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Background The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation and the production of memory B cells; however, its role in the later stages of B cell differentiation is unclear. Objective Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model. Methods Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2, and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting. RNA-seq was also performed on in vitro differentiating cells. Results Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells that are predominantly naïve generate phenotypically normal plasma cells with expected patterns of differentiation-associated genes and normal levels of CXCR4. Differentiated CD19-deficient cells were capable of responding to CXCL12; however, plasma cells derived from naïve B cells, both CD19-deficient and sufficient, had relatively diminished signaling compared to those generated from total B cells. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation. Conclusion CD19 is not required for generation of antibody-secreting cells or the responses of these populations to CXCL12, but may alter the response other ligands that require CD19 potentially affecting localization, proliferation, or survival. The observed hypogammaglobulinemia in CD19-deficient individuals is therefore likely attributable to the lack of memory B cells.

Over recent years countless epigenetic studies have been undertaken to investigate DNA methylation (DNAme) patterns in health and disease, frequently using Illumina Methylation arrays. Here we focus on the reliability of Infinium type II probes (~85% of all probes) on this platform and demonstrate that Illumina MethylationEPIC BeadChip v1.0 data from paired whole blood samples taken six weeks apart display an unexpectedly high level of variability on probes with low normalized mean intensities (MI). We propose a method for estimating probes ‘unreliability’ scores by simulating the β-value distribution influenced by technical noise and averaging the spread over all samples. We show that these unreliability scores negatively correlated with MI, which is associated with the number of C-bases in the probe sequence. To validate our results, we demonstrate that also for two publicly available HumanMethylation450K BeadChip datasets unreliability increases with increasing variability measured between replicates. Furthermore, we find that removing low MI type II probes during initial preprocessing can improve precision of DNAme derived cell type composition and age predictors, and that most of these less reliable probes are still present on the newer version (v2.0) of the MethylationEPIC BeadChip. Finally, to advance existing pre-processing pipelines, we implemented an R package for estimating MI and probe ‘unreliability’ scores.

XMEN (X-linked immunodeficiency with magnesium defect) is caused by loss-of-function mutations in MAGT1 which is encoded on the X chromosome. The disorder is characterised by CD4 lymphopenia, severe chronic viral infections and defective T-lymphocyte activation. XMEN patients are susceptible to Epstein-Barr virus infections and persistently low levels of intracellular Mg2+. Here we describe a patient that presented with multiple recurrent infections and a subsequent diffuse B-cell lymphoma. Molecular genetic analysis by exome sequencing identified a novel hemizygous MAGT1 nonsense mutation c.1005T>A (NM_032121.5) p.(Cys335*), confirming a diagnosis of XMEN deficiency. Follow-up immunophenotyping was performed by antibody staining and flow cytometry; proliferation was determined by 3H-thymidine uptake after activation by PHA and anti-CD3. Cytotoxic natural killer cell activity was assessed with K562 target cells using the NKTESTTM assay. While lymphocyte populations were superficially intact, B cells were largely naive with a reduced memory cell compartment. Translated NKG2D was absent on both NK and T cells in the proband, and normally expressed in the carrier mother. In vitro NK cell activity was intact in both the proband and his mother. This report adds to the growing number of identified XMEN cases, raising awareness of a, still rare, X-linked immunodeficiency.

The NLRP3 inflammasome is a vital mediator of innate immune responses. There are numerous NLRP3 mutations that cause NLRP3-associated autoinflammatory diseases (NLRP3-AIDs), mostly in or around the NACHT domain. Here, we present a patient with a rare leucine-rich repeat (LRR) domain mutation, p.Arg920Gln (p.R920Q), associated with an atypical NLRP3-AID with recurrent episodes of sore throat and extensive oropharyngeal ulceration. Unlike previously reported patients, who responded well to anakinra, her oral ulcers did not significantly improve until the PDE4 inhibitor, apremilast, was added to her treatment regimen. Here, we show that this mutation enhances interactions between NLRP3 and its endogenous inhibitor, NIMA-related kinase 7 (NEK7), by affecting charge complementarity between the two proteins. We also demonstrate that additional inflammatory mediators, including the NF-кB and IL-17 signalling pathways and IL-8 chemokine, are upregulated in the patient’s macrophages and may be directly involved in disease pathogenesis. These results highlight the role of the NLRP3 LRR domain in NLRP3-AIDs and demonstrate that the p.R920Q mutation can cause diverse phenotypes between families.

Background: Elevated levels of interferon-gamma (IFNγ) have been found in COVID-19 infection, however its role in this setting remains poorly understood. Cases of non-tuberculous mycobacterium (NTM) infections due to anti-IFNγ autoantibodies (Ab) were first reported in 2004. NTM and COVID-19 co-infection in a patient with acquired IFNγ deficiency has not previously been described. The impact of anti-IFNγ Ab on the severity of COVID-19 has not been previously explored. Objective: We report a case of COVID-19 infection in a patient hospitalised with NTM infection due to acquired IFNγ deficiency caused by anti-IFNγ Ab. We also explore effects of IFNγ Ab on the severity of COVID-19 infection. Methods: Detailed immunological investigations were performed. Bio-rad, Bio-Plex methodology was used to detect anti-IFNγ Ab, titration, IFNγ recovery assay and SARS-CoV-2 serology. Anti-IFNγ Ab were tested in patients with severe (COV-Pat) and health care workers with mild/asymptomatic COVID-19 infection (COV-Asx). Results: Mycobacterium avium intracellulare was diagnosed following bone marrow examination and culture. High titre anti-IFNγ Ab were detected in patient’s serum. The autoantibodies neutralized both endogenously produced and exogenously administered IFNγ. SARS-COV-2 infection was identified during routine inpatient testing. Despite prolonged SARS-COV-2 infection the patient showed only minimal additional symptoms, never developed any significant inflammatory complications and eventually mounted an adequate IgG antibody response to the SARS-COV-2 trimeric S-protein. Elevated titres of anti-IFNγ Ab were detected in COV-Pat and COV-Asx, compared to non-infected healthy controls. The titres were broadly similar between COV-Pat and COV-Asx groups, but much lower compared to patients with acquired IFNγ Ab deficiency. Conclusion: IFN-γ is known to play a central role in hyperinflammatory disease states such as macrophage activation syndrome This study illustrates the potential value of inhibiting IFNγ to prevent pathological inflammatory response to COVID-19.

ObjectivesTo assess antibody and T cell responses to SARS-CoV-2 vaccination in patients with rheumatoid arthritis (RA) on disease-modifying antirheumatic drugs (DMARDs).MethodsThis prospective study recruited 100 patients with RA on a variety of DMARDs for antibody and T cell analysis, pre-vaccination and 4 weeks post-vaccination. Positive antibody response was defined as sera IgG binding to ≥1 antigen. Those that remained seronegative after first vaccination were retested 4 weeks after second vaccination; and if still seronegative after vaccination three. A T cell response was defined an ELISpot count of ≥7 interferon (IFN)γ-positive cells when exposed to spike antigens. Type I IFN activity was determined using the luminex multiplex assay IFN score.ResultsAfter vaccine one, in patients without prior SARS-CoV-2 exposure, 37/83 (45%) developed vaccine-specific antibody responses, 44/83 (53%) vaccine-specific T cell responses and 64/83 (77%) developed either antibody or T cell responses. Reduced seroconversion was seen with abatacept, rituximab (RTX) and those on concomitant methotrexate (MTX) compared to 100% for healthy controls (pConclusionPatients with RA on DMARDs have reduced vaccine responses, particularly on certain DMARDs, with improvement on subsequent vaccinations but with approximately 10% still seronegative after three doses.

Background: The human CD19 antigen is expressed throughout B cell ontogeny with the exception of neoplastic plasma cells and a subset of normal plasma cells. CD19 plays a role in propagating signals from the B cell receptor and other receptors such as CXCR4 in mature B cells. Studies of CD19-deficient patients have confirmed its function during the initial stages of B cell activation, however its role in the later stages of B cell differentiation is unclear. Objective: Using B cells from a newly identified CD19-deficient individual, we investigated the role of CD19 in the generation and function of plasma cells using an in vitro differentiation model.Methods: Flow cytometry and long-read nanopore sequencing using locus-specific long-range amplification products were used to screen a patient with suspected primary immunodeficiency. Purified B cells from the patient and healthy controls were activated with CD40L, IL-21, IL-2 and anti-Ig, then transferred to different cytokine conditions to induce plasma cell differentiation. Subsequently, the cells were stimulated with CXCL12 to induce signalling through CXCR4. Phosphorylation of key downstream proteins including ERK and AKT was assessed by Western blotting.Results: Long-read nanopore sequencing identified the homozygous pathogenic mutation c.622del (p.Ser208Profs*19) which was corroborated by the lack of CD19 cell surface staining. CD19-deficient B cells generate phenotypically normal plasma cells with normal levels of CXCR4, however they have an altered response to CXCL12 resulting in a loss of ERK signalling. Additionally, CD19 ligation on normal plasma cells results in AKT phosphorylation.Conclusion: CD19 is not required for generation of antibody secreting cells but may alter responses of these populations to CXCL12 and direct CD19 ligands, potentially affecting localisation, proliferation, or survival.

Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.

BackgroundInducible T cell co-stimulator (ICOS) deficiency has been categorized as a combined immunodeficiency often complicated by enteropathies, autoimmunity, lymphoproliferation, and malignancy. We report seven new patients and four novelICOSmutations resulting in a common variable immunodeficiency (CVID)–like phenotype and show that dysregulated IL-12 release, reduced cytotoxic T lymphocyte–associated protein 4 (CTLA4) expression, and skewing towards a Th1-dominant phenotype are all associated with inflammatory complications in this condition.MethodsA combination of whole exome and Sanger sequencing was used to identify novel mutations. Standard clinical and immunological evaluation was performed. FACS and ELISA-based assays were used to study cytokine responses and ICOS/ICOSL/CTLA4 expression following stimulation of whole blood and PBMCs with multiple TLR ligands, anti-CD3, and PHA.ResultsFour novel ICOS mutations included homozygous c.323_332del, homozygous c.451C>G, and compound heterozygous c.58+1G>A/c.356T>C. The predominant clinical phenotype was that of antibody deficiency associated with inflammatory complications in 4/7 patients. Six out of seven patients were treated with immunoglobulin replacement and one patient died from salmonella sepsis. All patients who were tested showed reduced IL-10 and IL-17 cytokine responses, normal IL-1β, IL6, and TNF release following LPS stimulation and highly elevated IL-12 production in response to combined LPS/IFNγ stimulation. This was associated with skewing of CD4+T cells towards Th1 phenotype and increased expression of ICOSL on monocytes. Lastly, reduced CTLA4 expression was found in 2 patients. One patient treated with ustekinumab for pancytopenia due to granulomatous bone marrow infiltration failed to respond to this targeted therapy.ConclusionsICOS deficiency is associated with defective T cell activation, with simultaneously enhanced stimulation of monocytes. The latter is likely to result from a lack of ICOS/ICOSL interaction which might be necessary to provide negative feedback which limits monocytes activation.

The importance of donor-specific antibodies (DSAs) in renal transplantation has long been recognized, but the significance of human leukocyte antigen (HLA)-DP antibodies remains less clear. We performed a retrospective single center study of renal transplants with pre-existing isolated HLA-DP-DSAs to assess clinical outcomes.Twenty-three patients with isolated HLA-DP-DSAs were compared with 3 control groups as follows: standard immunological risk (calculated reaction frequency [cRF] 85%, no current or historical DSA, no repeat mismatched antigens with previous transplants,Over a median follow-up of 1197 days, 65% of HLA-DP-DSA patients had ABMR on indication biopsies, and 30% of HLA-DP-DSA patients lost their graft. Pre-existing HLA-DP DSAs remained the single factor associated with ABMR after multivariable analysis (hazard ratio [HR] = 9.578,Transplants with pre-existing HLA-DP-DSAs should be considered high risk. Routine laboratory tests are unable to further risk stratify these patients. Recipients should be considered for intensified immunosuppression and closely monitored.

CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient’s T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells.

The Recombination Activating Genes, RAG1 and RAG2, are essential for V(D)J recombination and adaptive immunity. Mutations in these genes often cause immunodeficiency, the severity of which reflects the importance of the altered residue or residues during recombination. Here, we describe a novel RAG1 mutation that causes immunodeficiency in an unexpected way: The mutated protein severely disrupts binding of the accessory protein, HMGB1. Although HMGB1 enhances RAG cutting in vitro, its role in vivo was controversial. We show here that reduced HMGB1 binding by the mutant protein dramatically reduces RAG cutting in vitro and almost completely eliminates recombination in vivo. The RAG1 mutation, R401W, places a bulky tryptophan opposite the binding site for HMG Box A at both 12- and 23-spacer recombination signal sequences, disrupting stable binding of HMGB1. Replacement of R401W with leucine and then lysine progressively restores HMGB1 binding, correlating with increased RAG cutting and recombination in vivo. We show further that knockdown of HMGB1 significantly reduces recombination by wild-type RAG1, whereas its re-addition restores recombination with wild-type, but not the mutant, RAG1 protein. Together, these data provide compelling evidence that HMGB1 plays a critical role during V(D)J recombination in vivo.

With the onset of the SARS-CoV-2 pandemic and subsequent vaccination programme, a need has arisen to check for the development of T lymphocyte immunity against the virus. The SARS CoV-2 T-SPOT.COVID test measures the level of T cell immunity and has been used extensively in our laboratory over the last 6 months. Whilst this kit has been designed to be used on freshly isolated human peripheral blood mononuclear cells (PBMC), the use of frozen cells would improve clinical utility. To this end we have directly compared the use of fresh and frozen PBMC in this assay. Using healthy control blood along with renal and liver transplant patient samples we have shown that results with frozen cells are generally comparable to those from fresh cells in many, but not all samples tested, and that it is important to assess PBMC cell number and viability in thawed samples before proceeding in order to be able to interpret these results correctly.

The SARS-CoV-2 pandemic has provided the stimulus for the rapid development of a variety of diagnostic testing methods. Initially these were deployed as screening tools to evidence spread of the virus within populations. The recent availability of vaccines against the virus and the need to better understand the parameters of post-infection protective immunity requires development of methods, suitable for use in the routine diagnostic laboratory, capable of characterising the viral immune response in greater detail. Such methods need to consider both cellular and humoral immunity. Toward this aim we have investigated use of a commercial multiplex assay (COVID Plus Assay, One Lambda), providing assessment of the SARS-CoV-2 response at structural level, and developed an in-house cell stimulation assay using commercially available viral peptides (Miltenyi). This paper reports our experience in use of these methods in extended investigation of a cohort of healthcare workers with prior screening results indicative of viral infection. The antibody response generated is shown to be both qualitatively and quantitatively different in different individuals. Similarly a recall response to SARS-CoV-2 antigen involving the T cell compartment can be readily demonstrated in recovered individuals but is of variable magnitude.

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.

Human transitional B cells express relatively high IL-10 and low TNF-α levels, which correlate with B regulatory activity in vitro. Herein, we aim to further define B regulatory phenotype and determine whether B regulatory activity can serve as a prognostic marker for renal allograft dysfunction (graft loss or 2-fold fall in estimated glomerular filtration rate). Transitional B cells can be divided into T1 and T2 subsets based on surface phenotype. T1 cells express a significantly higher ratio of IL-10 to TNF-α than T2 cells or other B subsets. When analyzed in 45 kidney transplant recipients at the time of late for-cause biopsy, the T1/T2 ratio was independently associated with allograft dysfunction over the next 5 years. Next, the T1/T2 ratio was examined in an independent set of 97 clinically stable kidney transplant recipients 2 years after transplant. Again, the T1/T2 ratio was strongly and independently associated with allograft dysfunction over the ensuing 5 years. In these clinically quiescent patients, a low T1/T2 ratio identified a 41-patient subgroup in which 35% developed allograft dysfunction, with 25% losing their allografts. However, none of the 56 patients with a high ratio developed graft dysfunction. In both the initial study and validation groups, the T1/T2 ratio was a much stronger predictor of graft dysfunction than donor-specific antibodies or the estimated glomerular filtration rate. Thus, the T1/T2 ratio, a relative measure of expressing an anti-inflammatory cytokine profile, is a novel prognostic marker that might inform individualized immunosuppression.

The UK hand transplantation programme is hosted by the Department of Plastic and Reconstructive Surgery at Leeds Teaching Hospitals under the leadership of Professor Simon Kay. Since programme launch in 2013, ten procedures in six individuals have been performed involving unilateral or bilateral transplants. The multi-disciplinary team that delivers the programme includes the transplant immunology service. The laboratory experience in programme support is reported here.

Loss of function or null mutations of Stromal interaction molecule 1 (STIM1) are known to cause early-onset combined immunodeficiency (CID) disease with recurrent and chronic infections, autoimmunity, haemolytic anaemia, ectodermal dysplasia, muscular weakness and myalgia. here we report of novel STIM1 deficiency and 6 year follow up of two previous cases associated with mild immunological phenotype

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.

G6PC3 deficiency typically causes severe congenital neutropenia, associated with susceptibility to infections, cardiac and urogenital abnormalities. However, here we describe two boys of Pakistani origin who were found to have G6PC3 deficiency due to c.130 C > T mutation, but who have clinical phenotypes that are typical for a systemic autoinflammatory syndrome. The index case presented with combination of unexplained fevers, severe mucosal ulcers, abdominal symptoms, and inflammatory arthritis. He eventually fully responded to anti-TNF therapy. In this study, we show that compared with healthy controls, neutrophils and monocytes from patients have reduced glycolytic reserve. Considering that healthy myeloid cells have been shown to switch their metabolic pathways to glycolysis in response to inflammatory cues, we studied what impact this might have on production of the inflammatory cytokines. We have demonstrated that patients' monocytes, in response to lipopolysaccharide, show significantly increased production of IL-1β and IL-18, which is NLRP3 inflammasome dependent. Furthermore, additional whole blood assays have also shown an enhanced production of IL-6 and TNF from the patients' cells. These cases provide further proof that autoinflammatory complications are also seen within the spectrum of primary immune deficiencies, and resulting from a wider dysregulation of the immune responses.

Background Approximately 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. Methods We 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. Results Levels of B, T and NK cells were significantly reduced 2 weeks after chemotherapy (p

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.

Synthetic modifications on a 6-furanylquinazoline scaffold to optimize the dual ErbB-1/ErbB-2 tyrosine kinase inhibition afforded consistent SAR whereby a 4-(3-fluorobenzyloxy)-3-haloanilino provided the best enzyme potency and cellular selectivity. Changes made to the 6-furanyl group had little impact on the enzyme activity, but appeared to dramatically affect the cellular efficacy. The discovery of lapatinib emerged from this work.

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 naive 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 naive 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.

Background: Immune reconstitution after AlloSCT is a highly complex process influenced by both graft & recipient-related factors including post-graft therapies. Delayed T-cell reconstitution remains a source of morbidity & mortality and may contribute to limited anti-tumour effect. We sought to evaluate the immune reconstitution kinetics in the setting of post-graft Lenalidomide (Revlimid®) to augment the graft-versus-myeloma effect, by defining key immune biomarkers, in the NCRI LenaRIC phase II trial. Material & Methods: All patients recruited into the study underwent a reduced intensity conditioned (RIC) AlloSCT using Fludarabine & 2Gy TBI with ATG (Fresenius) as in vivo T-cell depletion, following a high dose melphalan ASCT (planned tandem SCT programme). Patients were scheduled to receive Lenalidomide (5mg-10mg/day) on day+35 if stable donor engraftment in the absence of GvHD. Lenalidomide discontinuation was planned at 12 months post-transplant, with donor lymphocyte infusions (DLI) given to patients with evidence of residual disease, relapse or mixed chimerism. Sequential peripheral blood samples were drawn at predetermined time frames before (baseline) and after graft infusion (D+30,+60,+90,+180,+270 & D+360) in all trial registrants. Using multi-parameter flow cytometry (MFC), a comprehensive panel of immune subset & functional/activation markers were used to define characteristics of T-cell, B-cell, regulatory T-cell & NK cells. The inflammatory proteome (IL-1b, IL-4, IL-5, IL-6, IL-10, IFNg, TNFa, CXCL8 & GM-CSF) and soluble immune checkpoints (PD-1 & ICOS) were analyzed using LUMINEX assays on serial serum samples at parallel points. PB cell subset-specific Complete Donor Chimerism (CDC) was analysed by single tandem repeat PCR at day (D)+100,+180,+270 and D+365. Results: From January 2011 to May 2015, 40 patients with myeloma ≥VGPR after 1st or 2nd ASCT, were recruited into the LenaRIC protocol. The median age was 49 years (range 35-65) with PB as the graft source (Matched Related Donor n=16, 10/10 Matched Unrelated Donor n=23; 1 patient not transplanted). Viable samples were received from 37/40 patients (92.5%), with no cycles of Lenalidomide received by 3/37 (8.1%) patients, 1-6 cycles received by 13/37 (35.1%) patients & >6 cycles received by 21/37 (56.8%) patients. 5 patients completed 12 cycles as per protocol treatment; of those completing >= 10 cycles, 5/16 received DLI. Only 10/37 (27%) of patients achieved a CD4+ cell count >200/µl in a median of 180 days compared with 30/37 (81%) of patients who achieved a CD8+ cell count >200/µl in a median of 60 days. In patients who did not receive any doses of Lenalidomide, higher levels of CD8+ cells (p=0.013) but lower NK cell levels (p=0.071) were evident at D+30 compared with those eligible to proceed with Lenalidomide. No difference was noted between Lenalidomide exposure subgroups in relation to CD8+ or CD4+ cell recovery. Treg cells were increased at D=270 & D+365 in those who received >6 cycles of Lenalidomide, though this corresponded with a higher level of HLA-DR expression on CD8+ T cells representing global immune activation. Significant differences between Lenalidomide exposure subgroups were evident in the inflammatory proteome readout at D+90. CXCL8 levels peaked at D+270, especially in those who received >6 cycles of Lenalidomide. Of 30 evaluable patients, T-cell CDC was seen in 63.3% at D100, with 84% of evaluable patients having CDC at D365. No difference between subgroups was evident in relation to rapidity of CDC and stability of CDC. Conclusions: The sequential monitoring of immune biomarkers in the setting of post-graft Lenalidomide following a T-deplete RIC-AlloSCT highlights the rapid and sustained quantitative and functional reconstitution of donor immune homeostasis. The clinical significance of these findings requires correlation with both the anti-tumour immune effect and theimmunopathy ofAlloSCT (GvHD). Unrestricted educational grants were provided by Cancer Research UK and byCelgene.Lenalidomideprovided free of charge byCelgene. The support and time of participating patients and their families is gratefully acknowledged Figure 1 a) Changes in circulating CD8+ T-cells according to Lenalidomide exposure following RIC-Allo SCT for myeloma b) Changes in circulating CD4+CD25+FoxP3+ T-Reg cells according to Lenalidomide exposure following RIC-Allo SCT for Myeloma Figure 1. a) Changes in circulating CD8+ T-cells according to Lenalidomide exposure following RIC-Allo SCT for myeloma. / b) Changes in circulating CD4+CD25+FoxP3+ T-Reg cells according to Lenalidomide exposure following RIC-Allo SCT for Myeloma Disclosures Cook: Glycomimetics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Brock:AstraZeneca: Equity Ownership; GlaxoSmithKline: Equity Ownership. Cavenagh:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Cook:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria; Cancer Research UK: Research Funding; Takeda: Honoraria.

We have examined the ability of several serine proteases (granzymes) isolated from the granules of the rat natural killer cell line, RNK, to generate antigenic peptides of ovalbumin (Ova) that are capable of being recognized by Ova-specific CD8 + T cells. The mouse MHC class I-restricted cytotoxic T-cell clone, GX-1, which recognizes a trypsinized fragment of Ova in the context of H-2 b , was able to lyse EL4 (H-2 b ) target cells in the presence of Ova and the granzymes but not in the presence of Ova or granzymes alone. Similar results were obtained using the murine Ova-specific CD8 + T cell hybridomas, RF33.70 and CD8OVA. In all cases, the T cells’ responses were MHC class I-restricted as Ova:granzyme mixtures failed to mediate the lysis of the MHC-disparate target cell, P815 (H-2 d ). The purified rat granzyme preparations contained three distinct enzymatic specificities: asp-ase, met-ase, and tryptase. Aprotinin, a protease inhibitor that only inhibits tryptase activity in vitro, completely abolished the CD8 + T-cell responses to Ova. These results, along with peptide loading studies using the RMA-S cell line, suggest that the granzyme treatment of Ova can generate the proper antigenic fragments which facilitate class I-restricted CTL responses both in vivo and in vitro. We believe that enzymes produced and released by NK or cytotoxic T cells within a tissue microenvironment may enhance the cleavage of target cell antigens as well as soluble antigens resulting in the improved uptake and processing of soluble antigens.

AimsCommon 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.MethodsImmunophenotyping 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.ResultsCVID 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.ConclusionsThese 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.

Common 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.The number and frequency of Treg cells (CD4(+), CD25(hi), Foxp3(+)) were evaluated in patients and controls, and Foxp3 expression in different subgroups of CVID patients with common clinical manifestations was compared.CVID patients had significantly fewer Treg cells than controls, and low frequency of Treg cells was associated with expansion of CD21(lo) 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.Foxp3 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.

A novel series of [4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)-2-pyrimidine-based cyclooxygenase-2 (COX-2) inhibitors, which have a different arrangement of substituents compared to the more common 1,2-diarylheterocycle based molecules, have been discovered. For example, 2-(butyloxy)-4-[4-(methylsulfonyl)phenyl]-6-(trifluoromethyl)pyrimidine (47), a member of the 2-pyrimidinyl ether series, has been shown to be a potent and selective inhibitor with a favourable pharmacokinetic profile, high brain penetration and good efficacy in rat models of hypersensitivity.

Respiratory syncytial virus (RSV) is the cause of one-fifth of all lower respiratory tract infections worldwide and is increasingly being recognized as representing a serious threat to patient groups with poorly functioning or immature immune systems. Racemic 1,4-benzodiazepines show potent anti-RSV activity in vitro. Anti-RSV evaluation of 3-position R- and S-benzodiazepine enantiomers and subsequent optimization of this series resulted in selection of a clinical candidate. Antiviral activity was found to reside mainly in the S-enantiomer, and the R-enantiomers were consistently less active against RSV. Analogues of 1,4-(S)-benzodiazepine were synthesized as part of the lead optimization program at Arrow and tested in the XTT assay. From this exercise, (S)-1-(2-fluorophenyl)-3-(2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)-urea, 17b (RSV-604) was identified as a clinical candidate, exhibiting potent anti-RSV activity in the XTT assay, which was confirmed in secondary assays. Compound 17b also possessed a good pharmacokinetic profile and has now progressed into the clinic.

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections worldwide, yet no effective vaccine or antiviral treatment is available. Here we report the discovery and initial development of RSV604, a novel benzodiazepine with submicromolar anti-RSV activity. It proved to be equipotent against all clinical isolates tested of both the A and B subtypes of the virus. The compound has a low rate of in vitro resistance development. Sequencing revealed that the resistant virus had mutations within the nucleocapsid protein. This is a novel mechanism of action for anti-RSV compounds. In a three-dimensional human airway epithelial cell model, RSV604 was able to pass from the basolateral side of the epithelium effectively to inhibit virus replication after mucosal inoculation. RSV604, which is currently in phase II clinical trials, represents the first in a new class of RSV inhibitors and may have significant potential for the effective treatment of RSV disease.

Described herein is the design and synthesis of indazolylaminopyridopyrimidines and quinazolines as inhibitors of the class 1 tyrosine kinase receptor family. Data is presented for N(4)-(1-benzyl-1H-indazol-5-yl)-N(6),N(6)-dimethylpyrido[3,4-d]pyrimidine-4,6-diamine 3B. This compound inhibited EGFr and c-erbB-2 enzymes selectively over other kinases. It inhibited the proliferation of a range of tumour cell lines in vitro and the growth of BT474 xenografts in SCID mice.

IntroductionWe have previously demonstrated an association between post-operative wound complications and systemic breast cancer recurrence (p Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4132.

Abstract 2228 Poster Board II-205 B lymphocyte homeostasis may have a role in regulating the immune response, both stimulatory & regulatory/tolerogenic, in Allogeneic Stem Cell Transplantation (AlloSCT). The predictive relationship of donor B-cell reconstitution with relapse risk, chronic Graft versus Host Disease (cGvHD) & overall survival (OS) was investigated. We performed prospective comprehensive immune reconstitution studies in 71 patients undergoing AlloSCT (Matched Related Donor=33, Matched Unrelated Donor =34, Umbilical Cord Blood =3, Haplo-identical Related Donor =1) for haematological malignancy & marrow failure syndromes who were available for follow-up beyond Day+100. 27 patients received full intensity conditioning & 44 reduced intensity conditioning with 34 patients receiving Alemtuzumab & 8 patients receiving ATG as in vivo T-cell depletion with a median PAM score of 22 (range 8-36). The grafts were bone marrow in 23, peripheral blood stem cells in 40 and umbilical cord blood in 3 with a median CD34+cell dose of 4.3×106/kg (range 0.8-10.5) & median duration of immune-suppression of 7.1 months (range 2, 49). Samples were analysed for lymphocyte subsets by multi-parameter flow cytometry & cell subset-specific Complete Donor Chimerism (CDC) by single tandem repeat PCR at day (D)+100, D+180, D+270 and D+365. The proportion of patients with a CD4+, CD8+, CD19+ & NK cell count within the normal range at D+100 & D+180 were: 10% & 27% for CD4+, 28% & 57% for CD8+, 24% & 57% for B-cells, 68% & 83% for NK cells, respectively. The proportion of patients with a CD4+, CD8+, CD19+ & NK cell CDC at D+100 & D+180 were: 80% & 78% for CD4+, 86% & 80% for CD8+, 89% & 91% for B-cells, respectively. 28 patients (38%) experienced cGvHD (22/28 extensive) with a median time to onset of 4 months (range 3-12) & median duration of immune suppression of 14.5 months (range 6.9-48.3). In univariate analysis, only the B-cell CDC at D+100 was associated with a higher incidence of cGvHD (mean 99.8%±0.2% vs. 90.4%±4.4 CDC, respectively; p=0.044) though a trend was seen in regard to D+180 B-cell CDC. No influence of mature/immature B-cell reconstitution at D+100 or D+180 was detected. 24 patients (34%) have relapsed at a median of 5.8 months (range 3-34). A trend to lower absolute B-cell counts at D+100 & D+180 was seen in those who relapsed with significantly lower B-cell CDC at D+100 and D+180 in those who relapsed compared to those who did not (D+100: mean 87.9%±2.4% vs. 98.3%±1.5 CDC, respectively; p=0.038; D+180: mean 80±5.6 vs. 100%±0 CDC, respectively; p=0.013). With a median follow-up of 16.7 months (range 5.1-51.8), 27 (38%) have died (disease progression n=12, infection n=4, GvHD-related n=6). In univariate analysis, no effect on OS was demonstrated by the absolute B-cell count or B-cell CDC at D+100 or D+180, though a trend was seen to lower levels in those who did not survive. In conclusion, delayed donor B-cell reconstitution was associated with a higher relapse risk. On the other hand, cGvHD was associated with higher early donor B-cell reconstitution, which suggests that residual recipient B-cells after AlloSCT may protect from cGvHD. This data provides further clinical evidence for a role of B-cells in the immune-modulation associated with the graft-versus-tumour/graft-versus-host disease paradigm of AlloSCT. Disclosures: No relevant conflicts of interest to declare.

Abstract #5045 Background: The factors leading to breast cancer recurrence are incompletely understood. We carried out a retrospective study of outcome for 1065 breast cancer patients for which we examined factors correlating with cancer recurrence. We found that infection of wounds after surgery for primary disease positively correlated with cancer recurrence (Murthy et al, BJC 2007). Patients with wound complications were 3 fold more likely to have systemic recurrences than those without (p Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5045.

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.