decreased CD4⁺ naïve and increased CD4⁺ effector memory T cell frequencies were associated with improved clinical outcomes. Though immunologic parameters are drastically altered with ATG induction, long-term clinical benefits remain unclear in pediatric patients.     

Kinetics of peripheral blood lymphocyte subpopulations predicts the occurrence of opportunistic infection after kidney transplantation

Serial monitoring of peripheral blood lymphocyte subpopulations (PBLSs) counts might be useful in predicting post‐transplant opportunistic infection (OI) after kidney transplantation (KT). PBLSs were prospectively measured in 304 KT recipients at baseline and post‐transplant months 1 and 6. Areas under receiver operating characteristic curves were used to evaluate the accuracy of different subpopulations in predicting the occurrence of overall OI and, specifically, cytomegalovirus (CMV) disease. We separately analyzed patients not receiving (n = 164) or receiving (n = 140) antithymocyte globulin (ATG) as induction therapy. In the non‐ATG group, a CD8⁺ T‐cell count at month 1 <0.100 × 10³ cells/μl had negative predictive values of 0.84 and 0.86 for the subsequent occurrence of overall OI and CMV disease, respectively. In the multivariate Cox model, a CD8⁺ T‐cell count <0.100 × 10³ cells/μl was an independent risk factor for OI (adjusted hazard ratio: 3.55; P‐value = 0.002). In the ATG group, a CD4⁺ T‐cell count at month 1 <0.050 × 10³ cells/μl showed negative predictive values of 0.92 for the subsequent occurrence of overall OI and CMV disease. PBLSs monitoring effectively identify KT recipients at low risk of OI, providing an opportunity for individualizing post‐transplant prophylaxis practices.     

Effect of Mammalian Target of Rapamycin Inhibition on Activated Regulatory T-Cell Expansion in Kidney Transplantation

BackgroundThe mammalian target of rapamycin (mTOR) plays a critical role in the host immune response in organ transplantation. This study evaluates the regulatory benefits of mTOR inhibitors in kidney transplant recipients (KTRs).MethodsThe mTOR-dependent immune-regulating effects in KTRs were evaluated by examining T-cell subsets among peripheral blood mononuclear cells from 79 KTRs. Recipients included an early introduction of everolimus (EVR) and reduced-exposure tacrolimus group (n = 46) and a standard tacrolimus-based without EVR (non-EVR) group (n = 33).ResultsTrough concentrations of tacrolimus at 3 months and 1 year were significantly lower in the EVR group than the non-EVR group (both P < .001). In addition, the respective proportions of patients without estimated glomerular filtration rate < 20% in the EVR and non-EVR groups were 100% and 93.3% at 1 year, 96.3% and 89.7% at 2 years, and 96.3% and 89.7% at 3 years after blood collection, respectively (P = .079). The frequencies of CD3⁺ T cells and CD4⁺ T cells among peripheral blood mononuclear cells were comparable between groups. Total CD25^highCD127⁻CD4⁺ regulatory T (Treg) cells were similar in the EVR and non-EVR groups. In contrast, circulating CD45RA⁻CD25^highCD127⁻CD4⁺ activated Treg cells were significantly higher in the EVR group (P= .008).ConclusionThese results suggest that the early introduction of mTOR benefits long-term kidney graft function and circulating activated Treg-cell expansion in KTRs.     

Ex Vivo Expansion of Human Tregs by Rabbit ATG Is Dependent on Intact STAT3‐Signaling in CD4+ T Cells and Requires the Presence of Monocytes

The addition of low, nondepleting doses of rabbit antithymocyte globulin (ATG) to human peripheral blood mononuclear cells has been shown to expand functional CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) in vitro. This report is the first to elucidate the exact cellular mechanisms of ATG‐mediated Treg expansion. CD4⁺ T cells require monocytes, but not other antigen presenting cell subsets, to be present in coculture to expand Tregs. However, T cells do not require direct cell–cell contact with monocytes, suggesting the importance of soluble factors. Moreover, ATG initially “reprograms” CD4⁺ T cells, but not monocytes, and induces STAT3 and STAT5 signaling in CD4⁺ cells. These reprogrammed CD4⁺ T cells subsequently secrete GM‐CSF and IL‐10 only in case of intact STAT3 signaling, which in turn promote the generation of tolerogenic CD14⁺CD11c⁺ dendritic cells characterized by enhanced IL‐10 and decreased IL‐12 production. Treg expansion following ATG treatment is accompanied by enhanced gene expression of both GM‐CSF and Bcl‐2, but not TGF‐β, in peripheral blood mononuclear cells. These results demonstrate that ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD4⁺ T cells in a STAT3‐dependent but TGF‐β‐independent manner, leading to the generation of monocyte‐derived dendritic cells with a tolerogenic cytokine profile.     

Thymic function, anti-thymocytes globulins, and cancer after renal transplantation

Background

Prolonged CD4 T cell lymphopenia after polyclonal antithymocyte globulins (ATG) is associated with an increased rate of cancers. Here, we examined whether pre-transplant thymic function estimated by TREC levels is predictive of cancer occurrence following ATG treatment.

Patients and methods

The impact of TREC on cancer occurrence was analyzed in 115 consecutive incident renal transplant recipients having received ATG.

Results

Mean follow-up was 7.5 ± 2.6 years. After ATG induction, patients with the lowest pre-transplant TREC values had lower post-transplant CD4⁺ and CD4⁺ CD45RA⁺ CD45RO⁻ T cell counts, and a higher frequency of T cells with a regulatory phenotype (CD127⁺CD4⁺CD25⁺Foxp3⁺). Log-transformed pre-transplant TREC values were significantly lower in patients who developed cancer after transplantation (p < 0.0001). The cumulative incidence of cancer was higher in patients having the lowest pre-transplant TREC values (T1 [low]: 47.4%, T2 [medium]: 12.5%, and T3 [high]: 2.7%; p < 0.0001). In multivariate analysis, pre-transplant TREC value was the only predictive factor of cancer (HR, 0.39; 95% CI, 0.16 to 0.97, for one log (TREC/10⁶ PBMC); p = 0.046).

Conclusions

Pre-transplant thymic function is associated with an increased rate of post-transplant cancer in patients having received ATG. Omitting ATG in recipients with low pre-transplant TREC values should be considered.     

Increased levels of CD4+ and CD8+ T cells expressing CCR1 in patients developing allograft dysfunction; a cohort study

BackgroundLeukocyte infiltration into the graft has pivotal effects on kidney transplantation outcome. The present study sought to determine whether the expression of sequential chemokine receptors on CD4⁺ and CD8⁺ T cells in human renal allograft can predict clinical episodes.MethodsBlood samples from 52 consecutive renal transplant patients were evaluated at the time of transplantation and at three times (2, 90 and 180 days) after transplantation to analyze the expression of CCR1 and CXCR3 on CD4⁺ and CD8⁺ T cells by flowcytometry. A total of 30 biopsies, including protocol biopsy (n = 24) and cause biopsy (n = 6), were investigated according to the Banff criteria.ResultsThe mean percentage of CD4⁺ and CD8⁺ T cells expressing CCR1 was significantly increased in patients with allograft dysfunction (n = 25) (p = 0.006, p = 0.004). The mean fluorescence intensity of CXCR3 on CD4⁺ and CD8⁺ T cells were found to be significantly higher in graft dysfunction than that in well-functioning grafts. (p < 0.001, p = 0.007). Receiver Operating Characteristic (ROC) Curve Analysis showed that the calculated AUC was 0.86 at the third month for CD4⁺ CCR1⁺ and CD8⁺ CCR1⁺ (p < 0.001). Multiple logistic regression analysis showed that an increase in CD4⁺ expressing CXCR3 leads to a lower risk of graft dysfunction (OR = 0.37), while an increase in CD8⁺ expressing CCR1 results in a higher risk of graft dysfunction (OR = 3.66).ConclusionDuring renal transplantation, CD4⁺ and CD8⁺ T cells expressing CCR1 were increased in patients who developed graft dysfunction. These findings may prospectively predict allograft dysfunction, and help elucidate the underlying pathogenic mechanisms.     

Plasmacytoid dendritic cells mediate the tolerogenic effect of CD8+regulatory T cells in a rat tolerant liver transplantation model

BackgroundTolerance is more easily induced in liver transplant models than in other organs; CD8⁺CD45RC^lowregulatory T cells (Tregs) have been shown to induce tolerance in heart allografts. Whether CD8⁺CD45RC^lowTregs could induce tolerance in a liver transplant model and how dendritic cells (DCs) mediate the CD8⁺CD45RC^lowTregs effect remains to be investigated.MethodsA rat liver transplantation model was established and used to test tolerance and acute rejection compared to control groups. Liver function and histopathological changes of allograft were examined by enzyme-linked immunosorbent assay (ELISA) and haematoxylin and eosin (H&E) staining, respectively. The distribution and proportion of CD8⁺CD45RC^lowTregs and plasmacytoid dendritic cells (pDCs) in the allografts and spleen were determined using flow cytometry. Cytokine secretion levels were determined using ELISA and real-time quantitative PCR (qRT-PCR).ResultsThe rat liver transplantation model was well established, with a success rate of 93.3% (28/30). The mean survival time of the tolerant and acute-rejection rats were 156 and 14 days, respectively. The proportions of CD8⁺CD45RC^lowTegs were higher in the allografts of tolerant rats than in those of acute-rejection rats (33.1 ± 4.3 and 12.4 ± 4.6, respectively; P = 0.04). Significant accumulation of pDCs was observed in tolerant liver graft rats compared to that in acute-rejection rats (1.46 ± 0.23 and 0.80 ± 0.20, respectively; P = 0.02). Importantly, CD8⁺CD45RC^lowTregs were positively associated with the frequency of pDCs (P = 0.001, r² = 0.775). The protein and mRNA expression of IL-10 and TGF-β in the allograft group were increased, possibly being responsible for tolerance induction.ConclusionCD8⁺CD45RC^lowT cells interact with pDCs through the induction of IL-10 and TGF-β expression and are responsible for inducing immune tolerance in rat liver transplantation.     

Increased frequency of CD39+CD73+ regulatory T cells and Deltex-1 gene expression level in kidney transplant recipients with excellent long-term graft function

BackgroundThe ability of regulatory T cells (Tregs) to limit inflammatory responses has been demonstrated. However, different subpopulations of this cell have varying abilities to suppress alloreactive immune responses. The primary goal of this study was to assess the frequency of CD4⁺FOXP3⁺CD39⁺CD73⁺ Tregs and Deltex-1 gene expression on long-term renal transplant function.MethodsA total of 49 subjects were divided into 3 groups: (i) the excellent long-term graft function (ELTGF) group, (ii) the chronic graft dysfunction (CGD) group, and (iii) the healthy control (HC) group. Following sample collection, peripheral blood mononuclear cells (PBMCs) were isolated, and the Deltex-1 gene expression level and the frequency of CD4⁺FOXP3⁺CD39⁺CD73⁺ Tregs were evaluated.ResultsThe ELTGF group had more CD4⁺FOXP3⁺ Tregs than the CGD group, but the difference was not statistically significant (P = 0.07). However, the frequency of CD4⁺FOXP3⁺CD39⁺CD73⁺ Tregs and the ratio of these cells to total CD4⁺ lymphocytes significantly increased in the ELTGF group than in the CGD group (P = 0.04 and P = 0.02 respectively). In addition, the expression level of the Deltex-1 gene was significantly lower in the CGD group than in the other 2 groups (P = 0.01 and P = 0.04 respectively).ConclusionsGiven the increased frequency of CD4⁺FOXP3⁺CD39⁺CD73⁺ Tregs and the expression level of the Deltex-1 gene in the ELTGF group, it appears that these factors probably improved function and long-term survival of the transplanted organ through the suppression of alloreactive responses and reduction of inflammation. In other words, one of the immunological mechanisms involved in the CGD group may be a deficiency in Tregs.     

Risk factors for impaired CD4+ T‐cell reconstitution following rabbit antithymocyte globulin treatment in kidney transplantation

To describe long‐term CD4⁺ T‐cell reconstitution after rabbit antithymocyte globulin (rATG) treatment and identify predictive factors following kidney transplantation. A single‐center retrospective study analyzed lymphocyte subsets in rATG‐treated kidney transplant recipients (1986–2009). 589 patients were analyzed (maximum follow‐up 21 years). A comparator group (n = 298) received an anti‐IL‐2 receptor monoclonal antibody. CD4⁺ T‐cell lymphopenia (<200/mm³) was present in 48.5%, 9.2%, 6.7%,2.0%, and 0% of patients at one, three, five, 10, and 20 years post‐transplant, respectively. CD4⁺ T‐cell count increased during the first 10 years but remained below the pretransplant count even after 20 years. At 1, 3, and 6 months post‐transplant, mean CD4⁺ T‐cell count was significantly lower in patients with CD4⁺ T‐cell lymphopenia at 12 months versus patients without lymphopenia. On multivariate analyses, significant independent predictors for long‐term impaired CD4 T‐cell reconstitution were recipient age, pretransplant CD4⁺ T‐cell count, 12‐month CD4⁺ T‐cell count, and tacrolimus or MMF therapy. Recipient age >40 years was identified as a cutoff point. CD4⁺ T‐cell reconstitution following rATG treatment remains impaired even after 21 years. Most risk factors for long‐term impaired CD4⁺ T‐cell reconstitution may be evaluated pretransplant or are modifiable post‐transplant.     

ATG‐Induced Accelerated Immune Senescence: Clinical Implications in Renal Transplant Recipients

Persistent ATG‐induced CD4⁺ T cell lymphopenia is associated with serious clinical complications. We tested the hypothesis that ATG induces accelerated immune senescence in renal transplant recipients (RTR). Immune senescence biomarkers were analyzed at transplant and one‐year later in 97 incident RTR ?62 patients receiving ATG and 35 receiving anti‐CD25 mAb (α‐CD25). This consisted in: (i) thymic output; (ii) bone marrow renewal of CD34⁺ hematopoietic progenitor cells (CD34⁺HPC) and lymphoid (l‐HPC) and myeloid (m‐HPC) progenitor ratio; (iii) T cell phenotype; and (iv) measurement of T cell relative telomere length (RTL) and telomerase activity (RTA). Clinical correlates were analyzed with a 3 year follow‐up. Thymic output significantly decreased one‐year posttransplant in ATG‐treated patients. ATG was associated with a significant decrease in l‐HPC/m‐HPC ratio. Late stage differentiated CD57⁺/CD28^? T cells increased in ATG‐treated patients. One‐year posttransplant T cell RTL and RTA were consequently lower in ATG‐treated patients. ATG is associated with accelerated immune senescence. Increased frequency of late differentiated CD4⁺ T cell frequency at transplantation tended to be predictive of a higher risk of subsequent opportunistic infections and of acute rejection only in ATG‐treated patients but this needs confirmation. Considering pretransplant immune profile may help to select those patients who may benefit from ATG to prevent severe infections and acute rejection.

     

Vitamin D3 combined with antibody agents suppresses alloreactive memory T-cell responses to induce heart allograft long-term survival

BackgroundThe pre-stored memory T cells in organ transplant patient carry a high risk of allograft rejection. The current study aimed to determine whether the allogenic response of adoptively transferred memory T cells in mice was suppressed by vitamin D3 monotherapy alone or in combination with monoclonal antibody treatment.MethodsPrior to vascularized heterotopic heart transplantation, naïve C57BL/6 mice were primed with memory T cells. Recipient mice were administered vitamin D3 alone or in combination with monoclonal antibodies (anti-CD40L/ anti-LFA-1). Memory T cells and CD4⁺ forkhead box P3⁺ T cells in recipient spleens were measured using flow cytometry. Additionally, the expression of cytokines was measured by ELISA and quantitative PCR. Inflammatory factors in the grafts were identified by hematoxylin and eosin staining.ResultsVitamin D3 in conjunction with anti-CD40L/ anti-LFA-1 antibodies were administered according to the median survival time from 6.5 to 80 days. The results revealed that grafts were protected through the prevention of inflammatory cell infiltration. Combined treatment decreased the mRNA levels of IL-2, IFN-γ and IL-10 and increased the mRNA levels of IL-4, Foxp3 and TGF-β in the allograft. Rejection was suppressed by a reduction of CD4⁺CD44^high CD62L⁺ and CD8⁺ CD44^high CD62L⁺ memory T cells, the induction of regulatory T cells in the recipient spleen and a reduction of serum IL-2, IFN-γ and IL-10 levels.ConclusionVitamin D3 efficiently protected allografts from memory T-cell allo-responses when combined with anti-CD40L/anti-LFA-1 antibodies therapy.     

Decreased levels of perforin-positive lymphocytes are associated with posttraumatic complications in patients with major trauma

ObjectivePosttraumatic immune disorder can cause complications including systemic inflammatory response syndrome (SIRS) and multiple-organ dysfunction syndrome (MODS). Cytotoxic granules containing perforin and granzyme-B (GrB) are released by cytotoxic CD8⁺ T lymphocytes, NK and γδT cells after major trauma. This prospective clinical study was designed to analyze the association between these immune components and complications after major trauma.MethodsWe retrospectively studied 48 patients aged between 16 and 65 years admitted within 90 min of major trauma (Injury Severity Score > 16) and surviving beyond 7 days, and 20 healthy controls. Blood samples were drawn on admission and after 1, 3 and 7 days. CD8⁺ T, NK and γδT cell counts in peripheral blood and the levels of perforin and GrB in these cells were analyzed by flow cytometry. Clinical aspects of MODS and SIRS were recorded daily.ResultsCD8⁺ T cell counts were not significantly different in patients with SIRS or uncomplicated group, but were depressed in the MODS group after trauma. However, NK cell counts in patients with MODS were significantly depressed only at day 7 after injury, and γδT cell counts were significantly depressed after trauma. Perforin levels in CD8⁺ T, NK and γδT cells in patients with MODS were depressed after trauma. GrB levels in NK, CD8⁺ T and γδT cells in patients with MODS were significantly depressed at 3 and 7 days post trauma.ConclusionPosttraumatic MODS is associated with early, sustained, and severe depression of lymphocytes.     

Metal ion levels and lymphocyte counts: ASR hip resurfacing prosthesis vs. standard THA: 2–year results from a randomized study

Background and purpose Wear particles from metal–on–metal arthroplasties are under suspicion for adverse effects both locally and systemically, and the DePuy ASR Hip Resurfacing System (RHA) has above–average failure rates. We compared lymphocyte counts in RHA and total hip arthroplasty (THA) and investigated whether cobalt and chromium ions affected the lymphocyte counts.

Method In a randomized controlled trial, we followed 19 RHA patients and 19 THA patients. Lymphocyte subsets and chromium and cobalt ion concentrations were measured at baseline, at 8 weeks, at 6 months, and at 1 and 2 years.

Results The T–lymphocyte counts for both implant types declined over the 2–year period. This decline was statistically significant for CD3⁺CD8⁺ in the THA group, with a regression coefficient of –0.04 × 10⁹cells/year (95% CI: –0.08 to –0.01). Regression analysis indicated a depressive effect of cobalt ions in particular on T–cells with 2–year whole–blood cobalt regression coefficients for CD3+ of –0.10 (95% CI: –0.16 to –0.04) × 10⁹ cells/parts per billion (ppb), for CD3+CD4+ of –0.06 (–0.09 to –0.03) × 10⁹ cells/ppb, and for CD3⁺CD8⁺ of –0.02 (–0.03 to –0.00) × 10⁹ cells/ppb.

Interpretation Circulating T–lymphocyte levels may decline after surgery, regardless of implant type. Metal ions—particularly cobalt—may have a general depressive effect on T– and B–lymphocyte levels.

Registered with ClinicalTrials.gov under # NCT01113762     

Recombinant anti-monkey CD3 immunotoxin depletes peripheral lymph node T lymphocytes more effectively than rabbit anti-thymocyte globulin in naïve baboons

T cell depletion is an important procedure for both experimental and therapeutic immune modulation. Rabbit anti-thymocyte globulin (ATG), which is a commonly used T cell depletion antibody in clinical organ and cell transplantation protocols, is effective in temporarily depleting peripheral blood T lymphocytes but only moderately effective in depleting peripheral lymph node T cells which comprise the majority of T lymphocytes. A recombinant anti-CD3 immunotoxin, A-dmDT390-scfbDb (C207), has been developed and shown in an initial study to retain the lymph node depleting properties of conjugated CD3 immunotoxin. This agent could potentially be used synergistically with or as a replacement for rabbit ATG in preclinical primate models of transplantation. We directly compared the peripheral blood and lymph node depleting abilities of this recombinant anti-CD3 immunotoxin and rabbit ATG in naïve animals at clinically tolerated doses. Baboons were treated with a full course of either rabbit ATG (n = 2) or CD3 immunotoxin (n = 3). Peripheral blood and lymph node T lymphocytes were measured before and following treatment. Peripheral blood CD3 + cells fell below 100 cells/μL in every animal. In the two animals receiving ATG, CD3 + cells represented 53% and 68% of lymph node cells two days following a full course of rabbit ATG. In contrast, CD3 + cells represented 3%, 5%, and 38% in lymph nodes following a full course of CD3-IT. Thus, recombinant anti-monkey CD3 immunotoxin showed improved peripheral lymph node T lymphocyte depletion to rabbit ATG and spared other immune cells.     

Study of intercurrent infection pattern in hepatitis C seropositive renal transplant recipients,relationship with T-cell function

Background: We assessed the effect of hepatitis C seropositivity on the percentage of various T-cells in living donor renal transplant recipients (LDRTRs) and their association with intercurrent infections post renal transplantation (post-Tx).

Methods: One hundred and thirty-three matching LDRTRs [A (seronegative) (68 patients) and B (seropositive) (65 patients) by ELISA] were studied prospectively 10 days, 6 months and 12 months post-Tx for intercurrent infections, acute rejection and T-cell% by flow cytometry.

Results: CD4⁺, CD8⁺, CD4/CD8 were significantly higher 10 days post-Tx in Group B compared to Group A, p < 0.001. A significant increase in CD8% was seen 6-month post-Tx among Group B compared to Group A. No difference was detected between groups in (CD4⁺, CD8⁺, CD4/CD8, CD3–CD16/65⁺%), rate and severity of intercurrent infection, rate of acute rejection, 12 months post-Tx. A significantly higher rate of severe infections particularly urinary tract infections (UTI) was noted in Group B compared to Group A the first 3 months post-Tx particularly in those who received the combination of antithymocyte globulin (ATG) or basiliximab, tacrolimus, steroids, mycophenolate mofetil (MMF). CD4⁺% correlated negatively with intercurrent infections in Group B 6 months post-Tx.

Conclusion: HCV⁺?patients are more prone to intercurrent infections the first 3 months post-Tx. Infection rate correlates positively with pre-transplant HCV seropositivity and immunosuppressive regimen.     

Clinical significance of tumor-infiltrating lymphocytes in neoplastic progression and lymph node metastasis of human breast cancer

To investigate the clinical significance of tumor-infiltrating lymphocytes (TILs) within the tumor milieu, we quantitatively measured and compared the subpopulations of TILs in 24 patients with stage I–III breast carcinoma. Peripheral blood mononuclear cells (PBMCs), normal breast parenchyma-infiltrating lymphocytes (NILs), and TILs were isolated from tissue specimens and quantified by flow cytometry. The results showed that increased proportion of CD8⁺ T cells, with decreased proportion of CD4⁺ T cells, was significant in gated CD3⁺ TILs as compared to autologous NILs or PBMCs (P < 0.001). The tumor-infiltrating CD8⁺ T cells significantly increased with stage progression, reflected in a more strongly decreased CD4/CD8 percentage (P = 0.003). The CD4/CD8 percentage of TILs was strongly correlated with lymphovascular permeation and subsequent lymph node metastasis (P < 0.001). Increased percentages of tumor-infiltrating CD8⁺ T cells with decreased CD4/CD8 percentages are of prognostic importance for cancer progression in human breast cancer.     

Gut microbiota intervention by pre and probiotics can induce regulatory T cells and reduce the risk of severe acute GVHD following allogeneic hematopoietic stem cell transplantation

BackgroundAcute graft-versus-host disease (aGVHD) is one of the leading causes of limitation and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Numerous studies have shown that changes in the gut microbiome diversity increased post-transplant problems, including the occurrence of aGVHD. Probiotics and prebiotics can reconstitute the gut microbiota and thus increase bacterial metabolites such as short-chain fatty acids (SCFAs) that have immunomodulatory effects preventing aGVHD in recipients of allo-HSCTs.Methods/Study DesignWe conducted a pilot randomized clinical trial to investigate whether oral synbiotics are associated with the prevention or reduction in occurrence/severity and mitigate complications of aGVHD following allo-HSCT. A commercially available synbiotic mixture containing high levels of 7 safe bacterial strains plus fructo-oligosaccharides as a prebiotic was administered to allo-HSCT recipients. Out of 40 allo-HSCT patients, 20 received daily a synbiotic 21 days prior to transplantation (days −21 to day 0). In contrast, in the control group 20 recipients of allo-HSCT did not receive a symbiotic therapy.ResultsWithin first 100 days of observation, the incidence of severe (grade III/IV) aGVHD in the a synbiotic-therapy group was 0% (0 out of 20 patients), whereas it was 25% (5 out of 20 patients) in the control group (P = 0.047). The median percentage of CD4 + CD25 + Foxp3+ regulatory T cells (Tregs) among CD4+ lymphocytes on day 28 after HSCT in the synbiotic group was higher (2.54%) than in control group (1.73%; P = 0.01). There was no difference in Treg cells on day 7 after HSCT between two groups. However, the median percentage and the absolute count of Tregs in patients who experience aGVHD was significantly lower on days 7 and 28 after HSCT (both P < 0.05). The overall 12-month survival (OS) rate was higher (90%) in the symbiotic-treated patients than in the control group (75%), but the difference was not statistically significant (P = 0.234).ConclusionOur preliminary findings suggest that synbiotic intake before and during the conditioning regimen of allo-HSCT patients may lead to a reduction in the incidence and severity of aGVHD through the induction of CD4 + CD25 + Foxp3+ regulatory T cells, thus contributing to the improvement of transplant outcomes. Much larger studies are needed to confirm our observations.     

Pre-transplant infusion of donor-derived dendritic cells maintained at the immature stage by sinomenine increases splenic Foxp3+ Tregs in recipient rats after renal allotransplantation

ObjectiveThe immunosuppressive mechanism of sinomenine in organ allotransplantation was investigated, especially its effect of blocking dendritic cell (DC) maturation, which might influence the frequency of regulatory T cells (Tregs).MethodsBone marrow cells from male donor Wistar rats were induced to differentiate into DCs in vitro in the presence or absence of sinomenine, and characterized by flow cytometry. These two groups of DCs were respectively injected into male recipient Sprague-Dawley rats via the tail vein, at both high and low doses. Sprague-Dawley rats receiving saline injection were used as controls. Seven days later, renal transplantation was performed from donor Wistar rats to the recipient Sprague-Dawley rats. Seven days after transplantation, spleens were collected from the recipients. The proportions of Tregs and Foxp3⁺ Tregs to CD4⁺ T cells were determined using flow cytometry.ResultsWith sinomenine treatment, the frequency of mature DCs was reduced, as indicated by lower expression of the surface markers CD80, CD86, and RT1B. In recipient Sprague-Dawley rats that received sinomenine-treated DCs before renal allotransplantation, the proportions of splenic Tregs and Foxp3⁺ Tregs were significantly higher than in control recipients receiving saline or DCs without sinomenine treatment (all p < 0.05). A high dose of sinomenine-treated DCs (10⁶ cells) had a more obvious effect in increasing Tregs than the low dose (10⁵ cells) (p < 0.05).ConclusionPre-transplant infusion of donor-derived sinomenine-induced maturation arrested DCs could result in the increase of Foxp3⁺ Tregs in the spleens of recipients after renal allotransplantation.