Ola Winqvist

Pilot Study of Sentinel-Node-Based Adoptive Immunotherapy in Advanced Colorectal Cancer

BackgroundDespite optimal surgical treatment and modern adjuvant therapies, 50% of patients diagnosed with colorectal cancer die within 5xa0years. Immunotherapy offers an appealing complement to traditional chemotherapy, with possible long-term protection against tumor recurrences through immunological memory. We have conducted a pilot study of a novel adoptive immunotherapy, using autologous, in vitro expanded lymphocytes isolated from the tumor-draining sentinel lymph node.Study DesignSentinel nodes were recovered from 16 patients with disseminated or locally advanced, high-risk colorectal cancer. Single-cell suspensions of sentinel-node-acquired lymphocytes were clonally expanded in vitro in the presence of autologous tumor extract and returned as a transfusion. Patients were followed with clinical and radiological evaluations. Long-term survival was compared with traditionally treated controls.ResultsSentinel-node-acquired CD4+ Th1-lymphocytes could be clonally expanded in vitro and safely administered to all 16 patients without side-effects. In four out of nine stage IV patients, complete tumor regression occurred. Median survival time in the stage IV patients (nxa0=xa09) was 2.6xa0years, as compared with 0.8xa0years in conventionally treated controls. A dose-dependent effect with regards to reduced tumor burden and long-term survival was observed.ConclusionSentinel-node-based adoptive immunotherapy is feasible; the method has shown no apparent side-effects and appears to convey therapeutic antitumor effects. Further studies are justified to determine its efficacy and precise role in the treatment of colorectal cancer.

Different regulation of eosinophil activity in Crohn's disease compared with ulcerative colitis

The aim of this investigation was to study the involvement of eosinophil and neutrophil granulocytes in different stages of Crohns disease (CD) and ulcerative colitis (UC). Biopsy samples were taken from the right flexure of the colon and from the rectum in patients with active (n=12) and inactive colonic CD (n=7), patients with active (n=33) and inactive UC (n=24), and from control subjects (n=11). Cell suspensions from biopsies and blood were analyzed by flow cytometry with regards to activation markers and viability. Immunohistochemistry was used to evaluate cell number and degranulation. Blood eosinophils were cultured with Th1 and Th2 cytokines, and the expression of activity markers was assessed by flow cytometry. Eosinophil number, viability, and activity were increased during active CD and UC compared with controls. The activity, assessed as CD44 expression, tended to diminish during inactive CD but was increased further in quiescent UC. Neutrophil number and activity were increased only during inflammation in both diseases. Culture of blood eosinophils with IL‐5 and IL‐13 caused increased CD44 expression, whereas IL‐5 and IFN‐γ induced elevated CD69 expression. We observed different patterns of eosinophil activation in CD and UC, with the highest CD44 expression during quiescent UC. Our in vitro experiments with recombinant cytokines suggest that the diverse mechanisms of eosinophil activation in CD and UC are a result of different cytokine milieus (Th1 vs. Th2). In contrast, neutrophil activation reflects the disease activity in CD and UC, irrespective of Th cell skewing.

Intralymphatic immunotherapy of pollen-induced rhinoconjunctivitis: a double-blind placebo-controlled trial.

BackgroundAllergen-specific immunotherapy represents the only disease-modifying treatment for allergic diseases. We and others have previously demonstrated that intralymphatic immunotherapy (ILIT), a less time-consuming alternative to conventional subcutaneous immunotherapy (SCIT), is safe and effective. However, this has recently been disputed. The aim of this study was therefore to expand our previous trial, further assessing the safety and efficacy of ILIT.MethodsThirty-six patients with pollen-induced rhinoconjunctivitis were randomised to receive three intralymphatic inguinal injections of active allergen (1000 SQ-U birch- or grass-pollen) or placebo. Clinical effects, safety and circulating immunological markers were assessed before, 4xa0weeks after treatment and at the end of the consecutive pollen season.ResultsNo moderate or severe reactions were recorded following ILIT. Patients receiving active ILIT experienced a significant improvement in self-recorded seasonal allergic symptoms, as compared to placebo (pu2009=u20090.05). In a subgroup of these patients (“improved”), a reduction in nasal symptoms following nasal allergen provocation was also demonstrated. No changes in total IgE or IgG4 were found. However, the affinity of allergen specific IgG4 following active treatment was significantly increased, as compared to non-improved patients (pu2009=u20090.04). This could be correlated with clinical improvement, on an individual level.ConclusionsThis double-blinded placebo-controlled study confirms that ILIT is a safe and effective treatment for pollen-induced rhinoconjunctivitis, markedly reducing seasonal allergic symptoms.Trial registrationEudraCT: 2009-016815-39

Peripheral and site-specific CD4(+) CD28(null) T cells from rheumatoid arthritis patients show distinct characteristics.

Proinflammatory CD4+CD28null T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4+CD28null T cells from blood and synovial fluid in comparison with conventional CD28‐expressing CD4+ T cells. Forty‐four patients with RA, displaying a distinct CD4+CD28null T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN‐γ, TNF, IL‐17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4+CD28null T cells were significantly more hypomethylated in the CNS‐1 region of the IFNG locus than conventional CD4+CD28+ T cells and produced higher levels of both IFN‐γ and TNF after TCR cross‐linking. CD4+CD28null T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL‐17A production could hardly be detected in CD4+CD28null cells from the blood, a significant production was observed in CD4+CD28null T cells from synovial fluid. CD4+CD28null T cells were not only found to differ from conventional CD4+CD28+ T cells in the circulation, but we could also demonstrate that synovial CD4+CD28null T cells showed additional effector functions (IL‐17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28null population.

Clinical and immunological characteristics of Autoimmune Addison's disease : a nationwide Swedish multicenter study

Context: Studies of the clinical and immunological features of autoimmune Addison disease (AAD) are needed to understand the disease burden and increased mortality. Objective: To provide upgraded data on autoimmune comorbidities, replacement therapy, autoantibody profiles, and cardiovascular risk factors. Design, Setting, and Participants: A cross-sectional, population-based study that included 660 AAD patients from the Swedish Addison Registry (2008–2014). When analyzing the cardiovascular risk factors, 3594 individuals from the population-based survey in Northern Sweden, MONICA (monitoring of trends and determinants of cardiovascular disease), served as controls. Main Outcome Measures: The endpoints were the prevalence of autoimmune comorbidities and cardiovascular risk factors. Autoantibodies against 13 autoantigens were determined. Results: The proportion of 21-hydroxylase autoantibody-positive patients was 83%, and 62% of patients had ≥1 associated autoimmune diseases, more frequently coexisting in females (P < 0.0001). AAD patients had a lower body mass index (P < 0.0001) and prevalence of hypertension (P = 0.027) compared with controls. Conventional hydrocortisone tablets were used by 89% of the patients, with a mean dose of 28.1 ± 8.5 mg/d. The mean hydrocortisone equivalent dose normalized to the body surface was 14.8 ± 4.4 mg/m2/d. A greater hydrocortisone equivalent dose was associated with a greater incidence of hypertension (P = 0.046). Conclusions: Careful monitoring of AAD patients is warranted to detect associated autoimmune diseases. Contemporary Swedish AAD patients did not have an increased prevalence of overweight, hypertension, type 2 diabetes mellitus, or hyperlipidemia. However, high glucocorticoid replacement doses could be a risk factor for hypertension.

Phase I/II study of adjuvant immunotherapy with sentinel lymph node T lymphocytes in patients with colorectal cancer

Although the development of multi-disciplinary management has improved the survival of colorectal cancer (CRC), the prognosis of metastatic CRC patients remains poor. Accumulating evidence has demonstrated that immunotherapy with cancer vaccines and adoptive T cell transfusions may improve outcomes as an adjuvant to current standard CRC treatment. In this phase I/II study, 71 CRC patients who underwent radical surgery (stage I–III, nxa0=xa046) or palliative surgery (stage IV with non-resectable synchronous metastases, nxa0=xa025) were included. In the first part of this study, sentinel lymph nodes (SLNs) were intraoperatively identified in 55 patients (46 with stage I–III CRC and 9 with stage IV CRC). SLN-T lymphocytes were expanded ex vivo for a median of 28.5xa0days (range 23–33xa0days). Thereafter, a median of 153xa0×xa0106 cells (range 20.7–639.0xa0×xa0106) were transfused. No treatment-related toxicity was observed. In the second part of this study, the stage IV patients were routinely followed. The 24-month survival rate of the SLN-T lymphocyte group was significantly higher than that of the control group: 55.6 versus 17.5xa0% (pxa0=xa00.02). The median overall survival of the SLN-T lymphocyte and control groups was 28 and 14xa0months, respectively. Our study showed that adjuvant SLN-T lymphocyte immunotherapy is feasible and safe for postoperative CRC patients. Additionally, this therapy may improve the long-term survival of metastatic CRC. Further investigation of the clinical efficacy and anti-tumor immunity is warranted.

Detection of metastatic colon cancer cells in sentinel nodes by flow cytometry

In colon cancer the presence of metastases in the regional lymph nodes is an important prognostic factor. Currently, examination is based on histological and immunohistochemical evaluations of lymph node sections. However, these methods are time-consuming, labour intensive and micro-metastases might be missed. The sentinel node technique may be used to identify the direct tumour draining lymph node. In this study the possible use of flow cytometry for detection of sentinel node metastases in patients with colon cancer has been investigated. Peripheral blood mononuclear cells (PBMC) with the addition of DLD-1 colon cancer cells were stained intracellularly for cytokeratin 20 (CK20), and for the cell surface markers epithelial cell adhesion molecule (EpCAM) and carbohydrate antigen 19-9 (CA19-9). CK20 positive colon cancer cells were reliably detected with as few as 0.037% events. However, PBMCs from both colon cancer patients and from healthy individuals contained CK20 positive cells. Staining for EpCAM resulted in an almost complete recovery of tumour cells, and as few as 0.037% added cells were detected. Low intra-assay variability was determined for EpCAM (CV 8.8) and CA19-9 (CV 9.7) stainings. The results from staining for CK20 or for EpCAM and CA19-9 concorded, but the degree to which respective antigens were expressed varied. The use of flow cytometry for detection of metastatic colon cancer cells was verified in fourteen sentinel nodes specimens from patients with colon cancer. Herein we have explored the potential of flow cytometry to become a fast, sensitive and reliable method for detection of lymphatic metastases in patients with colon cancer using direct fluorophore-conjugated antibodies against multiple surface antigens. The method seems feasible and further testing is warranted.

AIRE expressing marginal zone dendritic cells balances adaptive immunity and T-follicular helper cell recruitment

Autoimmune polyendocrine syndrome Type I (APS I) results in multiple endocrine organ destruction and is caused by mutations in the Autoimmune regulator gene (AIRE). In the thymic stroma, cells expressing the AIRE gene dictate T cell education and central tolerance. Although this function is the most studied, AIRE is also expressed in the periphery in DCs and stromal cells. Still, how AIRE regulated transcription modifies cell behaviour in the periphery is largely unknown. Here we show that AIRE is specifically expressed by 33D1(+) DCs and dictates the fate of antibody secreting cell movement within the spleen. We also found that AIRE expressing 33D1(+) DCs expresses self-antigens as exemplified by the hallmark gene insulin. Also, as evidence for a regulatory function, absence of Aire in 33D1(+) DCs led to reduced levels of the chemokine CXCL12 and increased co-stimulatory properties. This resulted in altered activation and recruitment of T-follicular helper cells and germinal centre B cells. The altered balance leads to a change of the early response to a T cell-dependent antigen in Aire(-/-) mice. These findings add to the understanding of how specific DC subtypes regulate the early responses during T cell-dependent antibody responses within the spleen and further define the role of AIRE in the periphery as regulator of self-antigen expression and lymphocyte migration.

Decreased numbers of FoxP3-positive and TLR-2-positive cells in intestinal mucosa are associated with improvement in patients with active inflammatory bowel disease following selective leukocyte apheresis.

BackgroundImpaired immunological tolerance to commensal enteric flora is considered one possible pathogenic mechanism of inflammatory bowel disease (IBD). Given that regulatory T cells and Toll-like receptor (TLR)-positive cells are key actors in mucosal immune regulation, we aimed to identify the dynamics of these actors in the intestinal mucosa in relation to clinical improvement following selective leukapheresis treatment.MethodsTen patients with active IBD despite treatment with corticosteroids, immunomodulators, or anti-tumor necrosis factor therapy were assessed by immunohistochemical staining of colorectal mucosal biopsies obtained before and after five sessions (week 7) of granulocyte and monocyte adsorption apheresis (GCAP). The presence of FoxP3-positive regulatory T cells, macrophages, dendritic cells, and TLR-2 and-4 positive cells was determined in relation to short-(week 7) and long-term (week 52) clinical outcome data.ResultsFollowing GCAP, the number of FoxP3-(P = 0.012) and TLR-2 (P = 0.008)-positive cells significantly decreased in biopsies after 7 weeks, in parallel with both clinical improvement at week 7 and a longstanding response after 12 months.ConclusionsDownregulation of FoxP3 and TLR-2 cells in the colorectal mucosa mirrors both short-and long-term improvement in patients with active IBD responding to GCAP. This observation suggests a potential role of these cells in the pathogenesis of IBD and the induction of immunological tolerance in the mucosa.

Pilot study of adoptive immunotherapy with sentinel node-derived T cells in muscle-invasive urinary bladder cancer

Abstract Objective: The aim of this study was to determine by computed tomography (CT) whether treatment with tumor-draining lymph-node-derived expanded autologous T lymphocytes results in objective responses and/or improved survival in patients with metastatic urinary bladder cancer (UBC) and to record the toxicity of the treatment. Materials and methods: Eighteen patients with metastatic UBC were prospectively selected from two centers. The preoperative staging was T2–T4bN1–2 and/or M0–M1 or MX. Tumor-draining lymph nodes were harvested at intended cystectomy for the extraction of T lymphocytes. This was followed by expansion of the T lymphocytes in a cell culture, and subsequent reinfusion of these autologous tumor-specific T lymphocytes. Responses to therapy were evaluated by CT scans according to Response Evaluation Criteria In Solid Tumors (RECIST) and clinical follow-up, according to the research protocol. Results: Nine out of 18 patients were treated. Treatment was feasible and safe. In two out of nine immunologically treated patients, objective responses were detected in terms of diminished or obliterated nodal metastases. When excluding three patients with disseminated osseous metastases plus one with a T4b tumor left in situ, a success rate of two out of six treated patients was seen. The two responders had survival times of 35 and 11 months, respectively. No toxicity was recorded. Conclusions: Infusion of expanded autologous tumor-specific T lymphocytes is feasible and safe, and objective responses according to RECIST were recorded. One objective responder to immunotherapy displayed notably long overall survival.