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Cell-mediated cytotoxicity is a critical function of the immune system in mounting defense against pathogens and cancers. Current methods that allow direct evaluation of cell-mediated cytotoxicity suffer from a wide-range of drawbacks. Here, we present a novel strategy to measure cytotoxicity that is direct, sensitive, rapid, and highly adaptable. Moreover, it allows accurate measurement of viability of both target and effector cells. Target cells are fluorescently labeled with a non-toxic, cell-permeable dye that covalently binds to cell proteins, including nuclear proteins. The labeled target cells are incubated with effector cells to begin killing. Following the killing reaction, the cell mixture is incubated with another dye that specifically stains proteins of dead cells, including nuclear proteins. In the final step, cell nuclei are released by Triton X-100, and analyzed by flow cytometry. This results in four nuclear staining patterns that separate target and effector nuclei as well as nuclei of live and dead cells. Analyzing nuclei, instead of cells, greatly reduces flow cytometry errors caused by the presence of target-effector cell aggregates. Target killing time can often be reduced to 2 h and the assay can be done in a high throughput format. We have successfully validated this assay in a variety of cytotoxicity scenarios including those mediated by NK-92 cells, Chimeric Antigen Receptor (CAR)-T cells, and Tumor Infiltrating Lymphocytes (TIL). Therefore, this technique is broadly applicable, highly sensitive and easily administered, making it a powerful tool to assess immunotherapy-based, cell-mediated cytotoxicity.

The Bone Marrow Transplantation Program in Belarus was founded in 1992, and in 1993, a Bone Marrow Transplantation Centre was created in Minsk. From February 1994 to April 1996, 19 allogeneic bone marrow, 16 autologous bone marrow and 10 autologous peripheral blood stem cell transplantations were performed. Reasons for transplantation included chronic myeloid leukemia, multiple myeloma, severe aplastic anemia, acute myeloid leukemia, acute lymphoblastic leukemia, progressive myelofibrosis, Hodgkin's disease, non-Hodgkin's lymphoma, and neuroblastoma. Among the patients were two liquidators involved in the Chernobyl cleanup activity, both of whom underwent allogeneic bone marrow transplantation. A variety of ablative preparative regimens were used, and blood progenitor cells were mobilized by treatment with Cytoxan and granulocyte colony-stimulating factor. Therapy-related deaths resulted from graft-versus-host disease, septic shock, veno-occlusive disease bleeding and intestinal pulmonary fibrosis. Because the transplantation procedures were carried out on people who continued to be exposed to low-level irradiation, the post-transplantation period included a conservative strategy for prevention of graft-versus-host disease. There was nothing unusual about the post-transplantation period, although uncertainty about the continuing radiation dose should be taken into account when interpreting these data.

Ionizing radiation kills hematopoietic stem and progenitor cells. However, several cytokines, including tumor necrosis factor-alpha (TNF-alpha), protect the murine hematopoietic system if they are introduced before or immediately after irradiation. We examined the in vitro capacity of TNF-alpha to protect human hematopoietic stem cells and early progenitor cells from x-ray-induced death. Human CD34+ cells obtained from normal bone marrow were highly enriched for stem and progenitor cells. Pulse exposure of these cells to human TNF-alpha during the first hour immediately after x irradiation (doses of 0.45 Gy to 9 Gy) significantly improved further survival of true hematopoietic stem cells and early progenitors and the ability of CD34+ cells to produce mature hematopoietic cells in liquid culture with hematopoietic growth factors. The radioprotective effect of TNF-alpha was stronger at lower doses, when complete restoration of hematopoiesis was often observed. In contrast, the radioprotective effect of TNF-alpha was moderate at higher doses, with neither complete restoration of the number of stem and progenitor cells nor the production of mature cells. Our data suggest that TNF-alpha can protect human hematopoietic stem and early progenitor cells from ionizing radiation.

mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgene-mediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3+ CD8+, CD3+ CD4+, and natural killer (NK ) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3+CD8+ lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD/SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.

Transfection of human cells with DNA in biomedical applications carries the risk of insertional mutagenesis. Transfection with mRNA avoids this problem; however, in vitro production of mRNA, based on preliminary DNA template cloning in special vectors, is a laborious and time-consuming procedure. We report an efficient vectorfree method of mRNA production from polymerase chain reaction-generated DNA templates. For all cell types tested mRNA was transfected more readily than DNA, and its expression was highly uniform in cell populations. Even cell types relatively resistant to transfection with DNA could express transfected mRNA well. The level of mRNA expression could be controlled over a wide range by changing the amount of input RNA. Cells could be efficiently and simultaneously loaded with several different transcripts. To test a potential clinical application of this method, we transfected human T lymphocytes with mRNA encoding a chimeric immune receptor directed against CD19, a surface antigen widely expressed in leukemia and lymphoma. The transfected mRNA conferred powerful cytotoxicity to T cells against CD19+ targets from the same donor. These results demonstrate that this method can be applied to generate autologous T lymphocytes directed toward malignant cells.

CD30 is a transmembrane receptor which is overexpressed on Hodgkin lymphoma and large cell anaplastic lymphoma and represents an attractive target for therapy. Current methods of immunotherapy of CD30+ lymphomas include antibody based therapy (immunotoxins, bispecific molecules, radioimmunotherapy), and cellular immunotherapy. Here we describe an approach to cellular immunotherapy of CD30+ lymphomas using a synthetic mRNA of an anti CD30 chimeric immune receptor (anti CD30-CIR) introduced into activated T cells by electroporation. Anti-CD30 CIR consists of a single chain antibody directed against CD30, a transmembrane anchor to localize the protein in the cellular membrane, and the cytoplasmic domain of the TcR receptor chain. In these studies, the cDNA coding sequence was used as a template to produce anti CD30-CIR mRNA. Normal T cells expanded in vitro were electroporated with CIR mRNA and expression of anti CD30-CIR was detected by biotin-conjugated F(ab’)2 followed with Streptavidin-APC. To measure cytotoxicity of the CIR, target cells (HH, K562, Jurkat) were pulsed with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then were cultured with T cells transfected with anti-CD30 CIR mRNA or mock transfected T cells. Cytotoxicity was evaluated at different target::effector (T: E) ratios and normalized by standard algorithm. Twenty-four hours after electroporation almost all viable T cell (>90%) expressed anti-CD30 CIR and were able to specifically kill CD30+ targets in a 4h in vitro assay. These results demonstrate that anti-CD30-CIR mRNA modified T cells can be generated and are capable of killing CD30-expressing cells in vitro and therefore may be an effective strategy for the therapy of refractory CD30-expressing lymphomas. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1936.

Current treatment approaches for B-cell malignancies such as non-Hodgkin’s lymphoma and chronic lymphocytic leukemia are initially effective, but most patients ultimately relapse. Thus, there is a compelling need to develop novel therapies. One promising approach is immunotherapy using cytotoxic T cells (CTL). The major limitation of immunotherapy is the availability of large number of high-affinity autologous CTLs. Retargeting of T cells by genetic modification to introduce chimeric receptors can overcome this problem. Since infusion of permanently modified CTLs may not be desirable, we have developed a new method of transfecting CTLs using mRNA, which results in nearly universal but transient expression of a chimeric receptor targeting CD19. The chimeric immunoreceptor used for transfection was composed of anti-CD19 single chain antibody, CD3ζ and 4-1BB signaling domain (anti-CD19-CIR). mRNA was produced by in vitro transcription of PCR generated templates followed by poly(A) addition. CD8+ CTLs were isolated from expanded or resting T cells from healthy volunteers using magnetic beads. T cells were expanded using magnetic beads conjugated with anti-CD28 and anti-CD3. mRNA transfection was performed by electroporation using Amaxa nucleofector. The cytotoxicity was evaluated by a standard 51Cr release method. To test the in vivo function, CD19+ lymphoma cells expressing firefly luciferase (ffLuc) was injected to NOD/scid mice intraperitoneally. Intra peritoneal tumors have established by day 3, and anti-CD19-CIR or mock transfected T cells were injected intraperitoneally at days 3 and 6. The mice were imaged using IVIS biophotonic imaging system. When transfected with anti-CD19-CIR mRNA, more than 90% of the CD3+ T lymphocytes expressed the receptor on their surface. Both the CD4+ and the CD8+ subpopulations were transfected equally and the anti-CD19-CIR expression was sustained for a minimum of 3 days. T cells transfected with anti-CD19-CIR effectively lysed CD40L expanded autologous or allogeneic B cell lymphoblasts and multiple B-cell lymphoma cell lines (Daudi, SKI-DLCL-1 and CRL-2261). T cells expressing low, medium or high levels of anti-CD19-CIR effectively lysed the targets regardless of the level of expression. In vivo, anti-CD19-CIR transfected T cells were able to treat established tumors as demonstrated by non-invasive serial imaging of luciferase-mediated bioluminescence (Figure). Our data provide a rationale for developing methods for large-scale electroporation of chimeric receptors in CTLs, in support of clinical application of this treatment modality in patients with high-risk B-cell malignancies. Figure: In vivo activity of Anti-CD19-CIR transfectedCTLs.A) Pseudocolorimage representing light intensity and anatomic localization of the ffLuc-derived lymphoma signal in two representative mice. B) Longitudinal monitoring of the bioluminescent signals of ffLuc+ Daudi cells in NOD/scid mice. Points, mean photon flux (in p/s/cm2/sr); bars, SE. Figure:. In vivo activity of Anti-CD19-CIR transfectedCTLs.A) Pseudocolorimage representing light intensity and anatomic localization of the ffLuc-derived lymphoma signal in two representative mice. B) Longitudinal monitoring of the bioluminescent signals of ffLuc+ Daudi cells in NOD/scid mice. Points, mean photon flux (in p/s/cm2/sr); bars, SE.

Severe T cell depletion required for allogeneic hematopoietic stem cell transplantation from haplo-identical donors results in poor immune reconstitution and leads to high rates of mortality from infections, and relapse. One approach to overcome this problem is to infuse T cells depleted of alloreactivity. Selective depletion (SD) of alloreactive T cells is achieved by elimination of activated T cells after ex-vivo stimulation with recipient cells. To determine optimum selective depletion conditions, we have investigated the factors that modify alloreactivity of T cells. Methods: Alloreactivity was measured by one-way mixed lymphocyte reaction (MLR) using 3H thymidine uptake. PBMCs were used as responders and either irradiated expanded T cells (expT) or dendritic cells (DCs) as stimulators. T cells were expanded using anti-CD3 coated beads. DCs were generated from monocytes by GM-CSF and IL-4 stimulation. Selective depletion was performed by co-incubation of responder and stimulator cells for 72 hours and depletion of activated cells by an immunotoxin, LMB-2 (Anti-Tac (Fv)-PE-38), which was added to the culture at 24 and 48 hours. Effectiveness of the depletion was tested by a secondary MLR utilizing the original stimulator cells and unrelated third part cells. Results: Expansion of T cells has resulted in increase of HLA-DR, CD80 and CD86 expression compared to resting T cells (52.5% vs. 6%, 20.9% vs. 0.9%, and 32.9% vs. 20.9%, respectively), resulting in better stimulation in MLR (6505 cpm vs 1620 cpm). In one-way MLR using either PBMCs or CD25 depleted PBMCs as responders and expanded T cells and DCs as targets, with or without anti-CD28 in the culture media. DCs were better stimulators than expT cells (6636 vs. 4308). However, most dramatic effect was seen when anti-CD28 was added to the culture, increasing response to both expT cells and to a lesser extent DCs (40,169 and 19,303). Removal of CD25 positive cells also improved alloreactivity in all culture conditions (6636 in expT, 16,644 in DC, 57,363 in expT+CD28, and 30,943 in DC+CD28). To better define the effect of the target, we have performed Vbeta repertoire analysis of responding cells after expT cell, DC and expT cell+anti-CD28 stimulation. Flow cytometry revealed expansion of discrete Vbeta families, in addition to shared ones. We have then performed selective depletion using PBMCs or CD 25 depleted PBMCs as stimulators and expT cells, expT cell+anti-CD28, and DCs as stimulators. Residual alloreactivity after expT cell stimulation against original stimulators, DCs and third party cells were 7%, 147% and 99% respectively. Interestingly, after SD utilizing DCs as stimulators, there was substantial residual activity against expT cells (69%). When SD was performed using expT cells as stimulators with anti-CD28, combined with CD25 depletion, the depletion against both original stimulators and DCs was improved (2% and 54%, respectively). Conclusion: Depletion of regulatory T cells, and co-stimulation with ant-CD28 improves alloreactivity and selective depletion. Whether improvement in in-vitro selective depletion will result in better clinical outcome will be tested in a clinical trial.