Clinical Signs Of Autoimmunity Research Articles

Functional Human iPSC-Derived Thymic Epithelial Progenitor Cells Reconstitute T Cell Development and Function in an In Vivo Model of Thymic Aplasia

The thymus is an essential primary lymphoid organ in which T cells mature, proliferate, and acquire a broad and self-tolerant T cell receptor repertoire. In the thymus, specialized thymic epithelial cells (TECs) select T cells capable of recognizing foreign antigens, and eliminate self-reactive T cells to prevent autoimmunity. In addition, the thymus also contributes to the differentiation of regulatory T cells which confine immune responses to actively preserve central tolerance. During thymic development, the interactions between hematopoietic lymphoid progenitor cells and thymic epithelial progenitor cells (TEPCs) provides indispensable signals to the development of the thymic epithelium. In humans, unlike in mice, the development of the thymus is completed before birth. There is no evidence of thymic stem cells that could contribute to a sustained regeneration of the thymic epithelium throughout life. Accordingly, the thymus has no endogenous capacity to regenerate. The thymus peaks in size during infancy and involutes with age, leading to the weakened immune function of the elderly known as immune senescence. Allogeneic hematopoietic stem cell transplant (HSCT) is a curative strategy for many malignant and non-malignant hematopoietic diseases, however, often at the cost of causing severe thymic dysfunction due to conditioning medications and graft-versus-host disease (GVHD). This can severely impair T cell immune reconstitution post-HSCT leading to life-threatening complications. In addition, genetic forms of congenital athymia (e.g. 22q11 Deletion Syndrome, TBX1, TBX2, PAX1, and CHD7 deficiency) can result in absence of T cell development. Complete congenital athymia is fatal early in life unless immune reconstitution is achieved by allogeneic thymus transplantation. However, the shortage of transplantable donor thymus tissue and lack of HLA-matching between donor and recipient limits the use of allogeneic thymus transplantation to a small number of children with complete congenital athymia. Thus, there is a critical unmet clinical need to provide HLA-matched thymic tissues for a larger group of patients with irreversible thymic injury and severe thymic dysfunction. Creating a regenerative thymus replacement therapy by generating functional TEPCs from induced-pluripotent stem cells (iPSCs) has been a longstanding aspiration of the transplant community. We have taken a novel approach by developing a robust in vitro TEPC differentiation protocol that sequentially induces all stages of human thymic ontogeny including bipotent third (III) pharyngeal pouch endoderm (PPEIII), ventralization of the PPE (PPEv), and TEPC commitment, usually lymphoid dependent. Here, we show that TEPCs from various iPSC-lines (tissue source, donor age and sex) express key markers of thymic epithelial fate and function, including FOXN1, PAX1, PAX9, HLA-DR. To examine their functional capacity, we transplanted iPSC-derived TEPCs under the kidney capsule of humanized (hCD34+ HSCs engrafted) athymic NSG-nude mice (NOD.scid.Il2Rγcnull, Foxn1null knockout allele). We observed robust hCD3+ development (40-60% out of hCD45 population) with CD4+ and CD8+ T cells (±70% and ±25%, respectively) at different stages of maturation (17% CD45RA+ CD45RO-, naïve; 80% CD45RA- CD45RO+, memory). Similar levels of T cells were found in mice that received human fetal thymus transplants, while T cells were not detected in NSG-nude mice without any thymus graft (primary or iPSC-derived). Mice with iPSC-derived thymus grafts had no clinical signs of autoimmunity or GVHD. In vivo matured grafts express levels of FOXN1, PAX1, PAX9, DLL4, PSMB11 and CD205 protein that are comparable to primary thymus tissue grafts. Altogether, our protocol reliably produces functional TEPCs from a diverse set of iPSCs that support T cell differentiation in vivo. We are currently characterizing the transcriptomic and epigenomic profile of iPSC-derived TEPCs on a single cell level before and after transplant. In addition, we are examining in vivo T cell function by analyzing the TCR repertoire and response to T-cell dependent antigens. We foresee that iPSC-derived TEPCs will offer a safe alternative as an HLA-matched thymic replacement therapy that will benefit a large cohort of patients, i.e. patients undergoing HSCT and patients receiving solid organ transplants in addition to patients with congenital thymic aplasia.

Autoimmune Hepatitis During Ledipasvir/Sofosbuvir Treatment of Hepatitis C: A Case Report.

We report the case of a woman with chronic hepatitis C and idiopathic thrombocytopenic purpura (ITP) who developed autoimmune hepatitis (AIH) during antiviral therapy with ledipasvir (LDV)/sofosbuvir (SOF). The onset of acute hepatitis rose two weeks after starting treatment with LDV/SOF when HCV‐RNA tested negative, suggesting a link between rapid HCV clearance and de novo autoimmune diseases. Conclusion: This case report proposes new immunologic scenarios in patients with hepatitis C virus (HCV) with laboratory or clinical signs of autoimmunity during direct‐acting antiviral (DAA) therapy.

Abstract B021: Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice

Abstract Background: The adoptive transfer of T cell engineered with chimeric antigen receptors (CARs) is currently considered a highly promising therapeutic option for treating so far incurable malignant diseases. In order to reduce adverse reactions associated with CAR engineered T cells, a flexible modular universal chimeric antigen receptor (UniCAR) platform is developed, which allows a precise control of CAR T cell reactivity to manage side effects while preserving efficacy. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CARs into two individual components. The UniCAR is structurally similar to second-generation CARs, however the single chain variable fragment (scFv) does not recognize a cell surface antigen but a short peptide motif derived from a human nuclear autoantigen. Antigen-specificity and crosslinking of UniCAR expressing T cells to target cells is provided by a separate targeting module (TM) compromising a scFv, binding to specific cell surface antigens, fused to the aforementioned peptide motif. Thus, T cells engineered to express UniCARs remain inactivated after infusion, since the UniCAR target is not available for binding under physiological conditions. Only in the presence of the TM, antigen specific activation of UniCAR T cells occurs, which allows for a more sophisticated regulation. Methods: We developed a novel radio-immune target module (TM), which consists of an anti-PSCA (prostate stem cell antigen) scFv for redirecting of UniCARs to PSCA expressing tumor cells. Three different tags are fused in a row to the C-Terminus of the scFv to functionalize the molecule for combined immune- and radiotherapy. First, a ten amino acid (aa) long tag mediates the crosslink to T cells genetically modified with the UniCAR. Further downstream, a peptide tag (Rx) for enhanced conjugation of radionuclides and finally, a 6xhistidine (His) tag are located. TM-Rx efficacy was evaluated for target cell killing in-vitro and compared to a TM without Rx-tag. In-vivo efficacy of the TM-Rx was also investigated in a high and low-tumor burden mouse model. Results: Rx-TM and TM equivalently kill PSCA-expressing tumor cells in-vitro in presence of UniCARs in an E:T (Effector:Target) ratio of 1:1 after 24 hours. UniCAR T cell engrafted NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice show no clinical signs of autoimmunity neither in presence nor absence of the TM. One week treatment with TM delays subcutaneous tumor growth and prolongs survival of tumor bearing, UniCAR T cell engrafted NSG mice. Longterm infiltration of tumor tissue by UniCARs could be observed over 16 weeks in the high and low-tumor burden ectopic carcinoma model. Conclusion: The UniCAR technology shows efficacy both in-vitro and in-vivo. Extension of the TM with an additional tag does not impair functionality of the UniCAR platform. Short-treatment prolongs survival of tumor bearing mice but does not lead to cure, therefore combination with other treatment modalities e.g. radiation oncology seems reasonable. Citation Format: Elham Pishali Bejestani, Malte von Bonin, Marc Cartellieri, Armin Ehninger, Mechthild Krause, Michael Baumann, Gerhard Ehninger, Michael Bachmann. Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B021.

Autoimmune signals in non-segmental vitiligo patients are associated with distinct clinical parameters and toxic exposures

Although non-segmental vitiligo is commonly considered an autoimmune disease, the possible differences between non-segmental vitiligo patients with and without autoimmune signals have not been clearly established. To perform a comparison of non-segmental vitiligo patients with autoimmune signals (AIS) vs. those without autoimmune signals (NAIS) in regards to clinical characteristics and toxic/drug exposure. 112 vitiligo patients were selected for a sex and age matched (1 : 1) case control study at an university based dermatology outpatient hospital specialized in pigmentary disorders. Medical assessment was performed by dermatologists using the modified Vitiligo European Task Force form and serological and clinical signs of autoimmunity were evaluated. Disease duration, age of onset, patient history of cardiovascular disease, past smoking history, use of drugs, and consummation of goitrogenic foods were all significantly increased in the AIS group using McNemar's test for matched pairs. In our conditional regression model, the simultaneous presence of disease duration, use of prescription drugs, and consummation of goitrogenic foods were the best predictors of AIS vitiligo patients. The evaluation of non-segmental vitiligo patients according to the presence vs. the absence of autoimmune signals allows us to correlate patients exhibiting autoimmune phenomenon with certain clinical characteristics, namely long disease duration, use of prescription drugs, and consumption of goitrogenic substances. In the presence of the aforementioned clinical profile, we suggest an evaluation of autoimmune signals.

IMPACT study: A phase II-III clinical study with the immunomodulator MGN1703 in patients with advanced colorectal carcincoma.

633 Background: The synthetic DNA-based immunomodulator MGN1703 acts as an agonist of toll-like receptor 9. Based on promising data from a phase I study in patients with metastatic solid tumors including those with CRC, a phase II-III study was initiated in patients with advanced CRC having disease control after first-line therapy. The objective of the study is to assess efficacy and safety of the MGN1703 treatment in comparison to placebo. Methods: The IMPACT study is designed as a randomized double-blind placebo-controlled phase II-III study, which is conducted in patients with advanced CRC showing disease control after first-line therapy with standard chemotherapy regimen. The treatment is administered subcutaneously twice weekly in a ratio 2:1 (60 mg MGN1703 or placebo). The study is conducted in Germany, Austria, France, UK, Czech Republic and Russia, and 129 patients will be recruited into the study. The efficacy and safety of the study treatment will be evaluated based on extensive immunological tests, radiological assessment, safety laboratory results and assessments of the quality of life. The study treatment will be continued until tumor progression, intolerable toxicity, exclusion criteria or withdrawal of consent. Results: The majority of adverse events were assessed as not drug-related by the investigator. The remaining AEs include mild night sweat (not assessable), mild fever (at three occasions, possible related), and mild arthralgia (certain related) in one patient each. Three SAE have been reported so far of which one was assessed as probably drug-related – atypical pneumonia. Only in single patients local reactions such as mild redness and swelling at injection site were reported. No laboratory or clinical signs of autoimmunity or dose-limiting toxicities were reported, so far. Conclusions: With these preliminary safety results of the ongoing clinical study in patients with advanced CRC it could be shown that ttreatment with MGN1703 at the dosage of 60 mg is well tolerated and safe. Reported adverse events assessed as possibly drug-related belong to expected study drug reactions known for immune modulating drugs. These events were not accompanied by any signs of autoimmunity.

Immunity to the HER-2/neu oncogenic protein.

The study of oncogenic viruses led to the discovery that transforming retroviruses contain oncogenes homologous with and/or derived from cellular proto-oncogenes. In humans malignant transformation is often the result of the activation of proto-oncogenes. Normal proto-oncogenes can be activated to transforming proto-oncogenes by a variety of mechanisms including point mutation, translocation and amplification. Development of successful strategies for the immunotherapy of human cancers is an area of intense investigation. Part of the problem in developing cancer-specific immunotherapy has been the lack of well-defined tumour antigens. Our laboratory has focused on the question of whether oncogenic proteins expressed by transforming proto-oncogenes can serve as targets for immune attack. Some patients with HER-2/Neu-positive breast cancer have an existent immune response to the HER-2/neu protein with no clinical signs of autoimmunity, supporting the idea that overexpressed oncogenic proteins can be targeted in therapy without fear of destructive autoimmunity. The identification of candidate cytotoxic T lymphocyte epitopes might allow the generation of tumour-specific cytotoxic T lymphocytes for use in therapy and identify potential epitopes for peptide vaccines.

HLA-A0201-positive, IL-7/B7.1-cotransfected allogeneic tumor cells as a vaccine in metastatic renal cell cancer—A clinical phase-I trial

3072 Background: Tumor vaccination remains a promising experimental approach in RCC. HLA-A0201-restricted T cell immunity against RCC is well established. RCC26 is an allogeneic HLA-A0201+ human RCC cell line, a T cell clone specifically recognising RCC26 and other RCC in the context of HLA-A0201 has previously been generated, the TCR of this T cell clone was characterised. Furthermore, IL-7/B7.1 cotransfected tumor cells are a potent vaccine in animal models. Methods: RCC26 was transfected with pKEx-IL-7-IR-B7 coding for human IL-7 and B7.1 (CD80). 10 HLA-A0201+ patients with metastatic RCC and disease progression under cytokine therapy were included. 10 vaccinations with 2.5–40x106 gene-modified irradiated tumor cells which had been produced under GMP conditions were performed s.c. over 22 weeks. Primary endpoints of the study were feasibility, safety and immunological response, secondary endpoint was clinical response. The protocol was approved by the ethics committee, all patients gave informed consent. Results: Gene-modified RCC26 cells produced IL-7 (3.4 ng/106 cells/24h), more than 90% of the cells were CD80+. Vaccination was feasible and safe with no severe toxicity. Local DTH-reactions were observed in 4 patients. Skin biopsies of the vaccination site showed lymphocytic infiltrates dominated by CD4+T cells. In 8 patients vaccination induced HLA- and /or antinuclear antibodies without clinical signs of autoimmunity. Analysis of the T cell response against RCC-associated antigens is under way. No partial or complete responses could be documented. However, 50% of the patients had stable disease with the longest TTP being 69 weeks. Mean TTP in our cohort was 25 weeks (range 4 to 69 weeks). Conclusion: Our results show that vaccination with an allogeneic gene-modified tumor cell line is feasible and safe. Stable disease lasting up to 69 weeks in a substantial proportion of patients suggests immunological activity of the vaccine. Vaccination of patients with a low tumor burden is a promising strategy for the future, i.e. after surgery or treatment with novel multi-kinase inhibitors. No significant financial relationships to disclose.

A follow-up study in 28 patients treated with infliximab for severe recalcitrant psoriasis: evidence for efficacy and high incidence of biological autoimmunity

Infliximab, an antitumour necrosis factor-alpha chimeric monoclonal antibody, is effective for the treatment of severe psoriasis. While the induction of antinuclear antibodies (ANA) and antidouble-stranded-DNA antibodies (anti-dsDNA-ab) is frequently observed in patients with rheumatoid arthritis and Crohn disease receiving infliximab, the incidence of induced biological and clinical autoimmunity remains unknown in the context of psoriasis. To investigate biological and clinical signs of autoimmunity in 28 patients receiving infliximab for severe, recalcitrant forms of psoriasis, and the clinical response to treatment. Twenty-eight patients, 15 men and 13 women (median age 39.4 years) with psoriasis refractory to three or more systemic treatments were included. Twenty presented with plaque-type psoriasis [median Psoriasis Area and Severity Index (PASI) score 25.9; range 7.2-48], five with psoriatic erythroderma (median PASI score 54; range 48-72) and three with generalized pustular psoriasis (GPP). Psoriatic arthritis was present in 13 patients (46.4%). Infliximab 5 mg kg(-1) was given at week (W) 0, W2, W6 and every 8 weeks thereafter. Clinical data were assessed at baseline and before each infusion. Detection of ANA and of IgM and IgG anti-dsDNA-ab were performed at baseline and at W22 by immunofluorescence and enzyme-linked immunosorbent assay, respectively. The mean number of infliximab infusions was 5.5 (range 2-15). Among patients with plaque-type and erythrodermic psoriasis, 17 of 25 (68%) and three of five reached a PASI improvement of 75% or more, respectively, while rapid improvement of clinical and biological signs was observed in all three patients with GPP. The prevalence of positive detection of ANA raised from 12% at baseline to 72% at W22 (P = 0.0001), an increase which was also observed for IgM anti-dsDNA-ab (68% vs. 0%, P < 0.0001), while no significant change was observed for the IgG isotype (16% vs. 0%, P = 0.125). Three patients developed nonerosive polyarthritis, without any other criteria for systemic lupus. The incidence of biological autoimmunity is high in patients with refractory psoriasis receiving infliximab. The concomitant onset of polyarthritis in three cases raises the need to investigate the incidence of autoimmune manifestations in psoriatic patients receiving infliximab in further large-scale studies.

Infliximab Treatment Does Not Induce Organ-specific or Nonorgan-specific Autoantibodies Other than Antinuclear and Anti-Double-Stranded DNA Autoantibodies in Crohnʼs Disease

Treatment of Crohn's disease (CD) by infliximab has been associated with the induction of antinuclear (ANA) and anti-double stranded DNA (dsDNA) autoantibodies. As yet, little is known about the effect of the humoral response induced by infliximab on the production of other organ-specific or nonorgan-specific autoantibodies. Thirty-five patients with CD treated with repeated infusions of infliximab were prospectively studied. Thirty-two patients with CD who had never received infliximab served as controls. A large panel of autoantibodies directed against phospholipid, beta2-glycoprotein I, mitochondria, smooth muscle, liver--kidney microsomes, filaggrin, thyroid, adrenals, and rheumatoid factor was tested along with ANA and anti-dsDNA autoantibodies during 1 year of intermittent treatment with infliximab. Autoantibodies were detected using the appropriate methods (i.e., indirect immunofluorescence, a radioimmunological technique, and ELISA assays). Induction of ANA and anti-dsDNA autoantibodies was observed in 53% and 35% of infliximab-treated patients with CD, respectively. Overall, no other organ-specific or nonorgan-specific autoantibodies were detected during follow-up. A single patient who developed ANA and anti-dsDNA autoantibodies showed clinical features consistent with drug-induced lupus, which quickly resolved after discontinuation of infliximab. The other patients with CD receiving infliximab did not develop any clinical symptoms of autoimmunity. The humoral response induced by infliximab was restricted to ANA and anti-dsDNA autoantibodies, which persist for up to 1 year of follow-up. We confirmed the significant prevalence of such autoantibodies induced by infliximab in CD, but they are not generally associated with clinical signs of autoimmunity.

Treatment of a glioblastoma patient by vaccination with autologous dendritic cells pulsed with allogeneic major histocompatibility complex class I–matched tumor peptides

Dendritic cells (DCs) are antigen-presenting cells that play a central role in the initiation and modulation of antitumor immune responses. In this pilot study, we investigated the ability of autologous DCs pulsed ex vivo with allogeneic major histocompatibility complex class I-matched glioblastoma peptides to stimulate host antitumor immune responses when injected as a vaccine. A patient with recurrent brainstem glioblastoma multiforme (GBM) received a series of three intradermal immunizations of antigen-pulsed DCs on an outpatient basis following surgical debulking of her posterior fossa tumor. Dendritic cell vaccination was well tolerated, and no clinical signs of autoimmunity or experimental allergic encephalomyelitis were detected. She developed a measurable cellular immune response against the allogeneic glioblastoma peptides used in her vaccine preparation, as demonstrated by in vitro T-cell proliferation assays. In addition, increased T-cell infiltration was noted within the intracranial tumor site in the biopsy sample obtained following DC vaccination. An objective clinical response, however, was not evident, and this patient eventually died 21 months after her disease was diagnosed. To our knowledge, this is the first patient with brain cancer ever to be treated with DC-based immunotherapy. This case illustrates that vaccination with DCs pulsed with acid-eluted glioblastoma peptides is feasible and can induce systemic antigen-specific immunity in a patient with recurrent GBM. Additional studies are necessary to determine the optimum DC doses and antigen loading conditions that may translate into clinical effectiveness and survival benefit for patients with brain tumors. Phase I trials for malignant glioma are currently underway.

Autoantibody prevalence in chronic hepatitis B virus infection: Effect in interferon alfa

The most effective treatment of chronic hepatitis B virus (HBV) infection is interferon alfa (IFN-alpha), a potentially severe side effect of which is the induction of autoimmunity. To assess whether IFN- alpha causes clinical or serological autoimmune manifestations, we studied 61 children randomized to receive 5 MU/m2 of IFN-alpha three times per week for 12 weeks, with or without steroid priming or no treatment. Autoantibodies to antinuclei (ANA), smooth muscle (SMA), gastric parietal cell (GPC), liver kidney microsomal type 1, mitochondrial, liver cytosolic antigen, thyroid microsomal, and thyroid globulin were detected by standard techniques. Over a median of 4 years (range, 1-5 years) from randomization, no clinical signs of autoimmunity were observed. Autoantibody positivity for nuclei, smooth muscle, and/or gastric parietal cells was observed on at least one occasion in 42 of 61 children (69%), with no overall difference in the prevalence between patients treated with interferon alone (19 of 24 [79%]), steroids plus interferon (13 of 20 [65%]), or untreated controls (10 of 17 [59%]). There was also no difference in the autoantibody prevalence before, during, and at follow-up after cessation of treatment in both interferon-treated and interferon- untreated patients. Autoantibodies are common in chronic HBV infection, and their prevalence is uninfluenced by IFN-alpha. (Hepatology 1996 Sep;24(3):520-3)

Autoimmune phenomena in bronchial asthma with special reference to aspirin intolerance.

We assessed the autoimmune status of 185 adult patients with bronchial asthma and 46 healthy subjects of similar sex and age. The patients were divided into groups with: (1) aspirin-induced asthma (AIA) (n = 80); (2) intrinsic asthma with good aspirin tolerance (n = 46); and (3) atopic asthma (n = 59). Antinuclear antibodies (ANA) at a titer of > or = 1:40 were present in the serum of 55% of the patients with AIA, 41% of those with intrinsic asthma, 39% of those with atopic asthma, and 11% of the healthy subjects, with the difference between each patient group and the healthy subjects being statistically significant (p < 0.05). The fluorescence staining pattern of ANA on Hep-2 cells was mainly speckled, but the precise antigen specificity of the antibodies could not be identified with reference sera against extractable nuclear antigens. None of the subjects exhibited anti-double stranded deoxyribonucleic acid (anti-ds-DNA) or anti-neurtrophil cytoplasmic antibodies (ANCA). Positive ANA were associated with signs of complement activation, the presence of rheumatoid factor, and circulating immune complexes. Clinical signs of autoimmunity, evidenced by rheumatic symptoms, cold sensitivity, and Raynaud's phenomenon, were more common among the patients who tested positively for ANA. The assessment of autoimmunity may help in better characterizing patients with asthma and understanding various symptoms of the disease. Any causal relationship between asthma and autoimmunity remains to be established.