Donor-derived regulatory dendritic cell infusion results in host cell cross-dressing and T cell subset changes in prospective living donor liver transplant recipients.

Regulatory dendritic cells (DCreg) promote transplant tolerance following their adoptive transfer in experimental animals. We investigated the feasibility, safety, fate, and impact on host T cells of donor monocyte-derived DCreg infused into prospective, living donor liver transplant patients, 7 days before transplantation. The DCreg expressed a tolerogenic gene transcriptional profile, high cell surface programed death ligand-1 (PD-L1):CD86 ratios, high IL-10/no IL-12 productivity and poor ability to stimulate allogeneic T cell proliferation. Target DCreg doses (range 2.5-10 × 106 cells/kg) were achieved in all but 1 of 15 recipients, with no infusion reactions. Following DCreg infusion, transiently elevated levels of donor HLA and immunoregulatory PD-L1, CD39, and CD73 were detected in circulating small extracellular vesicles. At the same time, flow and advanced image stream analysis revealed intact DCreg and "cross-dressing" of host DCs in blood and lymph nodes. PD-L1 co-localization with donor HLA was observed at higher levels than with recipient HLA. Between DCreg infusion and transplantation, T-bethi Eomeshi memory CD8+ T cells decreased, whereas regulatory (CD25hi CD127- Foxp3+ ): T-bethi Eomeshi CD8+ T cell ratios increased. Thus, donor-derived DCreg infusion may induce systemic changes in host antigen-presenting cells and T cells potentially conducive to modulated anti-donor immune reactivity at the time of transplant.

Donor-derived regulatory dendritic cell infusion modulates effector CD8+ T cell and NK cell responses after liver transplantation.

Immune cell-based therapies are promising strategies to facilitate immunosuppression withdrawal after organ transplantation. Regulatory dendritic cells (DCreg) are innate immune cells that down-regulate alloimmune responses in preclinical models. Here, we performed clinical monitoring and comprehensive assessment of peripheral and allograft tissue immune cell populations in DCreg-infused live-donor liver transplant (LDLT) recipients up to 12 months (M) after transplant. Thirteen patients were given a single infusion of donor-derived DCreg 1 week before transplant (STUDY) and were compared with 40 propensity-matched standard-of-care (SOC) patients. Donor-derived DCreg infusion was well tolerated in all STUDY patients. There were no differences in postoperative complications or biopsy-confirmed acute rejection compared with SOC patients up to 12M. DCreg administration was associated with lower frequencies of effector T-bet+Eomes+CD8+ T cells and CD16bright natural killer (NK) cells and an increase in putative tolerogenic CD141+CD163+ DCs compared with SOC at 12M. Antidonor proliferative capacity of interferon-γ+ (IFN-γ+) CD4+ and CD8+ T cells was lower compared with antithird party responses in STUDY participants, but not in SOC patients, at 12M. In addition, lower circulating concentrations of interleukin-12p40 (IL-12p40), IFN-γ, and CXCL10 were detected in STUDY participants compared with SOC patients at 12M. Analysis of 12M allograft biopsies revealed lower frequencies of graft-infiltrating CD8+ T cells, as well as attenuation of cytolytic TH1 effector genes and pathways among intragraft CD8+ T cells and NK cells, in DCreg-infused patients. These reductions may be conducive to reduced dependence on immunosuppressive drug therapy or immunosuppression withdrawal.

Impact of Induction Therapy on Circulating T Follicular Helper Cells and Subsequent Donor-Specific Antibody Formation After Kidney Transplant.

INTRODUCTION: The cellular events that contribute to generation of donor-specific anti-HLA antibodies (DSA) post-kidney transplantation (KTx) are not well understood. Characterization of such mechanisms could allow tailoring of immunosuppression to benefit sensitized patients. METHODS: We prospectively monitored circulating T follicular helper (cTFH) cells in KTx recipients who received T-cell depleting (thymoglobulin, n = 54) or T-cell nondepleting (basiliximab, n = 20) induction therapy from pre-KTx to 1 year post-KTx and assessed their phenotypic changes due to induction and DSA occurrence, in addition to healthy controls (n = 13), for a total of 307 blood samples. RESULTS: Before KTx, patients displayed comparable levels of resting, central memory cTFH cells with similar polarization to those of healthy controls. Unlike basiliximab induction, thymoglobulin induction significantly depleted cTFH cells, triggered lymphopenia-induced proliferation that skewed cTFH cells toward increased Th1 polarization, effector memory, and elevated programmed cell death protein 1 (PD-1)int/hi expression, resembling activated phenotypes. Regardless of induction, patients who developed DSA post-KTx, harbored pre-KTx donor-reactive memory interleukin (IL)-21+ cTFH cells and showed higher % cTFH and lower % of T regulatory (TREG) cells post-KTx resulting in elevated cTFH:TREG ratio at DSA occurrence. CONCLUSION: Induction therapy distinctly shapes cTFH cell phenotype post-KTx. Monitoring cTFH cells before and after KTx may help detect those patients prone to DSA generation post-KTx.

Multicenter approach to recurrent acute and chronic pancreatitis in the United States: the North American Pancreatitis Study 2 (NAPS2).

BACKGROUND: Recurrent acute pancreatitis (RAP) and chronic pancreatitis (CP) are complex syndromes associated with numerous etiologies, clinical variables and complications. We developed the North American Pancreatitis Study 2 (NAPS2) to be sufficiently powered to understand the complex environmental, metabolic and genetic mechanisms underlying RAP and CP. METHODS: Between August 2000 and September 2006, a consortium of 20 expert academic and private sites prospectively ascertained 1,000 human subjects with RAP or CP, plus 695 controls (spouse, family, friend or unrelated). Standardized questionnaires were completed by both the physicians and study subjects and blood was drawn for genomic DNA and biomarker studies. All data were double-entered into a database and systematically reviewed to minimize errors and include missing data. RESULTS: A total of 1,000 subjects (460 RAP, 540 CP) and 695 controls who completed consent forms and questionnaires and donated blood samples comprised the final dataset. Data were organized according to diagnosis, supporting documentation, etiological classification, clinical signs and symptoms (including pain patterns and duration, and quality of life), past medical history, family history, environmental exposures (including alcohol and tobacco use), medication use and therapeutic interventions. Upon achieving the target enrollment, data were organized and classified to facilitate future analysis. The approaches, rationale and datasets are described, along with final demographic results. CONCLUSION: The NAPS2 consortium has successfully completed a prospective ascertainment of 1,000 subjects with RAP and CP from the USA. These data will be useful in elucidating the environmental, metabolic and genetic conditions, and to investigate the complex interactions that underlie RAP and CP.

Association between calcium sensing receptor gene polymorphisms and chronic pancreatitis in a US population: role of serine protease inhibitor Kazal 1type and alcohol.

AIM: To test the hypothesis that calcium sensing receptor (CASR) polymorphisms are associated with chronic pancreatitis (CP), and to determine whether serine protease inhibitor Kazal 1type (SPINK1) N34S or alcohol are necessary co-factors in its etiology. METHODS: Initially, 115 subjects with pancreatitis and 66 controls were evaluated, of whom 57 patients and 21 controls were predetermined to carry the high-risk SPINK1 N34S polymorphism. We sequenced CASR gene exons 2, 3, 4, 5 and 7, areas containing the majority of reported polymorphisms and novel mutations. Based on the initial results, we added 223 patients and 239 controls to analyze three common nonsynonymous single nucleotide polymorphisms (SNPs) in exon 7 (A986S, R990G, and Q1011E). RESULTS: The CASR exon 7 R990G polymorphism was significantly associated with CP (OR, 2.01; 95% CI, 1.12-3.59; P = 0.015). The association between CASR R990G and CP was stronger in subjects who reported moderate or heavy alcohol consumption (OR, 3.12; 95% CI, 1.14-9.13; P = 0.018). There was no association between the various CASR genotypes and SPINK1 N34S in pancreatitis. None of the novel CASR polymorphisms reported from Germany and India was detected. CONCLUSION: Our United States-based study confirmed an association of CASR and CP and for the first time demonstrated that CASR R990G is a significant risk factor for CP. We also conclude that the risk of CP with CASR R990G is increased in subjects with moderate to heavy alcohol consumption.

Phenotype, function, and differentiation potential of human monocyte subsets.

Human monocytes have been grouped into classical (CD14++CD16-), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1ß in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

Long-term effects of alemtuzumab on regulatory and memory T-cell subsets in kidney transplantation.

BACKGROUND: Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes. METHODS: We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied. RESULTS: We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4 memory T cells, and decreased proportion of CD4 regulatory T cells. Reactive patients exhibited higher proportions of CD4 effector memory T cells (TEM) and CD8 terminally differentiated TEM (TEMRA), with greater CD4 TEM and CD8 TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8 TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8 T cells. CONCLUSIONS: Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

Allospecific CD154+ T-cytotoxic memory cells identify recipients experiencing acute cellular rejection after renal transplantation.

BACKGROUND: The novel, recently described allo (antigen)-specific CD154+T cells were evaluated for their association with acute cellular rejection (ACR) in 43 adult renal transplant recipients receiving steroid-free tacrolimus after alemtuzumab induction. METHODS: Single blood samples corresponding to "for cause" allograft biopsies were assayed for CD154+naive or memory T-helper or T-cytotoxic cells in 16-hr mixed leukocyte reaction. RESULTS: Intra- and interassay variation was less than 10% for a variety of conditions. In logistic regression, leave-one-out cross-validation, and receiver-operating characteristic analyses, the rejection-risk threshold of allospecific CD154+T-cytotoxic memory cells (TcMs) associated best with biopsy-proven ACR with a sensitivity/specificity of 88% in 32 of 43 subjects. Sensitivity/specificity of 100%/88% was replicated in blinded prediction in the remaining 11 subjects. Allospecific CD154+TcM correlated inversely with CTLA4+TcM (Spearman r=-0.358, P=0.029) and increased significantly with increasing histological severity of ACR (P=2.99E-05, Kruskall-Wallis). CONCLUSIONS: The strong association between ACR and allospecific CD154+TcM may be useful in minimizing protocol biopsies among recipients at reduced rejection risk.

Alcohol consumption, cigarette smoking, and the risk of recurrent acute and chronic pancreatitis.

BACKGROUND: Recurrent acute pancreatitis (RAP) and chronic pancreatitis (CP) are associated with alcohol consumption and cigarette smoking. The etiology of RAP and CP is complex, and effects of alcohol and smoking may be limited to specific patient subsets. We examined the current prevalence of alcohol use and smoking and their association with RAP and CP in patients evaluated at US referral centers. METHODS: The North American Pancreatitis Study 2, a multicenter consortium of 20 US centers, prospectively enrolled 540 patients with CP, 460 patients with RAP, and 695 controls from 2000 to 2006. Using self-reported monthly alcohol consumption during the maximum lifetime drinking period, we classified subjects by drinking status: abstainer, light drinker (< or =0.5 drink per day), moderate drinker (women, >0.5 to 1 drink per day; men, >0.5 to 2 drinks per day), heavy drinker (women, >1 to <5 drinks per day; men, >2 to <5 drinks per day), or very heavy drinker (> or =5 drinks per day for both sexes). Smoking was classified as never, past, or current and was quantified (packs per day and pack-years). RESULTS: Overall, participants' mean (SD) age was 49.7 (15.4) years; 87.5% were white, and 56.5% were women. Approximately one-fourth of both controls and patients were lifetime abstainers. The prevalence of very heavy drinking among men and women was 38.4% and 11.0% for CP, 16.9% and 5.5% for RAP, and 10.0% and 3.6% for controls. Compared with abstaining and light drinking, very heavy drinking was significantly associated with CP (odds ratio, 3.10; 95% confidence interval, 1.87-5.14) after controlling for age, sex, smoking status, and body mass index. Cigarette smoking was an independent, dose-dependent risk factor for CP and RAP. CONCLUSIONS: Very heavy alcohol consumption and smoking are independent risks for CP. A minority of patients with pancreatitis currently seen at US referral centers report very heavy drinking.

Hereditary pancreatitis amlodipine trial: a pilot study of a calcium-channel blocker in hereditary pancreatitis.

OBJECTIVES: Hereditary pancreatitis (HP) is a form of recurrent acute pancreatitis (AP) mediated by mutations in cationic trypsinogen (PRSS1). Mutations cluster in the calcium-associated regulator regions of PRSS1. In rats, calcium-channel blockers (CCB) prevent hyperstimulation-associated AP. Because of the potential importance of hyperstimulation in triggering episodes of AP in HP, we designed a pilot study to evaluate the safety and potential benefit of CCB use in HP. METHODS: Subjects 6 years or older had a PRSS1 mutation, recurrent AP, and pain. Total study duration was 16 weeks. Amlodipine was given during weeks 0 to 11. Dose (2.5, 5, or 10 mg) was based on weight (range, 0.08-0.17 mg x kg(-1) x d(-1)). Subjects filled a daily diary including pain (0-10 scale) and blood pressure reading. Clinical assessments occurred at weeks -4, 0, 1, 2, 6, 10, 11, and 12. Subjects filled a Medical Outcomes Study Short-Form Survey version 2 (SF-10 for children <14 years old) at weeks -4, 0, 6, and 10. Data were compared for weeks -4 to 0 and 6 to 10. RESULTS: Nine subjects signed informed consent (4 males; 12-52 years old). Four were excluded during the screening phase. Drug was discontinued in one due to development of unilateral lower-extremity numbness. Four subjects (12-31 years old) completed the study. Mean blood pressure, laboratory tests, physical findings, and daily pain scores did not clinically significantly differ before and during drug therapy, but all reported reduced symptoms. Three reduced analgesic use. Three had improved scores on the Medical Outcomes Study Short-Form Survey version 2. CONCLUSIONS: Amlodipine is generally safe in subjects with HP and does not increase pain or episodes of AP. Further research into the mechanism of CCB on pancreatitis would be important to provide a pathophysiologic basis to support further trials in HP.