Plasma IFN-γ and IL-6 levels correlate with peripheral T-cell numbers but not toxicity in RCC patients treated with CAR T-cells.

Autologous T-cells genetically modified to express a chimeric antigen receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to twelve patients with CAIX-positive metastatic renal cell carcinoma. Here, we questioned whether plasma cytokine levels following treatment or in vitro cytokine production from the T-cell infusion products could serve as predictors for peripheral T-cell persistence or in vivo T-cell activity. We demonstrated that CAR surface as well as gene expression are down-regulated following T-cell infusion, and that peripheral numbers of CAR T-cells are best captured by flow cytometry and not by qPCR. Numbers of CAR T-cells in blood correlated with plasma levels of IFN-γ and IL-6, but not with any of the other cytokines tested. Plasma IFN-γ or IL-6 levels did not correlate with liver enzyme values. Thus, out of 27 cytokines tested, IFN-γ and IL-6 levels in plasma are potential surrogate markers for CAR T-cell persistence in solid tumors.

Changes in Ocean Heat, Carbon Content, and Ventilation: A Review of the First Decade of GO-SHIP Global Repeat Hydrography.

Global ship-based programs, with highly accurate, full water column physical and biogeochemical observations repeated decadally since the 1970s, provide a crucial resource for documenting ocean change. The ocean, a central component of Earth's climate system, is taking up most of Earth's excess anthropogenic heat, with about 19% of this excess in the abyssal ocean beneath 2,000 m, dominated by Southern Ocean warming. The ocean also has taken up about 27% of anthropogenic carbon, resulting in acidification of the upper ocean. Increased stratification has resulted in a decline in oxygen and increase in nutrients in the Northern Hemisphere thermocline and an expansion of tropical oxygen minimum zones. Southern Hemisphere thermocline oxygen increased in the 2000s owing to stronger wind forcing and ventilation. The most recent decade of global hydrography has mapped dissolved organic carbon, a large, bioactive reservoir, for the first time and quantified its contribution to export production (∼20%) and deep-ocean oxygen utilization. Ship-based measurements also show that vertical diffusivity increases from a minimum in the thermocline to a maximum within the bottom 1,500 m, shifting our physical paradigm of the ocean's overturning circulation.

T Cell Maturation Stage Prior to and During GMP Processing Informs on CAR T Cell Expansion in Patients.

Autologous T cells were genetically modified to express a chimeric antigen receptor (CAR) directed toward carboxy-anhydrase-IX (CAIX) and used to treat patients with CAIX-positive metastatic renal cell carcinoma. In this study, we questioned whether the T cell maturation stage in the pre-infusion product affected CAIX CAR expression and function in vitro as well as in vivo CAR T cell numbers and expansion. During the 14 days expansion of CAR T cells prior to administration, we observed shifts from a predominant CD4 to a CD8 T cell phenotype and from a significant fraction of naïve to central effector T cells. Surface expression of the CAR was equally distributed among different T cell subsets and T cell maturation stages. During T cell culture days 14-18 (which covered patient treatment days 1-5), T cells demonstrated a decline in CAR expression level per cell irrespective of T cell maturation stage, although the proportion of CAR-positive T cells and CAR-mediated T cell effector functions remained similar for both CD4 and CD8 T cell populations. Notably, patients with a higher fraction of naïve CD8 T cells at baseline (prior to genetic modification) or central effector CD8 T cells at 2 weeks of CAR T cell culture demonstrated a higher fold expansion and absolute numbers of circulating CAR T cells at 1 month after start of therapy. We conclude that the T cell maturation stage prior to and during CAR T cell expansion culture is related to in vivo CAR T cell expansion.

Long-term stability of T-cell activation and transduction components critical to the processing of clinical batches of gene-engineered T cells.

BACKGROUND AIMS: The generation of gene-modified T cells for clinical adoptive T-cell therapy is challenged by the potential instability and concomitant high financial costs of critical T-cell activation and transduction components. As part of a clinical trial to treat patients with metastatic renal cell cancer with autologous T cells engineered with a chimeric antigen receptor (CAR) recognizing carboxy-anhydrase-IX (CAIX), we evaluated functional stability of the retroviral vector, T-cell activation agent Orthoclone OKT3 (Janssen-Cilag, Beerse, Belgium) monoclonal antibody (mAb) and the transduction promoting agent RetroNectin (Takara, Otsu, Japan). METHODS: Carboxy-anhydrase-IX chimeric antigen receptor retrovirus-containing culture supernatants (RTVsups) were generated from two packaging cell lines, Phoenix-Ampho (BioReliance, Sterling, UK) and PG13, and stored at -80°C over 10 years and 14 years. For Orthoclone OKT3 and RetroNectin, aliquots for single use were prepared and stored at -80°C. Transduction efficiencies of both batches of RTVsups were analyzed using the same lots of cryopreserved donor peripheral blood mononuclear cells, Orthoclone OKT3 and RetroNectin over time. RESULTS: We revisit here an earlier report on the long-term functional stability of the RTVsup, observed to be 9 years, and demonstrate that this stability is at least 14 years. Also, we now demonstrate that Orthoclone OKT3 and RetroNectin are functionally stable for periods of at least 6 years and 10 years. CONCLUSIONS: High-cost critical components for adoptive T-cell therapy can be preserved for ≥10 years when prepared in aliquots for single use and stored at -80°C. These findings may significantly facilitate, and decrease the financial risks of, clinical application of gene-modified T cells in multicenter studies.

Systematic long-term observations of the global carbon cycle.

Imagine a meeting convened to avert a global financial crisis where none of the finance ministers had access to reliable information on changes in the stock market, national gross domestic product or international trade flows. It is hardly conceivable. Yet the infinitely more existence-threatening planetary social and ecological crisis we refer to as 'global change' (comprising the linked issues of biogeochemical, climate, biotic and human system change) is in an analogous situation. Our information on the profound and accelerating changes currently depends to an unacceptable degree on serendipity, individual passion, redirected funding and the largely uncoordinated efforts of a few nations. The thesis of this paper is that navigation of the very narrow 'safe passages' that lie ahead requires a comprehensive and systematic approach to Earth observations, supported by a globally coordinated long-term funding mechanism. We developed the argument based on observations of the carbon cycle, because the issues there are compelling and easily demonstrated, but we believe the conclusions also to be true for many other types of observations relating to the state and management of the biosphere.

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo.

BACKGROUND: We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)(+) T cells is reflected by changes of selected immune parameters measured in peripheral blood. METHODS: ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)(+) T lymphocytes prior to infusion, and were correlated with plasma cytokine levels. RESULTS: Prior to infusion, scFv(G250)(+) T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX(+) RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-gamma were produced, whilst production of TNF-alpha, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-gamma was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-gamma increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2. CONCLUSION: ScFv(G250)-receptor-mediated functions of the scFv(G250)(+) T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-gamma levels.

The Helicobacter pylori plasticity region locus jhp0947-jhp0949 is associated with duodenal ulcer disease and interleukin-12 production in monocyte cells.

Colonization with Helicobacter pylori always results in chronic gastritis, which is controlled by infiltration of mononuclear cells and the subsequent release of cytokines like interleukin (IL)-12. To identify H. pylori factors involved in inducing cytokine production in mononuclear cells, a random H. pylori mutant library was screened for the inability to induce IL-12 production in monocyte THP-1 cells. Of the 231 random mutants screened, one mutant (M1) showed a consistent twofold decrease in the amount of IL-12 induction compared to the parental strain 1061 (P <0.01). Further characterization of mutant M1 revealed that the kanamycin resistance cassette had integrated in the jhp0945 gene, which is situated in an H. pylori strain-specific plasticity region. Three reference strains possessing this plasticity region induced significantly higher amounts of IL-12 when compared to the H. pylori 26695 reference strain, which does not possess this plasticity region. The role in disease outcome of jhp0945 as well as the neighbouring plasticity region genes jhp0947 and jhp049 was assessed in a Dutch population cohort. Firstly, the presence of jhp0947 was completely linked with that of jhp0949 and was roughly associated with jhp0945 (P=0.072), but not with the cag pathogenicity island (PAI) (P=0.464). The presence of the jhp0947 and jhp0949 genes, but not of jhp0945, was significantly associated with duodenal ulcer disease when compared to gastritis (P=0.027). Therefore, the jhp0947-jhp0949 locus may be a novel putative H. pylori marker for disease outcome independent of the cag PAI.