Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses.

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.

Inflammation enhances the vaccination potential of CD40-activated B cells in mice.

Vaccination with antigen-pulsed CD40-activated B (CD40-B) cells can efficiently lead to the in vivo differentiation of naive CD8+ T cells into fully functional effectors. In contrast to bone marrow-derived dendritic cell (BMDC) vaccination, CD40-B cell priming does not allow for memory CD8+ T-cell generation but the reason for this deficiency is unknown. Here, we show that compared to BMDCs, murine CD40-B cells induce lower expression of several genes regulated by T-cell receptor signaling, costimulation, and inflammation (signals 1-3) in mouse T cells. The reduced provision of signals 1 and 2 by CD40-B cells can be explained by a reduction in the quality and duration of the interactions with naive CD8+ T cells as compared to BMDCs. Furthermore, CD40-B cells produce less inflammatory mediators, such as IL-12 and type I interferon, and increasing inflammation by coadministration of polyriboinosinic-polyribocytidylic acid with CD40-B-cell immunization allowed for the generation of long-lived and functional CD8+ memory T cells. In conclusion, it is possible to manipulate CD40-B-cell vaccination to promote the formation of long-lived functional CD8+ memory T cells, a key step before translating the use of CD40-B cells for therapeutic vaccination.

The Notch signaling pathway controls short-lived effector CD8+ T cell differentiation but is dispensable for memory generation.

Following an infection, naive CD8(+) T cells expand and differentiate into two main populations of effectors: short-lived effector cells (SLECs) and memory precursor effector cells (MPECs). There is limited understanding of the molecular mechanism and cellular processes governing this cell fate. Notch is a key regulator of cell fate decision relevant in many immunological pathways. In this study, we add to the role of Notch in cell fate decision and demonstrate that the Notch signaling pathway controls the MPEC/SLEC differentiation choice following both Listeria infection and dendritic cell immunization of mice. Although fewer SLECs were generated, Notch deficiency did not alter the rate of memory CD8(+) T cell generation. Moreover, we reveal that the Notch signaling pathway plays a context-dependent role for optimal cytokine production by effector CD8(+) T cells. Together, our results unravel critical functions for the Notch signaling pathway during effector CD8(+) T cell differentiation.

IL-6 production by dendritic cells is dispensable for CD8+ memory T-cell generation.

Following activation, naïve CD8(+) T cells will differentiate into effectors that differ in their ability to survive: some will persist as memory cells while the majority will die by apoptosis. Signals given by antigen-presenting cells (APCs) at the time of priming modulate this differential outcome. We have recently shown that, in opposition to dendritic cell (DC), CD40-activated B-(CD40-B) cell vaccination fails to efficiently produce CD8(+) memory T cells. Understanding why CD40-B-cell vaccination does not lead to the generation of functional long-lived memory cells is essential to define the signals that should be provided to naïve T cells by APCs. Here we show that CD40-B cells produce very low amount of IL-6 when compared to DCs. However, supplementation with IL-6 during CD40-B-cell vaccination did not improve memory generation. Furthermore, IL-6-deficient DCs maintained the capacity to promote the formation of functional CD8(+) effectors and memory cells. Our results suggest that in APC vaccination models, IL-6 provided by the APCs is dispensable for proper CD8(+) T-cell memory generation.

Notch signaling regulates PD-1 expression during CD8(+) T-cell activation.

Programmed cell death 1 (PD-1) is an inhibitory receptor involved in T-cell activation, tolerance and exhaustion. Little is known on how the expression of PD-1 is controlled during T-cell activation. Recent studies demonstrated that NFATc1 and IRF9 regulate Pdcd1 (PD-1) transcription and that T-bet acts as a transcriptional repressor. In this study, we have investigated the role of the Notch signaling pathway in PD-1 regulation. Using specific inhibitors of the Notch signaling pathway, we showed decreased PD-1 expression and inhibition of Pdcd1 transcription by activated CD8(+) T cells. Chromatin immunoprecipitation further showed occupancy of the Pdcd1 promoter with RBPJk and Notch1 intracellular domain at RBPJk-binding sites. Our results identify the Notch signaling pathway as an important regulator of PD-1 expression by activated CD8(+) T cells.

Murine superficial lymph node surgery.

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x10(6) cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

CD40-activated B cells can efficiently prime antigen-specific naïve CD8+ T cells to generate effector but not memory T cells.

BACKGROUND: The identification of the signals that should be provided by antigen-presenting cells (APCs) to induce a CD8(+) T cell response in vivo is essential to improve vaccination strategies using antigen-loaded APCs. Although dendritic cells have been extensively studied, the ability of other APC types, such as B cells, to induce a CD8(+) T cell response have not been thoroughly evaluated. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript, we have characterized the ability of CD40-activated B cells, stimulated or not with Toll-like receptor (TLR) agonists (CpG or lipopolysaccharide) to induce the response of mouse naïve CD8(+) T cells in vivo. Our results show that CD40-activated B cells can directly present antigen to naïve CD8(+) T cells to induce the generation of potent effectors able to secrete cytokines, kill target cells and control a Listeria monocytogenes infection. However, CD40-activated B cell immunization did not lead to the proper formation of CD8(+) memory T cells and further maturation of CD40-activated B cells with TLR agonists did not promote the development of CD8(+) memory T cells. Our results also suggest that inefficient generation of CD8(+) memory T cells with CD40-activated B cell immunization is a consequence of reduced Bcl-6 expression by effectors and enhanced contraction of the CD8(+) T cell response. CONCLUSIONS: Understanding why CD40-activated B cell immunization is defective for the generation of memory T cells and gaining new insights about signals that should be provided by APCs are key steps before translating the use of CD40-B cell for therapeutic vaccination.

Identification of differentially expressed genes in uveal melanoma using suppressive subtractive hybridization.

PURPOSE: Uveal melanoma (UM) is the most common primary cancer of the eye, resulting not only in vision loss, but also in metastatic death. This study attempts to identify changes in the patterns of gene expression that lead to malignant transformation and proliferation of normal uveal melanocytes (UVM) using the Suppressive Subtractive Hybridization (SSH) technique. METHODS: The SSH technique was used to isolate genes that are differentially expressed in the TP31 cell line derived from a primary UM compared to UVM. The expression level of selected genes was further validated by microarray, semi-quantitative RT-PCR and western blot analyses. RESULTS: Analysis of the subtracted libraries revealed that 37 and 36 genes were, respectively, up- and downregulated in TP31 cells compared to UVM. Differential expression of the majority of these genes was confirmed by comparing UM cells with UVM by microarray. The expression pattern of selected genes was analyzed by semi-quantitative RT-PCR and western blot, and was found to be consistent with the SSH findings. CONCLUSIONS: We demonstrated that the SSH technique is efficient to detect differentially expressed genes in UM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor.

Mutations in VANGL1 associated with neural-tube defects.

Neural-tube defects such as anencephaly and spina bifida constitute a group of common congenital malformations caused by complex genetic and environmental factors. We have identified three mutations in the VANGL1 gene in patients with familial types (V239I and R274Q) and a sporadic type (M328T) of the disease, including a spontaneous mutation (V239I) appearing in a familial setting. In a protein-protein interaction assay V239I abolished interaction of VANGL1 protein with its binding partners, disheveled-1, -2, and -3. These findings implicate VANGL1 as a risk factor in human neural-tube defects.

Functional characterization of the E399D DMT1/NRAMP2/SLC11A2 protein produced by an exon 12 mutation in a patient with microcytic anemia and iron overload.

DMT1 (Nramp2, Slc11a2) mediates iron uptake at the intestinal brush border and across the membrane of acidified endosomes. A single patient with severe microcytic anemia and iron overload was recently reported to carry a mutation in exon 12 of DMT1 (1285G>C). The mutation has two effects: it severely impairs splicing causing skipping of exon 12 and introduces an amino acid polymorphism (E399D) in the protein encoded by the remaining properly spliced transcript found in the patient. The functional properties and possible contribution to disease of the DMT1 E399D mutation are unknown and have been studied in independent mutants at that position (E399D, E399Q, E399A) expressed in LLC-PK1 kidney cells. The 3 mutants are shown to be fully functional with respect to stability, targeting and trafficking to the membrane, and are transport-competent. This indicates that DMT1G1285C is not a complete loss of function but rather that a modest amount of active DMT1 is produced in this patient. This activity may explain the distinguishing iron overload seen in this patient in addition to microcytic anemia that is absent in parallel rodent models of DMT1 deficiency.