Long-term in vivo microscopy of CAR T cell dynamics during eradication of CNS lymphoma in mice.

T cells expressing anti-CD19 chimeric antigen receptors (CARs) demonstrate impressive efficacy in the treatment of systemic B cell malignancies, including B cell lymphoma. However, their effect on primary central nervous system lymphoma (PCNSL) is unknown. Additionally, the detailed cellular dynamics of CAR T cells during their antitumor reaction remain unclear, including their intratumoral infiltration depth, mobility, and persistence. Studying these processes in detail requires repeated intravital imaging of precisely defined tumor regions during weeks of tumor growth and regression. Here, we have combined a model of PCNSL with in vivo intracerebral 2-photon microscopy. Thereby, we were able to visualize intracranial PCNSL growth and therapeutic effects of CAR T cells longitudinally in the same animal over several weeks. Intravenous (i.v.) injection resulted in poor tumor infiltration of anti-CD19 CAR T cells and could not sufficiently control tumor growth. After intracerebral injection, however, anti-CD19 CAR T cells invaded deeply into the solid tumor, reduced tumor growth, and induced regression of PCNSL, which was associated with long-term survival. Intracerebral anti-CD19 CAR T cells entered the circulation and infiltrated distant, nondraining lymph nodes more efficiently than mock CAR T cells. After complete regression of tumors, anti-CD19 CAR T cells remained detectable intracranially and intravascularly for up to 159 d. Collectively, these results demonstrate the great potential of anti-CD19 CAR T cells for the treatment of PCNSL.

Cellular and Molecular Mechanisms of CD8+ T Cell Differentiation, Dysfunction and Exhaustion.

T cells follow a triphasic distinct pathway of activation, proliferation and differentiation before becoming functionally and phenotypically "exhausted" in settings of chronic infection, autoimmunity and in cancer. Exhausted T cells progressively lose canonical effector functions, exhibit altered transcriptional networks and epigenetic signatures and gain constitutive expression of a broad coinhibitory receptor suite. This review outlines recent advances in our understanding of exhausted T cell biology and examines cellular and molecular mechanisms by which a state of dysfunction or exhaustion is established, and mechanisms by which exhausted T cells may still contribute to pathogen or tumour control. Further, this review describes our understanding of exhausted T cell heterogeneity and outlines the mechanisms by which checkpoint blockade differentially engages exhausted T cell subsets to overcome exhaustion and recover T cell function.

Features of Human CD3+CD20+ T Cells.

Monoclonal Abs against CD20 reduce the number of relapses in multiple sclerosis (MS); commonly this effect is solely attributed to depletion of B cells. Recently, however, a subset of CD3(+)CD20(+) T cells has been described that is also targeted by the anti-CD20 mAb rituximab. Because the existence of cells coexpressing CD3 and CD20 is controversial and features of this subpopulation are poorly understood, we studied this issue in detail. In this study, we confirm that 3-5% of circulating human T cells display CD20 on their surface and transcribe both CD3 and CD20. We report that these CD3(+)CD20(+) T cells pervade thymus, bone marrow, and secondary lymphatic organs. They are found in the cerebrospinal fluid even in the absence of inflammation; in the cerebrospinal fluid of MS patients they occur at a frequency similar to B cells. Phenotypically, these T cells are enriched in CD8(+) and CD45RO(+) memory cells and in CCR7(-) cells. Functionally, they show a higher frequency of IL-4-, IL-17-, IFN-γ-, and TNF-α-producing cells compared with T cells lacking CD20. CD20-expressing T cells respond variably to immunomodulatory treatments given to MS patients: they are reduced by fingolimod, alemtuzumab, and dimethyl fumarate, whereas natalizumab disproportionally increases them in the blood. After depletion by rituximab, they show earlier and higher repopulation than CD20(+) B cells. Taken together, human CD3(+)CD20(+) T cells pervade lymphatic organs and the cerebrospinal fluid, have a strong ability to produce different cytokines, and respond to MS disease modifying drugs.

HER2 chimeric antigen receptor T cell immunotherapy is an effective treatment for diffuse intrinsic pontine glioma.

Background: Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMG) of the thalamus and spinal cord are rare but devastating high-grade glial tumors of childhood with no curative treatment. Despite aggressive treatment attempts the prognosis has remained poor. Chimeric antigen receptor (CAR) T cell therapy has been identified as a promising new approach in the treatment of DMG tumors; however, additional targets are urgently required given known tumor heterogeneity and the prospect of antigen escape of this cancer. Methods: Using cell surface mass spectrometry, we detected high HER2 cell surface protein across a panel of patient-derived DIPG cells, thereby identifying an existing CAR T cell therapy for use in DIPG. Primary human T cells were transduced to express a second-generation HER2 CAR and interrogated for efficacy against patient-derived DIPG cells. Results: HER2 CAR T cells demonstrated potent and antigen-specific cytotoxicity and cytokine secretion when co-cultured with patient-derived DIPG cells. Furthermore, HER2 CAR T cells provided a significant regression in intracranial DIPG xenograft tumors. Conclusions: HER2 CAR T cells are already in clinic development and are well tolerated in pediatric patients. Here we provide strong preclinical evidence for the inclusion of DIPG patients in future pediatric CNS tumor HER2 CAR T cell clinical trials.

In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer.

Lung cancer patients are at risk for brain metastases and often succumb to their intracranial disease. Chimeric Antigen Receptor (CAR) T-cells emerged as a powerful cell-based immunotherapy for hematological malignancies; however, it remains unclear whether CAR T-cells represent a viable therapy for brain metastases. Here, we established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intracerebral two-photon laser scanning-microscopy. This approach enabled in vivo-characterization of fluorescent CAR T-cells and tumor cells on a single-cell level over weeks. Intraparenchymal injection of Lewis lung carcinoma cells (expressing the tumor cell-antigen EpCAM) was performed, and EpCAM-directed CAR T-cells were injected either intravenously or into the adjacent brain parenchyma. In mice receiving EpCAM-directed CAR T-cells intravenously, we neither observed substantial CAR T-cell accumulation within the tumor nor relevant anti-tumor effects. Local CAR T-cell injection, however, resulted in intratumoral CAR T-cell accumulation compared to controls treated with T-cells lacking a CAR. This finding was accompanied by reduced tumorous growth as determined per in vivo-microscopy and immunofluorescence of excised brains and also translated into prolonged survival. However, the intratumoral number of EpCAM-directed CAR T-cells decreased during the observation period, pointing toward insufficient persistence. No CNS-specific or systemic toxicities of EpCAM-directed CAR T-cells were observed in our fully immunocompetent model. Collectively, our findings indicate that locally (but not intravenously) injected CAR T-cells may safely induce relevant anti-tumor effects in brain metastases from lung cancer. Strategies improving the intratumoral CAR T-cell persistence may further boost the therapeutic success.

Novel high-affinity EGFRvIII-specific chimeric antigen receptor T cells effectively eliminate human glioblastoma.

OBJECTIVES: The increasing success of Chimeric Antigen Receptor (CAR) T cell therapy in haematological malignancies is reinvigorating its application in many other cancer types and with renewed focus on its application to solid tumors. We present a novel CAR against glioblastoma, an aggressive, malignant glioma, with a dismal survival rate for which treatment options have remained unchanged for over a decade. METHODS: We use the human Retained Display (ReD) antibody platform (Myrio Therapeutics) to identify a novel single-chain variable fragment (scFv) that recognises epidermal growth factor receptor mutant variant III (EGFRvIII), a common and tumor-specific mutation found in glioblastoma. We use both in vitro functional assays and an in vivo orthotopic xenograft model of glioblastoma to examine the function of our novel CAR, called GCT02, targeted using murine CAR T cells. RESULTS: Our EGFRvIII-specific scFv was found to be of much higher affinity than reported comparators reverse-engineered from monoclonal antibodies. Despite the higher affinity, GCT02 CAR T cells kill equivalently but secrete lower amounts of cytokine. In addition, GCT02-CAR T cells also mediate rapid and complete tumor elimination in vivo. CONCLUSION: We present a novel EGFRvIII-specific CAR, with effective antitumor functions both in in vitro and in a xenograft model of human glioblastoma.

In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer.

Brain metastases frequently occur in lung cancer and dramatically limit prognosis of affected patients. The influence of tumor-associated macrophages and microglia (TAM/M) and their receptor CX3CR1 on different steps of brain metastasis formation from lung cancer is poorly characterized. We established a syngeneic orthotopic cerebral metastasis model in mice by combining a chronic cranial window with repetitive intravital 2-photon laser scanning microscopy. This allowed in vivo tracking of fluorescence-expressing tumor cells and TAM/M on a single-cell level over weeks. Intracarotid injection of red tdTomato-fluorescent Lewis lung carcinoma cell was performed in transgenic mice either proficient or deficient for CX3CR1. After intracarotid cell injection, intravascular tumor cells extravasated into the brain parenchyma and formed micro- and mature macrometastases. We observed potential phagocytosis of extravasated tumor cells by TAM/M. However, during later steps of metastasis formation, these anti-tumor effects diminished and were paralleled by TAM/M accumulation and activation. Although CX3CR1 deficiency resulted in a lower number of extravasated tumor cells, progression of these extravasated cells into micro metastases was more efficient. Overall, this resulted in a comparable number of mature macrometastases in CX3CR1-deficient and -proficient mice. Our findings indicate that unspecific inhibition of CX3CR1 might not be a suitable therapeutic option to prevent dissemination of lung cancer cells to the brain. Given the close interaction between TAM/M and tumor cells during metastasis formation, other therapeutic approaches targeting TAM/M function may warrant further evaluation. The herein established orthotopic mouse model may be a useful tool to evaluate such concepts in vivo.

Erratum: Novel high-affinity EGFRvIII-specific chimeric antigen receptor T cells effectively eliminate human glioblastoma.

[This corrects the article DOI: 10.1002/cti2.1283.].

Clinical and psychological phenomenology of pain in autoinflammatory diseases.

BACKGROUND: Pain is the clinical hallmark of patients in patients with autoinflammatory diseases (AID) caused by variants of the NLRP3-, MEFV- or TNFRSF1A gene. However, no systematical analysis of the clinical and psychological presentation of pain has been performed to date. METHODS: Twenty-one symptomatic patients with variants in the NLRP3-, MEFV- and TNFRSF1A gene and clinical signs suggestive of an AID were retrospectively included in this monocentric cross-sectional case-series study. Patients were examined and interviewed using the German pain questionnaire. The hospital anxiety and depression scale (HADS) was applied to screen patients for anxiety and depression. RESULTS: Twenty out of 21 AID patients (95%) reported pain at the time of examination. Mean current pain intensity in all AID patients comprised 3.6 ± 1.3 and mean maximum pain intensity was 7.0 ± 1.6 on a 11-point numeric ranging scale (NRS). In 15 patients (71%), pain was present for more than 60 months. Ten patients (48%) experienced recurrent attacks with asymptomatic intervals and 7 patients (33%) suffered from constant pain, while 4 patients (19%) experienced both. Nociceptive pain including musculoskeletal and visceral affection was the most prominent type of pain (n = 20; 95%). Pain symptoms were treated continuously with analgesic or co-analgesic drugs in 10 patients (48%). Five patients (24%) have been positively screened for concomitant depression or anxiety. CONCLUSIONS: Early and prompt diagnosis is necessary to provide multimodal pain treatment and to avoid the development of chronic pain in patients with AID.

The Role of BAFF-R Signaling in the Growth of Primary Central Nervous System Lymphoma.

Primary CNS lymphoma (PCNSL) is an aggressive brain tumor. Despite improvements in therapeutic algorithms, long-term survival remains rare, illustrating an urgent need for novel therapeutic targets. BAFF-R is a pro-survival receptor expressed on most malignant B cells, including PCNSL. To date, its role in PCNSL growth remains elusive. Here, we have created a BAFF-R knockout lymphoma cell line (BAFF-R-KO) using CRISPR-Cas9. In serum-starved conditions, BAFF-R-KO cells exhibit decreased viability in vitro compared to BAFF-R+ cells. Combining an orthotopic mouse model of PCNSL with chronic cranial windows and intravital microscopy, we have demonstrated a significant delay in tumor growth in mice inoculated with BAFF-R-KO cells compared to BAFF-R+ PCNSL. Additionally, median survival of BAFF-R-KO mice was significantly prolonged. Altogether, our results indicate the high potential of BAFF-R as a novel treatment target for PCNSL.

APRIL and BAFF: novel biomarkers for central nervous system lymphoma.

BACKGROUND: Early diagnosis of CNS lymphoma (CNSL) is essential for successful therapy of this rapidly progressing brain tumor. However, in patients presenting with focal brain lesions, fast and reliable diagnosis of PCNSL remains a challenge. A proliferation-inducing ligand (APRIL) and B cell activating factor (BAFF) are important factors in the pathophysiology, diagnosis, and prognosis of systemic B cell malignancies. However, their utility as biomarkers for the diagnosis of CNSL and their effects on CNSL cells remain unclear. METHODS: In this prospective study, we analyzed the levels of APRIL and BAFF in the cerebrospinal fluid (CSF) of 116 patients with suspected focal brain lesions, including 53 CNSL patients. Additionally, we serially measured their levels during chemotherapy and relapse. Furthermore, we analyzed the effect of APRIL and BAFF on two B cell lymphoma cell lines using proliferation, viability, and chemotaxis assays. RESULTS: CSF levels of APRIL and BAFF reliably differentiated CNSL from other focal brain lesions (including primary and metastatic brain tumors, autoimmune-inflammatory lesions, and neuroinfectious lesions) with a specificity of 93.7% (APRIL, BAFF) and a sensitivity of 62.3% (APRIL) and 47.1% (BAFF). Serial CSF analysis of CNSL patients during chemotherapy and relapse demonstrates a close correlation of APRIL CSF levels and the course of this disease. In vitro, APRIL and BAFF showed anti-apoptotic effects during MTX treatment and mediated chemotaxis of malignant B cells. CONCLUSION: This study extends the spectrum of valuable diagnostic biomarkers in CNSL. In patients with focal brain lesions, measurement of APRIL in CSF could help accelerating the diagnosis of CNSL. Moreover, our results highlight an important role of APRIL and BAFF in the pathophysiology of CNSL.

Soluble TACI and soluble BCMA as biomarkers in primary central nervous system lymphoma.

BACKGROUND: B-cell survival is regulated through interactions of B-cell-activating factor and a proliferation-inducing ligand with their receptors transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA). We evaluated the diagnostic potential of soluble TACI (sTACI) and soluble BCMA (sBCMA) in CSF and serum as biomarkers in primary CNS lymphoma (PCNSL). METHODS: CSF (n = 176) and serum samples (n = 105) from patients with clinically or radiologically suspected PCNSL as well as from control patients were collected prospectively. Levels of sTACI and sBCMA were analyzed by enzyme-linked immunosorbent assay. Additionally, in patients with PCNSL, CSF was analyzed during disease course (time of diagnosis, n = 26; relapse, n = 10; remission, n = 14), and in 2 patients long-term longitudinal analysis was performed. RESULTS: Soluble TACI and sBCMA are significantly increased in patients with PCNSL (sTACI, median: 445 pg/mL; sBCMA, median: 760 pg/mL) compared with control patients (sTACI, median: 0 pg/mL; sBCMA, median: 290 pg/mL). At a cutoff value of 68.4 pg/mL, sTACI shows high sensitivity (87.9%) and specificity (88.3%) for the diagnosis of active PCNSL. Soluble BCMA is less sensitive (72.7%) and specific (71.8%) (cutoff: 460 pg/mL). When both markers are combined, specificity increases, however, at the cost of a lower sensitivity. In serum, both sTACI and sBCMA are not increased in PCNSL patients. Both soluble receptors correlate with clinical course and therapy response. CONCLUSIONS: Our results suggest that sTACI and sBCMA in the CSF are promising new biomarkers for diagnosis and therapy monitoring in PCNSL. However, our findings need to be validated in an independent cohort.