Clive Carter

Immunologic Human Renal Allograft Injury Associates with an Altered IL-10/TNF-α Expression Ratio in Regulatory B Cells

Human B cells with immunoregulatory properties in vitro (Bregs) have been defined by the expression of IL-10 and are enriched in various B-cell subsets. However, proinflammatory cytokine expression in B-cell subsets is largely unexplored. We examined the cytokine profiles of human PBMCs and found that subsets of CD24(hi)CD38(hi) transitional B cells (TrBs), CD24(hi)CD27(+) memory B cells, and naïve B cells express IL-10 and the proinflammatory cytokine TNF-α simultaneously. TrBs had the highest IL-10/TNF-α ratio and suppressed proinflammatory helper T cell 1 (Th1) cytokine expression by autologous T cells in vitro more potently than memory B cells did, despite similar IL-10 expression. Whereas neutralization of IL-10 significantly inhibited TrB-mediated suppression of autologous Th1 cytokine expression, blocking TNF-α increased the suppressive capacity of both memory and naïve B-cell subsets. Thus, the ratio of IL-10/TNF-α expression, a measure of cytokine polarization, may be a better indicator of regulatory function than IL-10 expression alone. Indeed, compared with TrB cells from patients with stable kidney graft function, TrBs from patients with graft rejection displayed similar IL-10 expression levels but increased TNF-α expression (i.e., reduced IL-10/TNF-α ratio), did not inhibit in vitro expression of Th1 cytokines by T cells, and abnormally suppressed expression of Th2 cytokines. In patients with graft dysfunction, a low IL-10/TNF-α ratio in TrBs associated with poor graft outcomes after 3 years of follow-up. In summary, these results indicate that B cell-mediated immune regulation is best characterized by the cytokine polarization profile, a finding that was confirmed in renal transplant patients.

Frequency of Treg Cells Is Reduced in CVID Patients with Autoimmunity and Splenomegaly and Is Associated with Expanded CD21lo B Lymphocytes

IntroductionCommon variable immunodeficiency is a heterogeneous antibody deficiency syndrome with autoimmune and inflammatory complications in a significant proportion of patients. The study was designed to evaluate the role of T regulatory (Treg) cells in common variable immunodeficiency (CVID) patients with autoimmunity.MethodsThe number and frequency of Treg cells (CD4+, CD25hi, Foxp3+) were evaluated in patients and controls, and Foxp3 expression in different subgroups of CVID patients with common clinical manifestations was compared.ResultsCVID patients had significantly fewer Treg cells than controls, and low frequency of Treg cells was associated with expansion of CD21lo B cells in patients. Patients with autoimmunity had significantly reduced frequency but normal numbers of regulatory T cells, whilst patients with splenomegaly had significant reduction in frequency and number of regulatory T cells.ConclusionFoxp3 is useful on its own or as an adjunct to classify CVID patients although the possibility of reduction in Treg cells as a secondary phenomenon cannot be excluded.

CVID patients with autoimmunity have elevated T cell expression of granzyme B and HLA-DR and reduced levels of Treg cells

Aims Common variable immunodeficiency (CVID) is a primary antibody immunodeficiency with approximately 20% of patients reporting additional autoimmune symptoms. The primary aim of this study was to compare the levels of activated and regulatory T cells (Treg cells) in CVID patients in an attempt to clarify their possible interactions leading to the generation of autoimmunity. Methods Immunophenotyping of T cells was performed by flow cytometry using a whole blood approach. Surface expression of human leukocyte antigen HLA class II DR and intracellular levels of granzyme B in T cell subsets were assessed; Treg levels were measured using CD4 CD25, FOXp3 and CTLA-4. Results CVID patients had higher levels of granzyme B and HLA-DR on CD8+ T cells compared with control values (mean of 59% vs 30% and 45% vs 21%, respectively). Patients also had reduced levels of Treg cells compared with control values (con mean=3.24% vs pat=2.54%). Patients with autoimmunity (5/23) had a similar level of T cell activation markers to the rest of the patients but with lower Treg cells (mean of 1.1%) and reduced CD25 and CTLA-4 expression. Patients with autoimmunity had a higher ratio of activated to Treg cells compared with patients with no autoimmune symptoms. Conclusions These results highlight that reduced levels of Treg cells were associated with elevated levels of activated T cells, suggesting that reduced Treg cells in these patients may have functional consequences in allowing exaggerated T cell responses.

An Analysis of Lymphocyte Phenotype After Steroid Avoidance With Either Alemtuzumab or Basiliximab Induction in Renal Transplantation

Several studies have analyzed the phenotype of repopulated T‐lymphocytes following alemtuzumab induction; however there has been less scrutiny of the reconstituted B‐cell compartment. In the context of a randomized controlled trial (RCT) comparing alemtuzumab induction with tacrolimus monotherapy against basiliximab induction with tacrolimus and mycophenolate mofetil (MMF) therapy in renal transplantation, we analyzed the peripheral B‐ and T‐lymphocyte phenotypes of patients at a mean of 25 +/− 2 months after transplantation. We examined the relationship between peripheral lymphocyte phenotype and graft function. Patients who received alemtuzumab had significantly higher numbers of B cells including naïve, transitional and regulatory subsets. In contrast, the CD4+ T‐cell compartment was dominated by a memory cell phenotype. Following either basiliximab or alemtuzumab induction patients with lower numbers of B cells or B subsets had significantly worse graft function. For alemtuzumab there was also a correlation between these subsets the stability of graft function and the presence of HLA‐specific antibodies. These results demonstrate that a significant expansion of regulatory type B cells is associated with superior graft function and that this pattern is more common after alemtuzumab induction. This phenomenon requires further prospective study to see whether this phenotype could be used to customize immunotherapy.

Lymphocyte depletion and repopulation after chemotherapy for primary breast cancer

BackgroundApproximately 30 % of breast cancer patients receive chemotherapy, yet little is known about influences of current regimens on circulating lymphocyte levels and phenotypes. Similarly, clinico-pathological factors that modify these influences, and implications for future immune health remain mainly unexplored.MethodsWe used flow-cytometry to assess circulating lymphocyte levels and phenotypes in 88 primary breast cancer patients before chemotherapy and at time-points from 2 weeks to 9 months after chemotherapy completion. We examined circulating titres of antibodies against pneumococcal and tetanus antigens using ELISAs.ResultsLevels of B, T and NK cells were significantly reduced 2 weeks after chemotherapy (p < 0.001). B cells demonstrated particularly dramatic depletion, falling to 5.4 % of pre-chemotherapy levels. Levels of all cells recovered to some extent, although B and CD4+ T cells remained significantly depleted even 9 months post-chemotherapy (p < 0.001). Phenotypes of repopulating B and CD4+ T cells were significantly different from, and showed no sign of returning to pre-chemotherapy profiles. Repopulating B cells were highly depleted in memory cells, with proportions of memory cells falling from 38 % to 10 % (p < 0.001). Conversely, repopulating CD4+ T cells were enriched in memory cells, which increased from 63 % to 75 % (p < 0.001). Differences in chemotherapy regimen and patient smoking were associated with significant differences in depletion extent or repopulation dynamics. Titres of anti-pneumococcal and anti-tetanus antibodies were both significantly reduced post-chemotherapy and did not recover during the study (p < 0.001).ConclusionBreast cancer chemotherapy is associated with long-term changes in immune parameters that should be considered during clinical management.

Downregulation of myeloma-induced ICOS-L and regulatory T cell generation by lenalidomide and dexamethasone therapy.

Multiple myeloma (MM) produces significant cellular and humoral immune defects. We have previously reported that MM induces CD4(+)CD25(+)FoxP3(+) cells (TRegs), via tumour expression of the immune checkpoint regulator, ICOS-L. We sought to define what impact the immunomodulatory drug lenalidomide, alone or with dexamethasone, has on TReg cell generation. Lenalidomide pre-treatment of MM cell lines reduced TReg generation and the concomitant TReg:TEff (CD4(+)CD25(+)FoxP3(-): effector T cells) ratio, as a consequence of reduced ICOSL transcription. Dexamethasone did not affect surface ICOS-L expression but did induce TReg cell apoptosis without impacting on TEff cell survival. Combined lenalidomide and dexamethasone significantly reduced both TReg induction and the TReg:TEff cell ratio. In vivo, serial analysis of the TReg:TEff ratio in MM patients on lenalidomide-dexamethasone therapy revealed a progressive reduction towards age-matched control values, though not complete correction. Our data demonstrate for the first time immune synergism to explain the observed immune-modulation associated with lenalidomide-dexamethasone therapy.

Perioperative reductions in circulating lymphocyte levels predict wound complications after excisional breast cancer surgery.

OBJECTIVE Postoperative wound complications after excisional surgery for primary breast cancer can result in patients requiring additional treatments and delay adjuvant therapy and are associated with worse prognoses.We investigated factors that might predispose patients to wound complications. BACKGROUND A number of patient characteristics have been associated with wound complications, but there is currently no quantitative measure of the risk of their occurrence. Our hypothesis was that wound complications are related, in part, to the immune status of patients. METHODS We recruited patients undergoing surgery for primary breast cancer and determined their circulating levels of various immune cells shortly before and after surgery as a measure of immune status. RESULTS One hundred seventeen patients were recruited; 16 (13.7%) developed wound complications. The following patient and tumor characteristics were associated with higher wound complication rates: diabetes (P = 0.02); larger tumors (T2/3 vs T1; P = 0.02); metastatic axillary nodes (P = 0.006). With respect to immune status, no significant differences in preoperative levels of circulating immune cells were detected between patients who developed wound complications and those who did not. However, patients who developed complications showed greater reductions in lymphocyte levels 4 hours postoperatively than those who did not (P <0.001). Multivariate analyses demonstrated that falls in lymphocyte levels of greater than 20% or 50% 4 hours postoperatively acted as a significant and independent predictor of wound complications (P < 0.005 and P < 0.0001,respectively). CONCLUSIONS Perioperative changes in lymphocyte levels could provide a practical predictive marker for wound complications on which selective antibiotic prophylaxis could be based.

The expression of activating natural killer cell receptors in patients with primary breast cancer.

Abstract #5047 Background: The effect of primary breast tumours and their subsequent treatment on immune system function is still poorly understood and may have critical implications with regard to disease recurrence and success of treatment with the new generation of biologicals. In line with our interest in the expression and role of natural killer (NK) cell activating receptors, we measured the expression of NK cell surface receptors NKp30, NKp46 and NKG2D in patients with primary breast cancer. NKp30 and NKp46 are members of the natural cytotoxic receptors (NCRs) and are expressed on the majority of NK cells of healthy individuals. NKG2D is a member of the C type lectin superfamily. These receptors activate NK cells upon stimulation and are involved in NK cell tumour recognition and triggering although their ligands on tumour cells remain elusive.
 Methods: Our experimental procedure involved obtaining serial blood samples prior to and post surgery (4 hours-6 months) in patients with primary breast cancer. This allowed us to study basal levels of NK cell receptors as well as to investigate the effect of surgery and post surgery treatment on receptor expression. NK receptor analysis was performed on whole blood by three colour flow cytometry using antibodies against CD3, CD56 and the NK receptors with samples analysed on a FACSCalibur flow cytometer (BD).The data was analysed using CELLQuest.
 Results and Discussion: NK cells were defined as CD3-, CD56+ lymphocytes and their frequency, as measured by the three colour staining protocol, was broadly in agreement with the levels found using four colour antibody staining and Trucount tubes (BD). The majority of patients expressed NKp30, NKp46 and NKG2D at levels consistent with that seen in age and sex matched control samples obtained from healthy individuals. However, in a number of patients (10/30), the levels of NKp30 and NKp46 expression were low at all timepoints tested. The levels of NKp30 were generally lower than NKp46 and were unrelated to surgery. NKG2D expression was less affected with the levels similar to that found in controls individuals. The significance of these findings with respect to NK cell target recognition is unclear as an array of activating and inhibitory receptors are involved in NK cell activation and target recognition. This staining approach has also allowed us to assess the relative frequency of CD56dim and CD56bright NK cells. In healthy individuals the majority of NK cells are CD56dim whilst around 5% are CD56bright. These are considered to represent a functionally distinct population. In a number of patients in this study, the expression of CD56 on NK cells was relatively low with the consequential decrease in the frequency of CD56bright cells. We are prospectively following the importance of these NK cell differences with respect to patient health, and disease free and overall survival. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5047.

Bialellic Mutations in Tetratricopeptide Repeat Domain 7A ( TTC7A ) Cause Common Variable Immunodeficiency-Like Phenotype with Enteropathy

TTC7A deficiency typically causes severe gastrointestinal manifestations such as multiple intestinal atresia or early-onset inflammatory bowel disease. In some cases, this is associated with severe combined immunodeficiency. Partial loss-of-function mutations appear to be associated with a milder phenotype resulting in common variable immunodeficiency-like condition with enteropathy.

A new case of Fas-associated death domain protein deficiency and update on treatment outcomes

In this study, we demonstrated that GW870086X inhibited the bronchoconstrictor response to inhaledAMPchallenge after both a single dose and at multiple time points after repeat dosing (up to 7 days). This study achieved the primary end point of a significant reduction in theAMPchallenge versus placebo at 2 hours on day 7. Serial challenges demonstrated a duration of effect up to 12 hours. However, at 26 hours on day 7, tolerance to inhaled AMP had occurred as shownby an increase in PC20 on placebo. Wewere unable to demonstrate a dose-response between the 1and3-mgdoses perhaps because Cmax concentrations measured systemically were similar in magnitude for both doses. Cmax measurements were determined for use as a surrogate for delivery. Similar to previous studies, the AMPand FENO data suggest that GW870086X appears to follow toU-shaped response, similar to observations in previous studies. There was no reduction in cortisol or osteocalcin after 7 days of repeat dosing with GW870086X. These data support the potential for reduced bone and metabolic AEs with GW870086X. The 1and 3-mg doses were selected empirically for this study because no cortisol suppression was demonstrated in a previous study (data on file, GlaxoSmithKline) at these doses and this findingwasagain confirmed in the present study.The novel dissociated inhaled glucocorticoid GW870086X was well tolerated after repeated dosing with a similar incidence and intensity of reported AEs between placebo and GW870086X. No serious AEs were reported. In conclusion, there was a significant protection against AMP challenge in subjects with asthma after single and multiple doses of GW870086X with a potential improved therapeutic index. The novel dissociated glucocorticoid concept merits further investigation in clinical studies of inflammatory lung disease with GW870086X. Brian R. Leaker, MD Brian O’Connor, MD Dave Singh, MD Peter J. Barnes, FRS From Respiratory Clinical Trials Ltd, London, the Medicines Evaluation Unit, University Hospital of South Manchester Foundation Trust, University of Manchester, Manchester, and National Heart & Lung Institute, Imperial College, London, United Kingdom. E-mail: Brian.Leaker@qasmc.com. GlaxoSmithKline UK funded this work. Disclosure of potential conflict of interest: B. R. Leaker has received research support from GlaxoSmithKline (GSK); has received consultancy fees from Daiichi-Sankyo; and has received research support from Pfizer, GSK, AstraZeneca, Chiesi, Sunovion, and Merck. B. O’Connor has received research funding from AstraZeneca. D. Singh has received research support from GSK and has received sponsorship to attend international meetings, honoraria for lecturing or attending advisory boards, and research grants from various pharmaceutical companies, including Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi, Genetech, GSK, Glenmark, Johnson and Johnson, Merck, NAPP, Novartis, Pfizer, Skypharma, Takeda, Teva, Therevance, Verona, CIPLA, and Forest. P. J. Barnes has received consultancy fees from AstraZeneca SAB, Chiesi, Novartis, Zambon, and Pfizer; has provided expert testimony for Watson; has served on Scientific Advisory Boards of AstraZeneca, Boehringer Ingelheim, Chiesi, Daiichi-Sankyo, GSK, Novartis, Takeda, Pfizer, Teva, and UCB; has received research support from Aquinox Pharmaceuticals, AstraZeneca, Boehringer Ingelheim, Chiesi, Daiichi-Sankyo, GSK, Novartis, Takeda, Pfizer, and Prosonix; has received lecture fees from Boehringer Ingelheim, Mundipharma, Teva, Chiesi, and AstraZeneca; and has received payment for the development of educational presentations from Teva. He is also a cofounder of RespiVert (now part of Johnson & Johnson), which has discovered novel inhaled antiinflammatory treatments for asthma and chronic obstructive pulmonary disease.