Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct. METHODS: The authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer-probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct. RESULTS: ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors' assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities. CONCLUSIONS: The authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test-retest reliability, and that specific assays can be developed for detection of unique constructs.

A quantitative polymerase chain reaction test to enumerate leukocytes in allograft tissue and the implications for donor eligibility testing.

A country-to-country analysis of infectious disease screening requirements for donated tissues or cells reveals they are not often harmonized. Transmission of one such infectious disease, human T-lymphotropic virus (HTLV), is related to the transfer of HTLV-infected, viable leukocytes of sufficient number. The ability to characterize allograft tissue as being absent of leukocytes, or containing relatively few leukocytes, by using a specific test has not been previously investigated. A quantitative polymerase chain reaction (qPCR) test was developed to interrogate protein tyrosine phosphatase, receptor type C (PTPRC) gene expression in tissue samples and was able to determine the number of leukocytes present in a tissue. The impact of a qualified leukocyte tissue testing method should be significant and lead to changes in donor eligibility regulations in certain countries. Human leukapheresis samples were used as a control to establish the amount of PTPRC in leukocytes. That value was used as a comparator to determine the number of leukocyte equivalents in tissues of interest. The qPCR test measured tissue leukocyte equivalents and the results were consistent with the relative abundance of leukocytes predicted for each tissue. Using qPCR to calculate leukocyte equivalents based upon PTPRC gene expression can be successfully employed to estimate the number of leukocytes in a tissue or allograft. This method could be used as a screen to rule out tissues that do not meet the criteria of being leukocyte rich and, therefore, do not need direct HTLV testing.

Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.

The lactating mammary gland synthesizes large amounts of triglyceride from fatty acids derived from the blood and from de novo lipogenesis. The latter is significantly increased at parturition and decreased when additional dietary fatty acids become available. To begin to understand the molecular regulation of de novo lipogenesis, we tested the hypothesis that the transcription factor sterol regulatory element binding factor (SREBF)-1c is a primary regulator of this system. Expression of Srebf1c mRNA and six of its known target genes increased ≥2.5-fold at parturition. However, Srebf1c-null mice showed only minor deficiencies in lipid synthesis during lactation, possibly due to compensation by Srebf1a expression. To abrogate the function of both isoforms of Srebf1, we bred mice to obtain a mammary epithelial cell-specific deletion of SREBF cleavage-activating protein (SCAP), the SREBF escort protein. These dams showed a significant lactation deficiency, and expression of mRNA for fatty acid synthase (Fasn), insulin-induced gene 1 (Insig1), mitochondrial citrate transporter (Slc25a1), and stearoyl-CoA desaturase 2 (Scd2) was reduced threefold or more; however, the mRNA levels of acetyl-CoA carboxylase-1α (Acaca) and ATP citrate lyase (Acly) were unchanged. Furthermore, a 46% fat diet significantly decreased de novo fatty acid synthesis and reduced the protein levels of ACACA, ACLY, and FASN significantly, with no change in their mRNA levels. These data lead us to conclude that two modes of regulation exist to control fatty acid synthesis in the mammary gland of the lactating mouse: the well-known SREBF1 system and a novel mechanism that acts at the posttranscriptional level in the presence of SCAP deletion and high-fat feeding to alter enzyme protein.

Alternative pathway of complement in children with diarrhea-associated hemolytic uremic syndrome.

BACKGROUND AND OBJECTIVES: Diarrhea-associated hemolytic uremic syndrome (D+HUS) is a common cause of acute kidney injury in children. Mutations in alternative pathway (AP) complement regulatory proteins have been identified in severe cases of thrombotic microangiopathy, but the role of the AP in D+HUS has not been studied. Therefore, we determined whether plasma levels of markers of activation of the AP are increased in D+HUS and are biomarkers of the severity of renal injury that predict the need for dialysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Patients were randomly selected from among participants in the HUS-SYNSORB Pk trial. Plasma samples were collected on days 1, 4, 7, and 10 after enrollment and day 28 after discharge from the hospital. Levels of two complement pathway products, Bb and SC5b-9, were determined by ELISA. RESULTS: Seventeen children (6 boys and 11 girls; age, 5.4 +/- 3.5 yr) were studied. Eight (47%) required dialysis support, and two had serious extrarenal events. On the day of enrollment, plasma levels of Bb and SC5b-9 were significantly increased in all patients compared with healthy controls (P < 0.01). The elevated concentrations normalized by day 28 after discharge. Circulating levels of complement pathway fragments did not correlate with severity of renal injury or occurrence of complications. CONCLUSIONS: Patients with acute-onset D+HUS manifest activation of the AP of complement that is temporally related to the onset of disease and that resolves within 1 mo. Therapies to inhibit the AP of complement may be useful in attenuating the severity of renal injury and extrarenal complications.

Thyrotropin receptor knockout mice: studies on immunological tolerance to a major thyroid autoantigen.

Graves' disease involves a breakdown in self-tolerance to the TSH receptor (TSHR). Central T cell tolerance is established by intrathymic deletion of immature T lymphocytes that bind with high affinity to peptides from autoantigens (like the TSHR) expressed ectopically in the thymus. In TSHR-knockout mice, tolerance cannot be induced to the TSHR, which should, therefore, be a foreign antigen for these animals. To test this hypothesis, TSHR-knockout mice and wild-type controls were vaccinated (three injections) with TSHR DNA or control DNA. TSHR antibodies, measured by ELISA, binding to TSHR-expressing eukaryotic cells, and TSH binding inhibition, developed in approximately 60% of TSHR-knockout mice, not significantly different from 80% in the wild-type mice. Antibody levels were also comparable in the two groups, and both strains recognized the same immunodominant linear antibody epitope at the amino terminus of the TSHR. Splenocyte responses to TSHR protein in culture, measured as interferon-gamma production, were similar in TSHR-knockout and wild-type mice. Moreover, T cells from both strains recognized the same two epitopes from a panel of 29 synthetic peptides encompassing the TSHR ectodomain and extracellular loops. This lack of difference in immune responses in TSHR-knockout and wild-type mice is unexpected and is contrary to observations in other induced animal models of autoimmunity. The importance of our finding is that the TSHR may not be similar to other model proteins used to define the concept of central immune tolerance.

TSH is a negative regulator of skeletal remodeling.

The established function of thyroid stimulating hormone (TSH) is to promote thyroid follicle development and hormone secretion. The osteoporosis associated with hyperthyroidism is traditionally viewed as a secondary consequence of altered thyroid function. We provide evidence for direct effects of TSH on both components of skeletal remodeling, osteoblastic bone formation, and osteoclastic bone resorption, mediated via the TSH receptor (TSHR) found on osteoblast and osteoclast precursors. Even a 50% reduction in TSHR expression produces profound osteoporosis (bone loss) together with focal osteosclerosis (localized bone formation). TSH inhibits osteoclast formation and survival by attenuating JNK/c-jun and NFkappaB signaling triggered in response to RANK-L and TNFalpha. TSH also inhibits osteoblast differentiation and type 1 collagen expression in a Runx-2- and osterix-independent manner by downregulating Wnt (LRP-5) and VEGF (Flk) signaling. These studies define a role for TSH as a single molecular switch in the independent control of both bone formation and resorption.