Regulatory Immunotherapy in Bone Marrow Transplantation

Every year individuals receive hematopoietic stem cell transplantation (HSCT) to eradicate malignant and nonmalignant disease. The immunobiology of allotransplantation is an area of ongoing discovery, from the recipient's conditioning treatment prior to the transplant to the donor cell populations responsible for engraftment, graft-versus-host disease, and graft-versus-tumor effect. In this review, we focus on donor-type immunoregulatory T cells, namely, natural killer T cells (NKT) and regulatory T cells (Treg), and their current and potential roles in tolerance induction after allogeneic HSCT

Regulatory immunotherapy in bone marrow transplantation

Every year individuals receive hematopoietic stem cell transplantation (HSCT) to eradicate malignant and nonmalignant disease. The immunobiology of allotransplantation is an area of ongoing discovery, from the recipient's conditioning treatment prior to the transplant to the donor cell populations responsible for engraftment, graft-versus-host disease, and graft-versus-tumor effect. In this review, we focus on donor-type immunoregulatory T cells, namely, natural killer T cells (NKT) and regulatory T cells (Treg), and their current and potential roles in tolerance induction after allogeneic HSCT

Abstract 3512: Expanded human regulatory iNKT cells exhibit direct cytotoxicity against hematolymphoid tumor targets

Abstract CD1d-restricted invariant natural killer T (iNKT) cells are rare but potent innate regulatory cells capable of immune modulation via robust production of Th1/Th2 cytokines, as well as tumor immunosurveillance via direct cytotoxicity. Protocols to expand iNKT cells and augment their cytotoxicity would broaden their application in allogeneic transplantation and adjuvant anti-tumor immunotherapy. We have optimized a protocol for ex vivo expansion of highly purified human CD3+CD4negVα24+ iNKT cells from human peripheral blood mononuclear cells (PBMC). PBMCs were stimulated with an iNKT-specific glycolipid (α-GalCer), recombinant IL-2 and IL-7 and sorted to &amp;gt; 98% purity at day 7. Sorted iNKT cells were further expanded in the presence of irradiated allogeneic PBMCs, Vα24-specific TCR stimulation, and low-dose IL-2 and IL-7 for 21 days. This results in roughly 10^3-fold expansion, with peak yields of at day 21-28 of expansion (range 1.5 x 10^5 - 10.6 x 10^6 iNKT cells from 1 x 10^8 - 3.5 x 10^8 starting PBMC). At 28 days, these iNKT cells secrete high levels of IL-13, TNF-α, GM-CSF, and IL-4, moderate IL-2 and IFN-γ in anti-CD2/CD3/CD28 bead-stimulated Luminex supernatant assay. Day 28 expanded iNKT cells are dose-dependent suppressors of sorted autologous CD3+CD8+ effector T cells in 72-hr mixed leukocyte reaction (MLR). Anti-CD2/CD3/CD28 activation of these iNKT cells induced expression of high levels of key cytolytic effector molecules, including granzyme B. We therefore measured cytotoxicity of activated day 28 iNKT cells against various hematolymphoid tumor targets following co-incubation of iNKT cells versus control effector populations with firefly luciferase-transduced RS4:11 and Nalm6 (B-ALL), U937 (monocytic), and K562 (CML) targets. iNKT cell effectors (E) demonstrated dose-dependent cytotoxicity against B-lymphoid tumor targets (T) (e.g.: Nalm6: 37.7% at E:T 2:1, 17.7% at E:T 1:1, 7.7% at E:T 0.5:1), with no significant cytotoxicity against myeloid targets (e.g.: K562: 11.8% at E:T 2:1, 6.7 at E:T 1:1, 1.8 at E:T 0.5:1). Our results indicate that human iNKT cells expressing high levels of Th2 and regulatory cytokines can be potently expanded ex vivo and manifest cytotoxicity against B-ALL targets, supporting their potential application in tailored immunotherapy in the pre- and post-transplant setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3512. doi:1538-7445.AM2012-3512</jats:p

A new cell culture model to genetically dissect the complete human papillomavirus life cycle

<div><p>Herein, we describe a novel infection model that achieves highly efficient infection of primary keratinocytes with human papillomavirus type 16 (HPV16). This cell culture model does not depend on immortalization and is amenable to extensive genetic analyses. In monolayer cell culture, the early but not late promoter was active and yielded a spliced viral transcript pattern similar to HPV16-immortalized keratinocytes. However, relative levels of the E8^E2 transcript increased over time post infection suggesting the expression of this viral repressor is regulated independently of other early proteins and that it may be important for the shift from the establishment to the maintenance phase of the viral life cycle. Both the early and the late promoter were strongly activated when infected cells were subjected to differentiation by growth in methylcellulose. When grown as organotypic raft cultures, HPV16-infected cells expressed late E1^E4 and L1 proteins and replication foci were detected, suggesting that they supported the completion of the viral life cycle. As a proof of principle that the infection system may be used for genetic dissection of viral factors, we analyzed E1, E6 and E7 translation termination linker mutant virus for establishment of infection and genome maintenance. E1 but not E6 and E7 was essential to establish infection. Furthermore, E6 but not E7 was required for episomal genome maintenance. Primary keratinocytes infected with wild type HPV16 immortalized, whereas keratinocytes infected with E6 and E7 knockout virus began to senesce 25 to 35 days post infection. The novel infection model provides a powerful genetic tool to study the role of viral proteins throughout the viral life cycle but especially for immediate early events and enables us to compare low- and high-risk HPV types in the context of infection.</p></div

E6 but not E7 is required for episomal genome maintenance in monolayer cells.

<p><b>(A)</b> E6 and E1^E4 transcript levels at 6 dpi of HFK with wt, E6-, and E7-TTL mutant virus. <b>(B)</b> E6 protein detection using the ArborVita Onco<i>E6</i>^<i>™</i> Cervical Test. The test is described in detail in Material and Methods. Note the absence of the E6-specific band in samples derived from control and E6-TTL mutant virus infected HFK. <b>(C)</b> E7 protein detection by western blot. Note the absence of E7 protein in samples derived from HFK infected with E7-TTL mutant virus. Samples from two different HPV16-immortalized HFK lines were analyzed. Ctrl: extracts from mock-infected HFK. <b>(D)</b> E6 transcript levels at 27 to 33 dpi with the indicated virus. The samples were taken at the last passage before control, E6-, and E7-TTL virus infected HFK underwent senescence. We also obtained samples from HFK infected with wt HPV16, which were the only cells to become immortalized, one passage later. *: indicates that these cells had senesced. <b>(E)</b> Quantification of viral genome by qPCR isolated from HFK at 27–33 dpi with respective virus and expressed as percent of ß-actin DNA levels. <b>(F)</b> Viral genome remains episomal in HFK infected with wt and E7-TTL but not with E6-TTL virus. DNA isolated from HFK infected with respective virus was subjected to exonuclease 5 treatment. Resistant DNA was quantified by qPCR. Primers for amplification of HPV16, mitochondrial (mt) DNA and 18S DNA were used.</p

Efficient infection of primary keratinocytes.

<p><b>(A)</b> HFK cells were infected with EdU-labeled HPV16 pseudovirus using ECM-to-cell transfer. At 40 hpi, cells were fixed and processed for the detection of EdU-labeled DNA (red), PML (green), lamin A/C (blue). A representative image of HFK cells infected with EdU-labeled pseudovirus is shown. <b>(B)</b> Quantification of HFK cells from two different donors containing nuclear EdU-labeled pseudogenome at 40hpi. EdU-labeled viral pseudogenome number was counted manually in z-stacks spanning the whole nucleus for each cell as expressed as percent of analyzed cells. 72 & 60 and 64 & 77 cells were analyzed for HFK1 and HFK2, respectively. <b>(C)</b> HFKs from three different donors were infected with HPV16 via ECM-to-cell transfer or by direct binding to cells. At 72 hpi, RNA was isolated and E1^E4 transcript was measured by RT-qPCR. The data are presented as fold changes relative to cells infected by direct virion binding. <b>(D and E)</b> E7 and E1^E4 transcript levels increase with prolonged exposure to virus-loaded ECM in primary HFK (D) and HTE (E). <b>(F)</b> RT-qPCR analysis of viral transcripts at 6 dpi of HFK with wt and E1-TTL mutant HPV16 virus. Unless otherwise noted, all results are based on three biological replicates. HFK were maintained and infected in the presence of 10 μM Y-27632 for experiments shown in panels A, B, D, and F.</p

Immunofluorescent detection of cellular markers in organotypic rafts grown from HPV16-infected HFK.

<p>Thin cuts of organotypic rafts were stained for MCM7 <b>(A)</b>, PCNA <b>(B)</b>, and p53 <b>(C)</b>. The lower panels show a merge with the Dapi stain to highlight nuclei.</p

Analysis of viral transcripts by next generation RNA sequencing.

<p><b>(A)</b> Relative expression levels of individual viral ORFs in HPV16-infected HFK at 2 and 7 dpi. Cells grown in the presence of 10 μM Y-27632 were infected with HPV16 quasivirions via ECM-to-cell transfer and transcripts were quantified by RT-qPCR. The data shown are fold changes normalized to E6 transcript levels at 2 dpi of HFK. <b>(B)</b> RT-qPCR analysis of viral transcripts isolated from HPV16-immortalized HFKs and normalized to transcript levels in HPV16-infected HFKs at 10 dpi. <b>(C)</b> Schematic representation of the HPV16 genome and its ORFs. <b>(D)</b> Read depth maps of viral transcripts isolated from HPV16-infected HFK at 2, 4 and 7 dpi and from HPV-immortalized HFK of representative samples. <b>(E)</b> Detailed analysis of splice junctions of viral transcripts isolated from HFK at 7dpi. Each curved line represents a splice junction derived from individual reads that connects splice donor and acceptor sites.</p