AU2007258279B2 - Co-culture lymphoid tissue equivalent (LTE) for an artificial immune system (AIS) - Google Patents

Abstract

The present invention relates to methods for preparing an artificial immune system. The artificial immune system comprises a cell culture comprising T cells, B cells and antigen-primed dendritic cells. The artificial immune system of the present invention can be used for in vitro testing of vaccines, adjuvants, immunotherapy candidates, cosmetics, drugs, biologies and other chemicals.

Description

WO 2007/146334 PCT/US2007/013871 TITLE CO-CULTURE.LYMPHOID TISSUE EQUIVALENT (LTE) FOR AN ARTIFICIAL IMMUNE SYSTEM (AIS) 5 CROSS REFERENCE TO RELATED CASES This application is a continuation-in-part of U.S. Application Serial No. 11/116,234, filed April 28, 2005, which claims the benefit of priority of U.S. Provisional Application Serial No. 60/565,846, filed April 28, 2004 and 60/643,175, filed January 13, 10 2005. This application also claims the benefit of priority of International Application No. PCT/US2005/014444, filed April 28, 2005. Each of these applications is hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION 15 Field of the Invention The present invention is directed to a method for constructing an integrated artificial human tissue construct system and, in particular, construction of an integrated human immune system for in vitro testing of vaccines, adjuvants, immunotherapy candidates, cosmetics, drugs, biologics, and other chemicals. The artificial immune system 20 of the present invention is useful for assessing the interaction of substances with the immune system, and thus can be used to accelerate and improve the accuracy and predictability of, for example, vaccine, drug, biologic, immunotherapy, cosmetic, and chemical development.

WO 2007/146334 PCT/US2007/013871 Backgrouad of the Technology Despite the advent and promise of recent technologies, including combinatorial chemistry, high-throughput screening, genomics, and proteomics, the number of new drugs 5 and vaccines reaching the market has not increased. In fact, the attrition rate within drug discovery programs exceeds 90%. The introduction of these new (and expensive) technologies has not reduced the lost opportunity costs associated with immunotherapy development; rather, these costs have increased. Indeed, it is now estimated that almost $1 billion is required to bring a 10 new drug to the market. The development and biological testing of human vaccines has traditionally relied on small animal models (e.g., mouse and rabbit models) and then non-human primate models. However, such small animal models are expensive and non-human primate models are both expensive and precious. Furthermore, there are many issues regarding the 15 value of such animal studies in predicting outcomes in human studies. A major problem remains the translation from test systems to human immunology. Successful transfer between traditional testing systems and human biology requires an intricate understanding of disease pathogenesis and immunological responses at all levels. Given worldwide health problems caused by known and emerging infectious agents and 20 even potential biological warfare pathogens, it is time for a fresh approach to understanding disease pathogenesis, the development and rapid testing of vaccines, and insights gathered from such work. 2 WO 2007/146334 PCT/US2007/013871 The body's distributed immune system can be roughly divided into four distinct compartments: tissues and blood, mucosal tissues, body cavities, and skin. Because of ease of study, most is known about the tissue and blood compartment and its lymphoid tissues, the spleen and lymph nodes. 5 The mammalian immune system uses two general adaptive mechanisms to protect the body against environmental pathogens. When a pathogen-derived molecule is encountered, the immune response becomes activated to ensure protection against that pathogenic organism. The first immune system mechanism is the non-specific (or innate) inflammatory 10 response. The innate immune system appears to recognize specific molecules that are present on pathogens but not within the body itself. The second immune system mechanism is the specific or acquired (or adaptive) immune response. Innate responses are fundamentally the same for each injury or infection; in contrast, acquired responses are custom-tailored to the pathogen in question. 15 The acquired immune system evolves a specific immunoglobulin (antibody) response to many different molecules, or antigens, derived from the pathogen. In addition, a large repertoire of T cell receptors (TCR) is sampled for their ability to bind processed peptides from the antigens that are bound by major histocompatibility complex (MHC) class I and II proteins on the surface of antigen-presenting cells (APCs), such as dendritic cells (DCs). 20 Acquired immunity is mediated by specialized immune cells called B and T lymphocytes (or simply B and T cells). Acquired immunity has specific memory for specific antigens; repeated exposure to the same antigen increases the memory response, which increases the level of induced protection against that particular pathogen. 3 WO 2007/146334 PCT/US2007/013871 Bcells produce and mediate their functions through the actions of antibodies. B cell-dependent immune responses are referred to as "humoral immunity" because antibodies are found in body fluids. T cell-dependent immune responses are referred to as "cell-mediated immunity," 5 because effector activities are mediated directly by the local actions of effector T cells. The local actions of effector T cells are amplified through synergistic interactions between T cells and secondary effector cells, such as activated macrophages. The result is that the pathogen is killed and prevented from causing diseases. The functional element of a mammalian lymph node is the follicle, which develops 10 a germinal center (GC) when stimulated by an antigen. The GC is an active area within a lymph node, where important interactions occur in the development of an effective humoral immune response. Upon antigen stimulation, follicles are replicated and an active human lymph node may have dozens of active follicles, with functioning GCs. Interactions between B cells, T cells, and FDCs take place in GCs. 15 Various studies of GCs in vivo indicate that the many important events occur there, including immunoglobulin (Ig) class switching, rapid B cell proliferation (GC dark zone), production of B memory cells, accumulation of select populations of antigen-specific T cells and B cells, hypermutation, selection of somatically mutated B cells with high affinity receptors, apoptosis of low affinity B cells, affinity maturation, induction of secondary 20 antibody responses, and regulation of serum immunoglobulin G (IgG) with high affinity antibodies. Similarly, data from in vitro GC models indicate that FDCs are involved in stimulating B cell proliferation with mitogens and it can also be demonstrated with antigen (Ag), promoting production of antibodies including recall antibody responses, producing 4 WO 2007/146334 PCT/US2007/013871 chemokines that attract B cells and certain populations of T cells, and blocking apoptosis of B cells. Similar to pathogens, vaccines function by initiating an innate immune response at the vaccination site and activating antigen-specific T and B cells that can give rise to long 5 term memory cells in secondary lymphoid tissues. The precise interactions of the vaccine with cells at the vaccination site and with T and B cells of the lymphoid tissues are important to the ultimate success of the vaccine. Almost all vaccines to infectious organisms were and continue to be developed through the classical approach of generating an attenuated or inactivated pathogen as the 10 vaccine itself. This approach, however, fails to take advantage of the recent explosion in our mechanistic understanding of immunity. Rather, it remains an empirical approach that consists of making variants of the pathogen and testing them for efficacy in non-human animal models. Advances in the design, creation and testing of more sophisticated vaccines have 15 been stalled for several reasons. First, only a small number of vaccines can be tested in humans, because, understandably, there is little societal tolerance for harmful side effects in healthy people, especially children, exposed to experimental vaccines. With the exception of cancer vaccine trials, this greatly limits the innovation that can be allowed in the real world of human clinical trials. Second, it remains challenging to predict which 20 immunodominant epitopes are optimal for induction of effective CD4+ and CD8' T cell responses and neutralizing B cell responses. Third, small animal testing, followed by primate trials, has been the mainstay of vaccine development; such approaches are limited by intrinsic differences between human and non-human species, and ethical and cost 5 WO 2007/146334 PCT/US2007/013871 considerations that restrict the use of non-human primates. Consequently, there has been a slow translation of basic knowledge to the clinic, but equally important, a slow advance in the understanding of human immunity in vivo. The artificial immune system (AIS) of the present invention can be used to address 5 this inability to test many novel vaccines in human trials by instead using human tissues and cells in vitro. The AIS enables rapid vaccine assessment in an in vitro model of human immunity. The AIS provides an additional model for testing vaccines in addition to the currently used animal models. Attempts have been made in modulating the immune system. See, for example, 10 U.S. Patent No. 6,835,550 BI, U.S. Patent No. 5,008,116, WO 2004/101773 Al, Suernatsu et al., [Nat Biotechnol, 22, 1539-1545, (2004)] and U.S. Patent Application No. 2003/0109042. Nevertheless, none of these publications describe or suggest an artificial (ex vivo) human cell-based, immune-responsive system comprising a vaccination site (VS) and a 15 lymphoid tissue equivalent (LTE). The present invention comprises such a system and its use in assessing the interaction of substances with the immune system. SUMMARY OF THE INVENTION The present invention is directed to artificial immune systems comprising cell 20 cultures of B cells, T cells and antigen-primed dendritic cells. The present invention is also directed to methods for detecting an immune response to an antigen using the cell cultures of the present invention. 6 H:\amt\Intenvoven\NRPortbh\DCC\AM1\6173730I.DOC10/04/2014 In one aspect, the present invention provides a cell culture comprising: T cells; B cells; and antigen-prepulsed dendritic cells. In another aspect, the present invention provides a method for testing an immune response to an antigen comprising: preparing a cell culture comprising: T cells; B cells; and serum-free cell culture media, wherein the T cells and B cells are present in an approximately 1:1 ratio; priming dendritic cells with an antigen; adding to the cell culture the antigen-primed dendritic cells; and analyzing the effect the antigen has on the T cells and/or B cells in the cell culture. 6A WO 2007/146334 PCT/US2007/013871 BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: Shows the detection of tetanus-specific antibody responses by ELISPOT and determination of the percentage of antigen-specific B cells using a 2D T and B cell co culture. 5 FIGURE 2: Depicts tetanus toxoid: B cell proliferation and comparison between PBMC and 2D T and B cell co-culture. FIGURE 3: Shows the flow cytometry data indicating B cell proliferation between 10 PBMC and 2D T and B cell co-culture for the same cell donor shown in Figure 2. Donor anti-TT ~64 pg/mL; plots are gated on CDI19+ Lymphocytes. FIGURE 4: Depicts tetanus toxoid-specific ELISPOT comparing PBMC to 2D T and B cell co-culture for the same cell donor shown in Figures 2 and 3. Left panel: PBMCs: 15 -6,200 B cells/well (determined by flow); 2D co-culture: -19,000 B cells/well (determined by flow). Right panel: PBMCs: -1,800 B cells/well (determined by flow); 2D co-culture: -5,800 B cells/well (determined by flow). FIGURE 5: Shows an in vitro system representative of the physiological state promotes 20 stronger B cell proliferative (Figure 5A) and tetanus toxoid-specific antibody responses(Figure 5B), using a 2D co-culture of T and B cells and TT-pulsed DCs. T, B, DC co-culture conditions for Figure 5B were as follows: T:B ratio 1:1; -500,000 7 WO 2007/146334 PCT/US2007/013871 lymphocytes; DC:T ratio: -1:60; X-VIVO media; cells harvested after 7 days; performed in duplicate. FIGURE 6: Depicts tetanus-specific antibody responses to a DTaP (diphtheria and 5 tetanus and acellular pertussis vaccine, adsorbed) vaccine and a simple tetanus toxoid Antigen, using a 2D co-culture of T and B cells and TT-pulsed DCs. The T, B, DC co culture conditions were as follows: -500,000 lymphocytes; T:B ratio =~1:1; DC:T ratio: -1:60; cells harvested after 7 days; X-VIVO media; donor -25 pg/mL anti-TT; performed in duplicate. 10 FIGURE 7: Shows the influence of vaccine versus antigen in a lymphoid tissue equivalent (LTE) for the same cell donor shown in Figure 6. The T, B, DC co-culture conditions were as follows: -500,000 lymphocytes; T:B ratio = -1:1; DC:T ratio: ~1:60; cells harvested after 7 days; X-VIVO media; donor -25 pg/mL anti-TT; performed in 15 duplicate. FIGURE 8: Depicts Strong B cell and T cell proliferative responses seen against C. albicans, associated with potent activation (HLA-DRhi', CD86"hi) of the dividing B cells using a 2D co-culture of T and B cells and TT-pulsed DCs. 20 FIGURE 9: C. albicans-specific ELISPOT data comparing TG-2D to PBMCs in which antigen-pulsed DCs were added to both. The figure shows specificity of the C. albican stimulated B cells demonstrated by ELIPSOT for the same donor in Figure 8. C. albicans 8 WO 2007/146334 PCT/US2007/013871 specific ELISPOT data comparing compares the 2D co-culture of T and B cells with PBMCs. ~100,000 cells plated per well; PBMCs: ~8,000 B cells per well (determined by flow); T-B 2D: -18,000 B cells per well (determined by flow). 5 FIGURE 10: Depicts antibody responses when some of the leukocytes are removed. FIGURE 11: Shows in vitro antigen-specific antibody response to influenza. Set-up: DCs were treated or untreated with H IN I (New Caledonia) influenza; 2D cultures of DCs and T and B cells were stimulated (or not) with 'soluble' HINI influenza. Results: 10 antigen-specific proliferation of T and B lymphocytes; generation of antigen-specific antibody secreting B lymphocytes (ELIspot data in figure); a synergistic effect fmm pulsing DCs and adding soluble antigen to the DC / T and B cell co-cultures in generation of influenza-specific antibody responses; co-culture appears superior to PBMC cultures. 15 FIGURE 12: Shows T and B cell proliferation induced by H INI influenza. DETAILED DESCRIPTION OF THE INVENTION The present invention concerns the development of accurate, predictive in vitro models to accelerate vaccine testing, allow collection of more informative data that will aid 20 in redesigning and optimizing vaccine formulations before animal or clinical trials, and raise the probability that a vaccine candidate will be successful in human trials, More specifically, the present invention comprises controlling the nature and state of the cells in 9 WO 2007/146334 PCT/US2007/013871 the lymphoid tissue equivalent (LTE, artificial lymph node) of the artificial immune system (AIS). The AIS can be used to test vaccines and other pharmaceuticals for immune reactivity in a manner that is more predictive than animal experiments. Consequently, it 5 can provide valuable pre-clinical data earlier in the research and development process. Antigenic molecules introduced to the AIS are acquired by dendritic cells (DCs) at the vaccination site (VS). The DCs are then transferred to the lymphoid tissue equivalent (LTE), where they present the antigen to T cells, activating their immune function. Activated helper T cells co-stimulate B cells to induce antibody production, while 10 activated cytotoxic T cells lyse antigen-bearing cells. Solubilized antigen(s) can also be introduced into the LTE to directly activate B cells for subsequent antibody production. While a number of published reports have demonstrated antigen-specific B cell responses (to C. albicans, TT, and other antigens) in vitro, these results are typically achieved by stimulating and restimulating cultures of whole PBMCs with antigen and 15 exogenous factors to boost B cell proliferation and/or activation. The present invention comprises the detection of antibody responses using defined cultures of B cells, T cells, and DCs and optionally follicular dendritic cells (FDCs), in 2 dimensional construct assay. The presence of secondary cells provides a more physiological environment for B cell activation and differentiation, such that artificial 20 factors in the cultures are not necessary to detect specific antibody responses. Using embodiments of the present invention, we have generated antigen-specific B cell responses using a 2-dimensional (2D) co-culture system comprising T cells, B cells, and antigen-pulsed DCs. In the examples, responses were generated against tetanus toxoid 10 WO 2007/146334 PCT/US2007/013871 (TT) and a whole protein extract of Candida albicans (C. albicans). The results from these examples show that culturing human T and B cells together in vitro at a -1:1 ratio, versus the ratio of T and B cells naturally found in the blood, gave stronger antigen responses, by both analysis of activation and proliferation (flow cytometry) and antibody production 5 (ELISPOT). Although the preferred ratio of T cells:B cells is -1:1, the ratio of T cells:B cells can range from 1:10 to -10:1. In the cultures of the examples, "T cells" included both CD4* and CD8' T cells. In peripheral blood, the T (total T cells):B cell ratio is -7:1. In the lymph node, the T (total T cells):B cell ratio is -1:1.6. In the germinal center, the T cell:B cell ratio is -1:8, and there the T cells are primarily CD4* T cells. 10 In the results of the experiments shown, engineered serum-free media (X-VIVO) was used, though we have also used serum (e.g., human, bovine) in other experiments (data not shown). Dendritic cells (DCs) were generated from CD14-purified monocytes that were cultured for -7 days in X-VIVO 15 media, supplemented with GM-CSF (-100 ng/ml) and IL4 (-25 ng/ml). The cytokine-derived DCs were pulsed with antigen 15 or vaccine and then cocultured with T and B cells. After adding the antigen-prepulsed dendritic cells to the cell culture, further soluble antigen can also be added to the cell culture. For PBMC cultures, either the antigen was added to the assay, or antigen-pulsed DCs were added to the assay. In Figures 1 to 9, antigen-pulsed DCs were added to the co culture of T and B cells, while soluble antigen was added to the PBMC cultures. Figure 9 20 shows a comparison of the co-culture to PBMCs, with antigen-pulsed DCs added to both systems. 11 WO 2007/146334 PCT/US2007/013871 Examples These experiments provide a direct comparison of PBMCs versus a co-culture of negatively selected T and B cells that were plated at a -1:1 ratio in - in these examples - a 96-well, round bottom plate. All assays were harvested on day 7 of in vitro culture. All 5 experiments were analyzed by ELISPOT for antibody production and by flow cytometry for proliferation, as determined by loss of CFSE. In the ELISPOT assays because there were different ratios of T and B cells in the PBMC culture compared with the TB-2D cultures, there were fewer B cells plated into the ELISPOT wells. However, in the experiment in Figure 4, the numbers of B cells used in the ELISPOT experiments for both 10 the PBMC and co-culture assays were approximately equal. We determined the approximate number of B cells in the ELISPOT wells by flow cytometry to enable comparisons. These results show that culturing human T and B cells together in vitro at a -1:1 ratio compared to the ratio of T and B cells naturally found in the blood give stronger 15 antigen responses, by both analysis of activation and proliferation (flow cytometry) and antibody production (ELISPOT). Example . B and T cell co-culture with tetanus toxoid, showing the ability to detect tetanus 20 specific antibody responses (Figure 1). Example 2a. 12 WO 2007/146334 PCT/US2007/013871 PBMC versus co-culture, using a tetanus toxoid antigen. Even though similar B cell proliferation responses were seen in PBMC and 2D T and B cell co-cultures (Figures 2, 3), an improved tetanus toxoid-specific antibody response was observed in a T and B cell co-culture LTE, as compared with PBMC cultures (Figure 4), 5 Example 2b. PBMC versus co-culture, using Candida albicans antigens. Figure 9 shows C. albicans-specific ELISPOT data, comparing TB-2D to PBMCs. In this experiment, DCs were pulsed with TT antigen only, but the ELISPOT was conducted on both TT- and C. 10 albicans-coated plates. Example 2c. PBMC versus co-culture (Figure 10). In this example we addressed the question of what happens if we take cells from an apparent "non-responder" and use only the GC cells 15 from the leukocytes. Note the response when some of the leukocytes are removed (Figure 10); non-responders in vitro now show an antibody response. Here, we used human CD4- T and B cells with FDCs and formed GCs in vitro and then examined whether IgG production could be obtained against a recall antigen. Specifically, we used tetanus toxoid (TT) in these experiments and isolated human B cells 20 and CD4* T cells from peripheral blood. We observed IgG recall responses using only the T cells, B cells, and FDCs that are typically found in GCs. In contrast, in the presence of PBL cells not normally in found in GCs, no antibody response was detectible in cells from some donors. These results show 13 WO 2007/146334 PCT/US2007/013871 that removing (not including) other cells, such NK cells, monocytes, and CD8' T cells, improved the IgG response. Example 3 5 In vitro system representative of the physiological state promotes higher B cell proliferative (Figure 5A) and tetanus toxoid-specific antibody responses (Figure 5B) following tetanus vaccination. The post tetanus toxoid experiment was conducted 5 weeks following vaccination. The tetanus antibody titer before vaccination was ~40ig/mL; after vaccination it was -30Opg/mL. T cells represent both CD4* and CD8* T cells. Peripheral 10 blood has a T:B ratio of-7:1 (total T cells). The lymph node has a T:B ratio of-1: 1.6 (total T cells). The germinal center has a T:B ratio of-1:8 (primarily CD4* T cells). Example 4 Use of a vaccine to elicit in vitro immune responses in a co-culture of T and B cells 15 (Figures 6 and 7). DCs were pulsed with the vaccine or the tetanus toxoid antigen and were then added to the co-culture of T and B cells. Tripedia* (diphtheria and tetanus toxoids and acellular pertussis vaccine, adsorbed; DTaP), for intramuscular use, is a sterile preparation of diphtheria and tetanus toxoids adsorbed, with acellular pertussis vaccine in an isotonic sodium chloride solution containing thimerosal (preservative) and sodium 20 phosphate (to control pH). After shaking, the vaccine is a homogeneous white suspension. Tripedia* vaccine is distributed by Aventis Pasteur Inc. The results in Figures 6 and 7 showed that there were no apparent adverse effects due to thimerosal, and that DTaP vaccine induced more T/B cell activation and antibody responses than TT alone. 14 WO 2007/146334 PCT/US2007/013871 Example 5 To detect antigen-specific antibody responses, we developed an ELISPOT approach to quantify B cell responses (antigen specificity) on a per cell basis. In this 5 example, T cells were cultured with B cells at a 1:1 ratio, with cytokine-derived DCs included at a DC:T and B (total) cell ratio of~-1:60. Soluble TT (~I pg/ml) or C. albicans (~10 g/ml) was included for the entire 7-day culture, while other wells received pokeweed mitogen (PWM; a strong, non-specific lymphocyte stimulator) for the final 3 days of the culture. 10 On the seventh day, the lymphocytes were examined for marker expression and CFSE profiles by flow cytometry and the frequency of TT and C. albican-specific B cells was calculated by ELISPOT. Briefly, -30x 103 total lymphocytes were plated in duplicate wells of an ELISPOT plate that had been pre-coated with TT, C. albicans, or anti immunoglobulin (Ig, to gauge total antibody production). 15 The cells were then serially diluted five times at a ~1:3 ratio and PWM was added to all wells to trigger antibody production. The cells were then incubated for -5 hr at 37*C in a 5% CO 2 incubator and washed away. Plate-bound antibody was detected using techniques similar to those required for ELISA. The results in Figure 8 demonstrate strong B cell and T cell proliferative responses 20 against C. albicans, associated with potent activation (HLA-DR t 'h, CD86O ) of the dividing B cells. Furthermore, a subset of the most divided B cells appears to have acquired a memory phenotype, indicated by increased CD27 expression. 15 WO 2007/146334 PCT/US2007/013871 The lack of a robust response against TT was consistent with the weak serum TT titer for this donor (-4tg/ml). As expected, PWM triggered potent T and B cell proliferative responses, though not as many divisions were seen as with specific antigen stimulation, likely because the cells were only cultured with the mitogen for 3 days. 5 The specificity of the C. albicans-stimulated B cells was demonstrated by ELIPSOT (Figure 9 [[2]]). This experiment suggests that a lx stimulation with C. albicans did give rise to a small population of antibody-producing cells (-0.2% of total B cells) that was not detected in untreated cultures or those stimulated with TT (left and middle wells). This discrepancy between the frequency of proliferating cells and C. 10 albicans-specific B cells detected by ELISPOT could be the result of several factors. A likely explanation is that we used a crude C. albicans whole antigen extract containing ~19% carbohydrates (by weight). While C. albicans polysaccharides are strong inducers of B cell responses, only protein antigen-specific responses would be detected in the ELISPOT assay. 15 Example 6 Tetanus-specific antibodies were detected in another ELISPOT experiment where the cell donor's serum anti-tetanus level was higher (63ig/ml), and DCs were cultivated in XVIVO- 15 medium. All other components, concentrations and ratios were left 20 unchanged, except that of the number of cells deposited per ELISPOT well was increased; the higher number used was -1x 10 cells/well. In this experiment, both TT- and C. albicans-specific antibodies were observed (up to 48 and 33 spots per well, respectively), although a high level of non-specific response, 16 H:\amt\Intenrvoven\NRPortbl\DCC\AMT6l64435_l.do-8/04/2014 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. 16A

Claims (14)

1. A cell culture comprising: T cells; B cells; and antigen-prepulsed dendritic cells.

2. The cell culture of claim 1, additionally comprising serum-free cell culture media.

3. The cell culture of claim 1 or claim 2, wherein the T cells and B cells are present in a ratio between approximately 1:10 and approximately 10:1.

4. The cell culture of any one of claims 1 to 3, further comprising follicular dendritic cells.

5. The cell culture of any one of claims 2 to 4, wherein said serum-free cell culture media is X-VIVO 15.

6. A method for testing an immune response to an antigen comprising: preparing a cell culture comprising: T cells; B cells; and serum-free cell culture media, wherein the T cells and B cells are present in an approximately 1:1 ratio; priming dendritic cells with an antigen; adding to the cell culture the antigen-primed dendritic cells; and analyzing the effect the antigen has on the T cells and/or B cells in the cell culture.

7. The method as claimed in claim 6, wherein said method additionally comprises adding soluble antigen to the cell culture. 17 i:\amt\lIntenvoven\NRPortbl\DCC\AMT\6164435_l.doc-8/04/2014

8. The method of claim 6 or claim 7, wherein said effect on the T cells and/or B cells is measured by measuring T cell and/or B cell activation.

9. The method of any one of claims 6 to 8, wherein said effect on the T cells and/or B cells is measured by measuring T cell and/or B cell proliferation.

10. The method of any one of claims 6 to 9, wherein said effect on the B cells is measured by measuring antibody production.

11. The method of any one of claims 6 to 10, wherein said antigen is selected from the group consisting of a vaccine, an adjuvant, an immunotherapy candidate, a cosmetic, a drug, a biologic, and a chemical compound.

12. The method of any one of claims 6 to 11, wherein the cell culture further comprises follicular dendritic cells.

13. The method of any one of claims 6 to 12, wherein said serum free cell culture media is X-VIVO 15.

14. The cell culture of any one of claims 1 to 5 or the method of any one of claims 6 to 13, substantially as herein described and with reference to any of the Examples and/or Figures. 18