**3. Results**

#### **3.1. Expansion of cells in serum-containing and serum-free medium**

CD34+ progenitors were cultured in Teflon bags using serum containing DMEM/10%FCS and serum-free CellGro SCGM/DC medium supplemented with cytokines as described above. Table 1 shows cell expansion and viability on day14. The total cell expansion is 7.9±0.8 fold for CellGro/SCGM/DC medium and 8.3±0.6 fold for DMEM/FCS medium. Both conditions gave high cell viability, but when human albumin was added to the serum-free medium, lower cell expansion and viability was observed.

CD34+ cells proliferate and differentiate very rapidly during the first week, while in the remaining culture period only a minor expansion took place. When different cell concentrations of CD34+ cells were seeded and cultured for 14 days, a cell concentration of 1x105 cells per ml kept through the culture period gave optimal growth conditions. Lower initial cell concentrations (1x104/ml) in the cultures gave no growth advantages (data not shown).

#### **3.2. Yield and phenotypes of immature and mature DCs**

Cultured cells lost their CD34 marker rapidly and no CD34 positive cells could be detected after 7 days of culturing. At day 14, DC purity was 35.9 ± 7.7 % as assessed by expression of CD86 and HLA DR antigens. Figure 2 (A and B) shows the phenotype of DCs in serum free medium on day 14 and 17. The CD86, CD83 and CD80 were up regulated greatly during maturation and the phenotypic profile obtained was comparable to DCs cultured in serum containing medium.

Different transfection parameters with regard to voltages and time of exposure were tested and the most suitable protocol was found to be 500V at 2ms. By the use of these parameters a transfection efficacy of >95% could be achieved and the mean fluorescence levels using EGFP (Enhanced green fluorescent protein) were increased to about 100-fold above background (figure 3). The percentages of surviving DC following mRNA tranfection using propidium iodide staining were 76%. A similar survival was obtained in the mock-transfected DC.

#### **3.3. T-cell responses to thawed transfected DCs**

To assess the function of matured DCs we use transfected DCs to stimulate autologous T cells four times weekly. Thereafter the T cells were tested in the ELISPOT assay, which give information of both transfection efficacy, processing and antigen stimulation capacity of transfected DCs. As shown in figure 4, after four times stimulation by thawed transfected DCs in vitro, a significant and specific T-cell response to transfected DCs as compared to the control experiment employing mock-transfected DCs was achieved.

Is Anticancer Vaccine Possible: Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched CD34+ Blood Progenitor Cells 83

82 Immunodeficiency

**3. Results** 

PBS/0.05% Tween and one additional time with PBS alone. Then, after adding 75 μl of substrate BCIP/NBT (Sigma B911) to each well the plate was incubated for 4-5 minutes. When spots appeared, water was added to stop the reaction. The number of spots per well was

CD34+ progenitors were cultured in Teflon bags using serum containing DMEM/10%FCS and serum-free CellGro SCGM/DC medium supplemented with cytokines as described above. Table 1 shows cell expansion and viability on day14. The total cell expansion is 7.9±0.8 fold for CellGro/SCGM/DC medium and 8.3±0.6 fold for DMEM/FCS medium. Both conditions gave high cell viability, but when human albumin was added to the serum-free

CD34+ cells proliferate and differentiate very rapidly during the first week, while in the remaining culture period only a minor expansion took place. When different cell concentrations of CD34+ cells were seeded and cultured for 14 days, a cell concentration of 1x105 cells per ml kept through the culture period gave optimal growth conditions. Lower initial cell

Cultured cells lost their CD34 marker rapidly and no CD34 positive cells could be detected after 7 days of culturing. At day 14, DC purity was 35.9 ± 7.7 % as assessed by expression of CD86 and HLA DR antigens. Figure 2 (A and B) shows the phenotype of DCs in serum free medium on day 14 and 17. The CD86, CD83 and CD80 were up regulated greatly during maturation and the phenotypic profile obtained was comparable to DCs cultured in serum containing medium. Different transfection parameters with regard to voltages and time of exposure were tested and the most suitable protocol was found to be 500V at 2ms. By the use of these parameters a transfection efficacy of >95% could be achieved and the mean fluorescence levels using EGFP (Enhanced green fluorescent protein) were increased to about 100-fold above background (figure 3). The percentages of surviving DC following mRNA tranfection using propidium iodide staining were 76%. A similar survival was obtained in the mock-transfected DC.

To assess the function of matured DCs we use transfected DCs to stimulate autologous T cells four times weekly. Thereafter the T cells were tested in the ELISPOT assay, which give information of both transfection efficacy, processing and antigen stimulation capacity of transfected DCs. As shown in figure 4, after four times stimulation by thawed transfected DCs in vitro, a significant and specific T-cell response to transfected DCs as compared to the

concentrations (1x104/ml) in the cultures gave no growth advantages (data not shown).

counted under a stereomicroscope and the frequency of reactive T cells was calculated.

**3.1. Expansion of cells in serum-containing and serum-free medium** 

medium, lower cell expansion and viability was observed.

**3.2. Yield and phenotypes of immature and mature DCs** 

**3.3. T-cell responses to thawed transfected DCs** 

control experiment employing mock-transfected DCs was achieved.

**Figure 3.** Immunophenotyping profile of: A) Immature DC; B) Mature DC generated from enriched monocytes. Overlay histograms show the expression of relevant antigens of immature (blue) and mature DC (green) versus isotype-matched control (red). The percentage of positive cells and mean fluorescence intensity value is shown too.

Is Anticancer Vaccine Possible: Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched CD34+ Blood Progenitor Cells 85

because of the danger of disease transmission but also because immune responses against FCS might result in high background responses obscuring the specific T cell immunity. Other studies have indicated that human serum may inhibit DC differentiation and therefore seems not to be a good alternative to replace FCS (13). Serum free conditions have been tested previously (13,14). In these studies cell expansion and DC yield were very low though the phenotype of the DCs were considered not to be affected. In this study we have demonstrated that there is no difference between serum-free and serum-containing medium with regard to ex-vivo expansion of both the total number of cells and the estimated content of matured DCs in the cell products. The addition of serum albumin to our cultures did not

**Figure 5.** Autologous T cells stimulated with four times ex vivo irradiated transfected DC. ELISPOT assay indicates that stimulated T cells are able to recognize transfected DC specifically by use of mock-

GM-CSF, TNF-α and IL-4 are cytokines that play an import role in DC differentiation (19,20) when serum-containing medium is being employed. However, our experiences and that of others indicate that this cytokine combination alone is not sufficient for ex vivo expansion of DCs from CD34+ cells. As described by others, ex-vivo culturing of CD34+ cells in the presence of SCF and Flt-3 (21) give an efficient expansion of total cell numbers without interfering with DC development. Since these early acting factors does not affect DC differentiation, but sustained the long-term expansion of CFU-DC, we chose to add them to our cultures. In contrast to monocyte- derived DCs, ex-vivo expansion of CD34 derived DCs usually occurred asynchronously over a 2 to 3 weeks period. Since INF-α can efficiently accelerate the course of maturation (22) we also included this cytokine in the cocktail the last 3 days of culture. This resulted in an up regulation of the maturation antigens CD86, CD83

result in any growth advantages.

transfected DC as conrol

and CD80.

**Figure 4.** Transfection efficacy of mRNA demonstrated by utilized EGFP mRNA (lower part) compared to non-transfected DC (upper part). Left panels are density plots indicating large cell population gated. Middle panels are density plots showing the viability of cells (non-transfected and EGFP mRNA transfected by application of propidium iodide PI stating FL3). Right panels are histogram plots showing GFP signal in living cells. The green fluorescence intensity is increased about 100-fold in transfected cells than in non-transfected cells.

#### **4. Discussion**

In our hospital, enriched CD34+ stem cells are routinely being prepared and used as progenitor stem-cell support to patients receiving high dose therapy (16). In the cases that such patients also are candidates for DC-based vaccine treatment, spared frozen CD34+ cells would be available as a source for DC production thereby avoiding new expensive procedures for production of monocyte-derived DCs (18).

The present study describes the establishment of a clinical ex- vivo culture system for expansion of mature DCs derived from CD34+ cells employing VueLifeTM FEP Teflon bags and serum free CellGro/SCGM/CellGro. Most methods applied for production of DC include FCS or pooled human serum. As a foreign protein, FCS is highly unwanted not only because of the danger of disease transmission but also because immune responses against FCS might result in high background responses obscuring the specific T cell immunity. Other studies have indicated that human serum may inhibit DC differentiation and therefore seems not to be a good alternative to replace FCS (13). Serum free conditions have been tested previously (13,14). In these studies cell expansion and DC yield were very low though the phenotype of the DCs were considered not to be affected. In this study we have demonstrated that there is no difference between serum-free and serum-containing medium with regard to ex-vivo expansion of both the total number of cells and the estimated content of matured DCs in the cell products. The addition of serum albumin to our cultures did not result in any growth advantages.

84 Immunodeficiency

**Figure 4.** Transfection efficacy of mRNA demonstrated by utilized EGFP mRNA (lower part) compared to non-transfected DC (upper part). Left panels are density plots indicating large cell population gated. Middle panels are density plots showing the viability of cells (non-transfected and EGFP mRNA transfected by application of propidium iodide PI stating FL3). Right panels are histogram plots showing GFP signal in living cells. The green fluorescence intensity is increased about 100-fold in

In our hospital, enriched CD34+ stem cells are routinely being prepared and used as progenitor stem-cell support to patients receiving high dose therapy (16). In the cases that such patients also are candidates for DC-based vaccine treatment, spared frozen CD34+ cells would be available as a source for DC production thereby avoiding new expensive

The present study describes the establishment of a clinical ex- vivo culture system for expansion of mature DCs derived from CD34+ cells employing VueLifeTM FEP Teflon bags and serum free CellGro/SCGM/CellGro. Most methods applied for production of DC include FCS or pooled human serum. As a foreign protein, FCS is highly unwanted not only

transfected cells than in non-transfected cells.

procedures for production of monocyte-derived DCs (18).

**4. Discussion** 

**Figure 5.** Autologous T cells stimulated with four times ex vivo irradiated transfected DC. ELISPOT assay indicates that stimulated T cells are able to recognize transfected DC specifically by use of mocktransfected DC as conrol

GM-CSF, TNF-α and IL-4 are cytokines that play an import role in DC differentiation (19,20) when serum-containing medium is being employed. However, our experiences and that of others indicate that this cytokine combination alone is not sufficient for ex vivo expansion of DCs from CD34+ cells. As described by others, ex-vivo culturing of CD34+ cells in the presence of SCF and Flt-3 (21) give an efficient expansion of total cell numbers without interfering with DC development. Since these early acting factors does not affect DC differentiation, but sustained the long-term expansion of CFU-DC, we chose to add them to our cultures. In contrast to monocyte- derived DCs, ex-vivo expansion of CD34 derived DCs usually occurred asynchronously over a 2 to 3 weeks period. Since INF-α can efficiently accelerate the course of maturation (22) we also included this cytokine in the cocktail the last 3 days of culture. This resulted in an up regulation of the maturation antigens CD86, CD83 and CD80.

The use of gas permeable bags for ex-vivo production has several advantages when compared to production in culture flasks. The bag system is closed and reduces the risk of contamination. DCs produced in Teflon bags do not attach to the surface and can easily be concentrated by centrifugation without any extra steps. It also facilitates large-scale production, which can be divided into aliquots containing cells with identical properties. We have shown that DCs can efficiently be produced in suspension using gas permeable Teflon bags. When CD34+ progenitors are cultured in flasks, usually the cell concentration is 104/ml. In our system we have shown that optimal cell concentration is 105/ml, which give a 10-fold reduction in the amount of medium and cytokines used.

Is Anticancer Vaccine Possible: Experimental Application of Produced mRNA Transfected Dendritic Cells Derived from Enriched CD34+ Blood Progenitor Cells 87

**Author details** 

**Acknowledgement** 

**5. References** 

*392:245.*

*121: 240-250*

Corresponding Author

 \*

Paula Lazarova1,2,\*, Gunnar Kvalheim1, Krassimir Metodiev3

preparation of frozen CD34+ progenitor cells.

*Immunological Methods 223:1-15*

*2Clinical laboratory, District Hospital "St. Anna", Varna, Bulgaria,* 

ready for clinical use. *J. Immunological Methods 245:15-*

peripheral blood in lymphoma patients. *Cytotherapy 2:95-104*

dendritic cells: immunological and clinical aspects.

medium. *Clinical & Exp. Immunology, 125*

*1Dept. Cellular Therapy, University Rikshospital-Radiumhospital, Oslo, Norway,* 

We warmly thank the staffs in stem cell laboratory (Radium-Oslo-Norway) for the

[1] Banchereau J. and R.M. Steinman. 1998. Dedritic cells and control of immunity. *Nature* 

[2] Banchereau J., F.Briere, C. Caux, J. Davoust, S. Lebecue, Y. Liu, B. Pulendran and K.

[3] Thurner B., C. Roder, D. Dieckmann, M. Heuer, M. Kruse, A. Glaser, P. Keikavoussi, E. Kampfen, A. Bender, G. Schuler. 1999. Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. *J.* 

[4] Feuerstein B., T.Berger, C. Maczek, C. Roder, D. Schreiner, U. Hirsch, I. Haendle, W. Leisgang, A. Glaser, O. Kuss, T. Diepgen, G. Schuler, B. Thurner. 2000. A method for the production of cryopreserved aliquots of antigen- preloaded, mature dendritic cells

[5] Maria R. Motta, M. Castellani, S. Rizzi, A. Curti, F. Gubinelli, M. Fogli, E. Ferri, C. Cellini, M. Baccarani and R. Lemoli. 2003 Generation of dendritic cells from CD14+ monocytes positively selected by immunomagnetic adsorption for multiple myeloma patients enrolled in a clinical trial of anti- idiotype vaccination. *British J. Haematology,* 

[6] Ramadan G., R. Schmidt and J. Schubert. 2001 In vitro generation of human CD86+ dendritic cells from CD34+ haematopoietic progenitors by PMA and in serum- free

[7] Strunk D., K. Rappersberger, K. Egger, et al. 1996. Generation of human dendritic cells/ Langerhans cells from circulating CD34+ hematopoietic progenitor cells. *Blood 87:1292* [8] Enomoto M., H Nagayama, K Sato, Y Xu, S Asano and TA Takahashi. 2000. In vitro generation of dendritic cells derived from cryopreserved CD34+ cells mobilized into

[9] Titzer S., O. Christensen, O. Manzke, H. Tesch, J. Wolf, B. Emmerich, C. Carsten, V. Diehl and H. Bohlen. Vaccination of multiple myeloma patients with idiotype-pulsed

Palucka. 2000. Immunobiology of dendritic cells. *Annu. Rev. Immunol. 18:767*

*3Dept. Preclinical & Clinical Sciences, Medical University, Varna, Bulgaria* 

DCs have been loaded with several antigens, such as tumor lysate, peptides, proteins, DNA and mRNA. As a source of antigens, the major limitation of using lysate, proteins or peptides isolated from patients' tumor cells is the amount of tumor tissue or the purity of the tumor specimens. The use of nucleic acids, either DNA or RNA, would overcome this practical limitation. As mRNA is a safer alternative due to its limited ability to cause permanent genetic alterations in the host, it appears to be more attractive to be used than DNA transfection (23). For this purpose, a vector-free transfection system based on square-wave electroporation to transfer mRNA into DCs has been developed (17). This method is currently successfully used in the clinic to produce mRNA-transfected monocyte-derived DCs (15). We here demonstrate that this method also resulted in efficient transfection of mRNA into immature CD34+ cell derived DCs without significantly affecting the survival of the cells.

Generally the antigen-stimulating capacity of DCs has been evaluated employing alloreactive T cells or responses against recall antigens. We have used priming of autologous T cell against antigens encoded by a prostate tumor cell line in order to evaluate the immunostimulatory role of the transfected DCs. The ELISPOT assay was used to detect and quantify of single T lymphocyte forming cytokine spots after antigen contact in vitro. The ELISPOT assay is a more stringent system for testing both the efficacy of transfection and the processing and antigen-stimulating capacity of transfected DCs. Our results show that CD34+ cells derived DCs, grown in serum free conditions in clinical scale productions reproducibly are capable of inducing a tumor specific immune response. These results are similar to what was seen in our previous study using monocyte-derived DCs

The finding and results from the present study allows us to proceed with a clinical protocol for application of CD34+ derived DCs for cancer vaccine. Possible attractive candidates for such an approach are relapsed Hodgkin's patients (16) and other patients that have previously been treated with auto transplantation and with spared frozen samples of CD34+ cells.


**Table 1.** Expansion fold and viability on Day 14
