TRANSINFECTED LYMPHOCYTES FOR ANTI-TUMOR THERAPY

Abstract

The present invention relates to lymphocytes, preferably that have been transinfected from dendritic cells with a bacterium, preferably CD4.sup.+ T cells, preferably Listeria monocytogenes, wherein said bacterium comprises a tumor peptide. It also relates to the use of lymphocytes for therapy and/or treatment of solid tumors, preferably melanoma, the kit or device which comprises for this purpose. Furthermore, it also refers to the transinfection method thereof.

Claims

1. Transinfected lymphocyte with a bacterium comprising a tumor antigen wherein the transinfected lymphocyte comprises the tumor antigen in the major histocompatibility complex I (MHC-I).

2. Lymphocyte according to claim 1 wherein the lymphocyte is CD4.sup.+ T cell and/or B cell.

3. Lymphocyte according to any of claim 1 or 2 wherein the bacterium is selected from the list consisting of: L. monocytogenes, Salmonella enterica, Mycobacterium bovis, Staphylococcus aureus and Escherichia coli.

4. Lymphocyte according to any of claims 1 to 3 wherein the transinfection is performed from an antigen presenting cell.

5. Lymphocyte according to claim 4 wherein the antigen presenting cell is a dendritic cell.

6. Lymphocyte according to any of claims 1 to 5 wherein the lymphocyte is heterologous or autologous.

7. Lymphocyte according to any of claims 1 to 6 wherein the lymphocyte is from a human.

8. Cell population comprising the lymphocyte according to any of claims 1 to 7.

9. Pharmaceutical composition comprising the lymphocyte according to any of claims 1 to 7 or the cell population according to claim 8.

10. Use of the lymphocyte according to any of claims 1 to 7 or of the cell population according to claim 8 for the manufacture of a medicament.

11. Use of the lymphocyte according to any of claims 1 to 7 or of the cell population according to claim 8 for the manufacture of a medicament for the prevention or treatment of tumor and/or stimulating the immune response against a tumor antigen.

12. Use according claim 11 wherein the tumor is selected from the list consisting of: melanoma, lymphoma, chronic lymphocytic leukemia, myeloma, breast, ovary, uterus, cervix, testis, prostate, colon, colorectal, pancreatic, stomach and gastrointestinal tumors, gastric cancer, liver tumor, kidney, bladder, mouth cancer, pharynx, larynx, esophagus, lung, thyroid, glioblastoma, glioma, sarcoma, encephalon, brain, neuroblastoma and marrow, head and neck blastoma, bone and connective tissue.

13. Kit or device comprising the lymphocyte according to any of claims 1 to 7 or the cell population according to claim 8.

14. Use of the kit or device according to claim 13 for prevention or treatment of tumor and/or stimulating the immune response against a tumor antigen.

15. Use according to claim 14 wherein the tumor is selected from the list consisting of: melanoma, lymphoma, chronic lymphocytic leukemia, myeloma, breast, ovary, uterus, cervix, testis, prostate, colon, colorectal, pancreatic, stomach and gastrointestinal tumors, gastric cancer, liver tumor, kidney, bladder, mouth cancer, pharynx, larynx, esophagus, lung, thyroid, glioblastoma, glioma, sarcoma, encephalon, brain, neuroblastoma and marrow, head and neck blastoma, bone and connective tissue.

16. In vitro method for transinfecting a lymphocyte comprising the following steps: a. Isolating an antigen-presenting cell and a lymphocyte from a biological sample; b. differentiating the antigen-presenting cell; c. infecting the antigen presenting cell of step (b) with a bacterium, where the bacterium comprises a tumor antigen; d. contacting the antigen-presenting cell infected in step (c) with the lymphocyte of step (a) at a temperature of 35-38? C. for 24-72 hours to transinfecting it.

17. Method of claim 16 wherein it further comprises a step (e) for the selection of the transinfected lymphocyte of step (d).

18. Method of claim 16 or 17 wherein the bacterium is selected from the list consisting of: L. monocytogenes, Salmonella enterica, Mycobacterium bovis, Staphylococcus aureus and Escherichia coli.

19. Method according to any of claims 16 to 18 wherein the antigen presenting cell is a dendritic cell.

20. Method according to any of claims 16 to 19 wherein the lymphocyte is CD4.sup.+ T cell and/or B cell.

21. Method according to any of claims 16 to 20 wherein the lymphocyte is autologous or heterologous.

22. Method according to any of claims 16 to 21 wherein in step (d) the temperature is 37? C. for 48 hours.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0062] FIG. 1. Transinfected CD4.sup.+ T cells (tiCD4.sup.+ T) present bacterial antigens via MHC-I to CD8.sup.+ T cells na?ve in vitro. a, CD4.sup.+ tiT transinfected with Listeria-WT (wild type strain; top panels) or Listeria-OVA (ovalbumin expressing strain; lower panels) were incubated with CD8.sup.+ na?ve T cells OT-I mice (which recognize a peptide of ovalbumin; OVAp-I 257-264; SIINFEKL (SEQ ID NO: 1) in the context of H-2K.sup.b). The histograms represent fluorescence of CD8.sup.+ T cells labeled with CellTrace? Violet. Cell division, a feature of activated cells, is observed as a leftward population (in each cell division dye is diluted). Gray filled histograms show fluorescence of nonactivated na?ve CD8.sup.+ T cells. Proliferation of CD8.sup.+ T cells was analyzed at 24 h, 48 and 72 after the contacts between CD4.sup.+ tiT/CD8.sup.+ cells. b, Histograms represent the expression of CD25 or CD69 of CD8.sup.+ T cells incubated with Listeria-WT (fine line) or Listeria-OVA (thick black line). Gray filled histograms show fluorescence of nonactivated na?ve CD8.sup.+ T cells. c, Histograms show the proliferation of CD8.sup.+ T cells activated with: anti-CD3 and CD28 antibodies, DCs loaded with soluble pOVA, DCs infected Listeria-OVA or tiCD4.sup.+ T cells transinfected with Listeria-OVA to day 2 or day 3. Note that the activation of CD8+ T cells is much greater when activated with tiCD4.sup.+ T transinfected with Listeria-OVA that when activated with DCs infected with Listeria-OVA. Histograms filled with gray show fluorescence of nonactivated CD8.sup.+ T cells. d, Histograms represent fluorescence (CellTrace? Violet) of CD8.sup.+ T cells from C57BL/6 WT mice or OT-I mice after conjugation with tiCD4.sup.+ transinfected with Listeria-OVA.

[0063] FIG. 2. tiCD4.sup.+ T cells process bacterial antigens. a, Proliferation of CD8.sup.+ T cells from mice OT-I, 2 or 3 days after incubation with CD4.sup.+ tiT transinfected with Listeria-WT or Listeria-OVA cells, which were captured from DCs loaded with the OVA peptide (OVAp-I). b, Proliferation of CD8.sup.+ T cells from mice OT-I incubated with CD4 fir cells (H-2K.sup.b? or H-2K.sup.b+) transinfected with Listeria-OVA captured from H-2K.sup.b+ DCs, or incubated with tiCD4.sup.+ T cells transinfected with Listeria-WT (gray). Note that these CD8.sup.+ T cells can only be activated when antigens are presented in H2K.sup.b (b+) haplotype of MHC-I. c, as in b, except transinfection, which is made from DCs that were H-2K.sup.b? (H-2K.sup.k/k), and the conjugates were allowed to form during 48 h.d, Expression of H-2K.sup.b/OVA (detected using a specific antibody) by tiCD4.sup.+ T cells (H-2K.sup.b+,black line, H-2K.sup.b?, gray) transinfected with Listeria-OVA that were captured by DCs from H-2K.sup.b?. e, f, Western blots showing the expression of Tap1 in na?ve CD4.sup.+ T cells, activated with DC loaded with soluble OVAp, or with tiCD4.sup.+ T cells transinfected with Listeria-OVA for 24 (E) or 48 h (F) after activation. ERK2 and laminB were used as controls. g, CD8.sup.+ T cell proliferation from mice OT-I, 3 or 4 days after conjugation with tiCD4.sup.+ T cells (from Tap1 KO mice, or its isogenic WT) transinfected with Listeria-OVA.

[0064] FIG. 3. tiCD4.sup.+ cells form immune synapses with na?ve CD8.sup.+ T cells. a, Histogram shows H-2K.sup.b expression in CD4.sup.+ T cells before (black line) and after transinfection (gray line). Filled gray histogram shows the fluorescence of the negative control. b, Expression of CD86 on CD4.sup.+ T cells before (black line) and after transinfection (gray line). Filled gray histogram shows the fluorescence of the negative control. c, Expression of H-2K.sup.b/OVAp-1 in tiCD4.sup.+ T cells transinfected with Listeria-OVA, 24 (line does not move) or 48 h (line moving right) post-transinfection and non-transinfected T cells (filled gray histogram). d and e, confocal images of tiCD4.sup.+ T cells transinfected with Listeria-WT (d) and Listeria-OVA (e) incubated with CD8.sup.+ na?ve T cells from OT-I mice for 1 h. It is shown CD3 fluorescence and actin. Bars represent 10 ?m. f, Relative specific cytotoxicity of CD8.sup.+ T effector cells (CTLs) activated by tiCD4.sup.+ T transinfected with Listeria-OVA (solid line) or splenocytes loaded with pOVA (dotted line). The specific cytotoxicity was measured using different ratios of EL-4 (target cells): CTLs (0.5:1, 1:1, 2:1, 5:1).

[0065] FIG. 4. tiCD4.sup.+ cells activate cytotoxic effector T cells in vivo. a, Histograms show fluorescence (CellTrace? Violet) of CD8.sup.+ CD45.1.sup.+ T cells injected in receptor mice (CD45.2), which were inoculated with tiT CD4.sup.+ cells transinfected with Listeria-WT or Listeria-OVA. 2?10.sup.6 tiCD4.sup.+ cells (left panels) or 5?10.sup.6 tiCD4.sup.+ cells (right panels). b, In vivo proliferation of CD8.sup.+ CD45.1.sup.+ T Cells, from OT-I mice, stained with CellTrace Violet injected in wild type C57BL/6 recipients (whose bone marrows have been reconstituted after irradiation with cells from H-2K.sup.k mice). CD8.sup.+ CD45.1.sup.+ T cells from OT-I mice (4?10.sup.6 cells/mouse) were transferred along with CD4.sup.+ T cells expressing H-2K.sup.b (right panels) or H-2K.sup.k (panels left) from AND mice (4?10.sup.6 cells/mouse) and MCC peptide (15 ?g/mouse). Mice also were infected with Listeria-OVA (10.sup.3 bacteria/mouse). 5 days after infection, spleens were isolated and proliferation of transferred CD8.sup.+ T cells was detected by flow cytometry (panels above). Panels below show CD8.sup.+ T cells transferred from OT-I mice (CD45.1.sup.+ CD4). c, (5?10.sup.6) B16 OVA melanoma cells (ovalbumin expressing) were injected subcutaneously into the right flank of recipient mice. All groups (9 mice/group) were inoculated with CD8.sup.+ na?ve T cells from OT-I mice (10.sup.3/mouse) together with phosphate buffered saline (PBS) in Group 1; 5?10.sup.5 tiCD4.sup.+ cells transinfected with Listeria WT, in Group 2; 5?10.sup.5 tiCD4.sup.+ transinfected with Listeria-OVA in group 3. The size of the tumors was monitored every two days (from day 5 to 13) and every 3 days thereafter.

[0066] FIG. 5. B cells capture bacteria by transinfection and cross-present bacterial antigens. a, Murine DC decorated + (or not ?) with Hen Egg Lysozyme (HEL) were infected with L. monocytogenes, and incubated with B cells from MD4 transgenic mice, whose B cells have B cell receptors (BCR) recognizing HEL, under conditions that allow the DC-B cell interactions (for 30 min), or in the presence of a polycarbonate barrier (transwell) which prevents such interactions. After formation of the conjugates, CD4.sup.+ T cells were re-isolated and conducted a classic gentamicin survival assay. CFU (colony forming unit; intracellular bacteria) are shown for each 50,000 CD4.sup.+ T cells. ? is direct infection of bacteria on B cells. b, DC infected with L. monocytogenes and decorated with HEL were incubated with B cells from MD4 mice. After formation of conjugates, B cells were re-isolated and it was quantified the survival (at different times) of intracellular bacteria by gentamicin resistance assays. Data were normalized to time 0. As control of bacterial fitness HeLa cells were infected in parallel. c, Expression of CD86 on B cells before (left panel; na?ve B cells) and one day after transinfection with Listeria-OVA (right panel). In both panels is shown IgM expression (FL-1 channel) vs expression of CD86 (channel FL-2). d, Proliferation of na?ve CD8.sup.+ T cells from OT-I mice labeled with CellTrace Violet, after 3 days of contact with tiB cells that have captured by transinfection Listeria-WT (top panels) or Listeria-OVA (below). Left panels show CellTrace Violet fluorescence vs CD25 expression (a marker of T cell activation). e, Specific relative cytotoxicity of CD8.sup.+ T cells (CTL) activated by tiB cells transinfected with Listeria-OVA (top line) or splenocytes decorated with OVAp-I peptide (bottom line). Different ratios of EL-4 (target cells):CTL were measured.

EXAMPLES

[0067] Below, the invention will be illustrated by assays performed by the inventors, which demonstrate the effectiveness of the product of the invention.

[0068] The fascinating discovery that T cells can capture bacteria from DCs infected through a process called transfection (Cruz-Adalia et al., 2014), prompted us to ask whether it was possible that transinfected T cells (ti) could act as true APCs. This was a risky question because, if true, it will break a dogma of immunology, the strict separation of roles between innate and adaptive immunity, and would be a huge advance in the basic understanding of how the immune system works.

[0069] To answer this question, we generated pure populations (by cell sorter) of CD4.sup.+ T cells transinfected (hereinafter called cells tiCD4.sup.+ T) with Listeria-OVA (L. monocytogenes expressing ovalbumin) or Listeria-WT (wild isogenic strain not expressing OVA) by using dendritic cells from bone marrow (BM-DCs) previously infected with the bacteria. The ability to present antigens of tiCD4.sup.+ T cells was tested by incubating them with na?ve CD8.sup.+ T cells isolated from transgenic OT-I mice. These CD8.sup.+ T cells recognize a peptide of ovalbumin (OVAp-I 257-264; SIINFEKL, SEQ ID NO: 1) in the H-2K.sup.b context. The flow cytometric analysis of CD8.sup.+ T cells stained with CellTrace? Violet shows a potent proliferation of CD8.sup.+ T cells that begins two days after contact with the tiCD4.sup.+ T cells (a movement to the left observed of the CellTrace? Violet fluorescence), but only in those CD8.sup.+ T cells that were in contact with CD4.sup.+ T cells transinfected with Listeria-OVA (FIG. 1a). This highly proliferative population expresses high levels of CD8 (CD8.sup.+ high) (FIG. 1a). However, when CD8.sup.+ T cells were incubated with tiCD4.sup.+ T cells transfected Listeria-WT they do not proliferate (FIG. 1a). According to these data, the expression of the activation markers CD69 and CD25 was detected only in CD8.sup.+ T cells incubated with tiCD4.sup.+ T cells transinfected with Listeria-OVA but not when incubated with tiCD4.sup.+ T cells transinfected with Listeria-WT (FIG. 1B). tiCD4.sup.+ T cells transinfected with Listeria-OVA induced the proliferation of CD8.sup.+ T cells in a more potent way than the induced by BM-DCs loaded with soluble OVA peptide, or even the proliferation induced by polyclonal activation with CD3/CD28 antibodies, and it was far more stronger than the produced by BM-DCs infected with Listeria-OVA (FIG. 1c). tiCD4.sup.+ T cells transinfected with Listeria-OVA on other hand are not able to induce proliferation of CD8.sup.+ T cells from wild mice, which do not recognize any OVA antigen (FIG. 1d), whereas they induce very potent proliferation of CD8.sup.+ T cells from OT-I mice (FIG. 1a, c, d), indicating that the observed effects are antigen-specific.

[0070] Taken together, these data suggest that tiCD4.sup.+ T cells are able to cross-present bacterial antigens to na?ve CD8.sup.+ T cells. At this point, we wondered to what extent tiCD4.sup.+ T cells could recapitulate different stages of the cross-presentation given in professional APC: endogenous antigen processing, antigen presentation via MHC-I, expression of co-stimulatory molecules, and interaction with target T cells through the formation of canonical immune synapses.

[0071] Antigen presentation of tiCD4.sup.+ T cells could be due to the capture of the MHC/antigen complexes from the surface of the DCs or by endogenous processing of the tiCD4.sup.+ cells themselves. To distinguish between these two options, CD4+ T cells were transinfected with Listeria-WT or Listeria-OVA from BM-DCs, previously loaded with the OVA peptide (OVAp/OT-I; cells that recognize CD8.sup.+ T cells from OT-I mice); later this tiCD4.sup.+ T cells (re-purified) were used to stimulate na?ve CD8.sup.+ T cells. In this configuration, antigen capturing from DCs cells would lead to a comparable activation of CD8.sup.+ T cells in both experimental conditions. However, it is observed that tiCD4.sup.+ cells transinfected with Listeria-OVA, but not with Listeria-WT induce a very strong activation of CD8.sup.+ T cells (FIG. 2a). Additional experiments using tiCD4.sup.+ T cells from transgenic mice expressing only the haplotype H-2K.sup.k (MHC-I), and therefore not able to stimulate CD8.sup.+ T cells from mice OT-I or their isogenic tiCD4.sup.+ T cells that express H-2K.sup.b, capturing both cell types bacteria from DCs expressing H-2K.sup.b, confirm that activation of CD8.sup.+ T cells is due in its vast majority by antigen processing inside tiCD4.sup.+ T cells (FIG. 2b).

[0072] A possible contribution to antigen presentation by the BM-DCs (eg contamination) was totally excluded by incubating CD8.sup.+ T cells from OT-I mice with CD4.sup.+ T cells (H-2Kb or H-2Kk) transinfected with Listeria-OVA from DCs expressing H-2K.sup.k only. Exclusively tiCD4.sup.+ T cells expressing H-2K.sup.b induced proliferation in CD8.sup.+ T cells (FIG. 2c) presenting OVA antigens in their MCH-I H-2K.sup.b molecules (FIG. 2d). Western blot analysis revealed in turn that CD4.sup.+ T cells activated and transinfected increase the expression of Tap1 (FIG. 2e, f), a protein involved in antigen presentation via MHC-I. Moreover, tiCD4.sup.+ T cells isolated from Tap1 KO mice showed very reduced capacity as cross-presenting cells after bacterial capture (FIG. 2g), i.e., the proliferation of CD8.sup.+ T cells was greatly reduced when antigen presenting cells were tiCD4.sup.+ from mice Tap1 KO compared to that observed when presenting cells were tiCD4.sup.+ from WT mice.

[0073] It was also found that after transinfection, tiCD4.sup.+ T cells increased the expression of MHC-I (H-2K.sup.b), H-2K.sup.b coupled to OVA antigen (of bacterial origin) and ligands of co-stimulatory molecules, such as CD86 (FIG. 3a-c), which supports a canonical antigen presentation on MHC-I. Activation of CD8.sup.+ T cells involved the formation of CD4.sup.+/CD8.sup.+ cell conjugates FIG. 1c), indicative of the formation of the immune synapse (IS), mark of T cell activation by APCs. The structure of a mature IS is composed of multimolecular concentric rings called supramolecular activation complexes (SMACs). Central SMACs (cSMAC) contain the MHC-TCR complex, and the peripheral zone (pSMAC) forms a ring structure containing adhesion molecules and F-actin (Saito and Batista, 2010). Immunofluorescence analysis confirms that na?ve CD8.sup.+ T cells form mature IS with tiCD4.sup.+ T cells transinfected with Listeria-OVA (FIG. 3e). CD4.sup.+/CD8.sup.+ cell conjugates contain CD3 molecules, part of the TCR complex, recruited in cSMAC, and show a massive accumulation of actin in the pSMAC; we found no evidence of the formation of these structures when using tiCD4.sup.+ T cells transinfected with Listeria-WT (in the few cell contacts that are detected) (FIG. 3d). Together, these data demonstrate that tiCD4.sup.+ T cells are professional APCs and activate CD8.sup.+ T cells by canonical cross-presentation.

[0074] Activation of CD8.sup.+ T cells by tiCD4+ T cells transinfected with Listeria-OVA gives them cytotoxic capacity because they can eliminate EL-4 target cells expressing OVAp/OT-I (FIG. 30. Furthermore we investigated whether tiCD4.sup.+ T cells are able to activate na?ve CD8.sup.+ T cells in vivo. For this purpose na?ve CD8.sup.+ T cells were isolated from OT-I/CD45.1 mice, stained ex vivo with CellTrace? Violet, transferred to a BL6/CD45.2 mouse, and tiCD4.sup.+ T cells were injected to elicit a response. We found that CD45.1 CD8.sup.+ T cells isolated from the spleen proliferate in response to tiCD4.sup.+ T cells transinfected with Listeria-OVA but not if they were transinfected with Listeria-WT (FIG. 4a). Moreover, we analyzed whether CD4.sup.+ T cells cross-present antigens in vivo during physiological activation of the immune system. To do so, the bone marrows of receptors C57BLJ6 mice were transplanted with stem cell progenitors with haplotype H-2K.sup.k (APCs from these mice will not be able to cross-present antigens to CD8.sup.+ T cells from OT-I mice). One month later, CD4.sup.+ T cells from AND mice (H-2K.sup.b or H-2K.sup.k) were transferred in recipient mice together with CD45.1.sup.+ CD8.sup.+ T cells from OT-I mice, and MCC peptide (to favor transinfection (Cruz-Adalia et al., 2014)). The recipient mice were also intravenously infected with Listeria-OVA. Only mice that received CD4.sup.+ T cells H-2K.sup.b were able to activate OT-I CD8.sup.+ CD45.1.sup.+ T cells (FIG. 4b), confirming the capacity for presenting antigens of CD4.sup.+ T cells during the course of a bacterial infection.

[0075] To further explore the therapeutic potential of CD8.sup.+ T cell activation by tiCD4.sup.+ T cells, we tested whether tiCD4.sup.+ T cells could protect against the formation of tumors using a mice model of aggressive melanoma. B16-OVA melanoma cell line (Borroto et al., 2014) was injected subcutaneously in C57BL/6 mice. One day later, na?ve OT-I CD8.sup.+ T cells were transferred in a single injection together with: Group 1: PBS; Group 2: tiCD4.sup.+ cells transinfected with Listeria-WT; and Group 3: tiCD4.sup.+ cells transinfected with Listeria-OVA. All mice in the control groups, vehicle treated cells or tiCD4.sup.+ T cells transinfected with Listeria-WT, developed tumors during the first 11 days after injection of B16-OVA cells (FIG. 4c). Treatment with tiCD4.sup.+ T cells transinfected with Listeria-OVA prevented tumor formation in 6 out of 9 mice and delayed tumor appearance in another mice, i.e. vaccination (single inoculation) with tiCD4.sup.+ cells working as antigen presenting cells conferred protection against melanoma in 7 of 9 cases (FIG. 4c). These results show that the cross-presentation by tiCD4.sup.+ T cells can be used in vivo to activate effector CD8.sup.+ T cells against tumors.

[0076] In conclusion, we show for the first time that cross-presentation of bacterial antigens, which was thought to be the exclusive task of APCs of the innate immune system, can be carried out effectively by CD4.sup.+ T cells, considered as the paradigm of the adaptive immune system. We also show evidence that this new way of antigen presentation can be exploited as a tool for cancer immunotherapy. The antitumor activity of tiCD4.sup.+ T cells is remarkable; note that a single injection provides protection against B-16, an extremely aggressive melanoma, whereas vaccines based on DCs require multiple inoculations, and even so they do not achieve such positive effects as model tiCD4.sup.+ (Mayordomo et al., 1995).

[0077] Moreover, we have also shown that B cells are also capable of capturing bacteria by transinfection. The process of bacterial capture by B cells from infected DC was quantitated on reisolating B cells after conjugate formation with infected DC, followed by classic gentamicin survival assays. This method allows us to analyze a large number of conjugates. The requirement of cell-cell contact was tested using a physical barrier (polycarbonate filters) that prevented DC-B cell contacts, and the role of antigen recognition by using DC decorated with hen egg lysozyme (HEL) (note that we use B cells isolated from MD4 transgenic mice, whose B cells have B cell receptors (BCR) t recognizing HEL). The largest uptake of bacteria by B cells was observed when physical contact between DC (infected) and B cells was allowed, and the DC were decorated with HEL (FIG. 5a), which is consistent with a transinfection via immunological synapse and is also similar to what observed in CD4.sup.+ T cells (Cruz-Adalia et al., 2014). When DC-B cell contact is prevented, bacterial capture by B cells is very low and similar to what has been observed in direct bacterial infections on B cells (negative control). We only took into account the experiments where B cell purity was greater than 97%, and colony forming units (CFUs) from contaminant cells were excluded from the results. To determine the fate of bacteria captured by B cells, HEL-decorated DC were infected with L. monocytogenes and conjugated with B cells from MD4 mice. After the formation of conjugates, B cells were reisolated and CFU (intracellular bacteria in B cells) were analyzed by gentamicin survival assays, where B cells were collected at different times. Unexpectedly, B cells killed very quickly (and efficiently) the bacteria captured (FIG. 5b). In the first hours after the formation of conjugates, over 90% of the internalized bacteria were destroyed. As control of bacterial fitness we measured the CFU after infecting HeLa cells (FIG. 5b).

[0078] Transinfection of Listeria-OVA induces an increased expression of MHC-I (H-2K.sup.b), accumulation of H-2K.sup.b coupled to OVAp-I antigen (not shown), and the expression of the CD86 co-stimulatory receptor in B cells (FIG. 5c.); these results are consistent with a role of antigen cross-presentation by transinfected B cells (tiB).

[0079] We generated a tiB cell population from MD4 mice, transinfected with Listeria-OVA or

[0080] Listeria-WT. We analyzed the ability to present antigens of tiB cells, incubating them with na?ve CD8.sup.+ T cells isolated from OT-I transgenic mice. Flow cytometric analysis showed a strong proliferation of CD8.sup.+ T cells 3 days after exposure to the tiB cells (FIG. 5d), but only when the tiB had captured Listeria-OVA. CD8.sup.+ T cells incubated with tiB that had captured Listeria-WT did not proliferate (FIG. 5d). Expression of activation marker CD25 was detected in CD8.sup.+ T cells incubated with tiB transinfected with Listeria-OVA, but not when used tiB transinfected with Listeria-WT (FIG. 5d). The tiB cells transinfected with Listeria-OVA induce proliferation of CD8.sup.+ T cells more potently than even the bone-marrow derived DCs (BM-DC) loaded with soluble peptide OVAp-I, infected with Listeria-OVA, or the polyclonal activation of CD8.sup.+ T cells themselves with anti CD3/CD28 antibodies. We also analyzed the ability of cytotoxic CD8.sup.+ T cells, and found that this cytotoxic activity was greater when stimulated with tiB cells transinfected with Listeria-OVA that when stimulated with splenocytes decorated with OVAp-I peptide (FIG. 5e). Cytotoxic activity was analyzed as the ability to remove EL-4 lymphoma cells decorated with OVAp-I. These data support the hypothesis that B cells behave similarly to the CD4.sup.+ T cells, capturing bacteria by transinfection and cross-presenting such antigens, inducing a potent activation of CD8.sup.+ T cells that are responsible for eliminating tumors expressing the recognized antigens.

Materials and Methods.

[0081] Bacteria: Listeria monocytogenes-OVA (pPL2-LLO-OVA), a Listeria strain that expresses OVA protein, and its WT isogenic strain L. monocytogenes 10403S.

[0082] Mice: (1) Wild-type C57BL/6 mice, (2) C57BL/6-Tg (TcraTcrb)425Cbn/J OT-II mice expressing a T cell receptor (TCR) specific for OVA peptide 323-339 in the context of MHC class II (I-Ab), (3) C57BL/6-Tg (TcraTcrb)1100Mjb/J OT-I mice expressing TCR specific for OVA peptide 257-264 in the context of H-2K.sup.b (4) AND-TCR transgenic mice recognizing moth cytochrome c 88-103 (ANERADLIAYLKQATK) (MCCp) on I-E.sup.k, expressing H-2K.sup.b, H-2K.sup.k or both haplotypes. (5) C57BL/6-Tg (IgheIMD4)4Ccg/J, (MD4) transgenic mice with more than 99% of their B cells expressing a B cell Receptor (BCR) specific for hen egg lysozyme (HEL). Male or female mice aged 8 to 12 weeks were used for experiments. Mice were kept in the specific-pathogen-free (SPF) unit at the CNB-CSIC animal facility. Experimental procedures were conducted in accordance with Spanish and European guideline, and under the FELASA guidelines.

Primary Cells:

[0083] Dendritic cells (DC) were generated as described in Inaba et al., 1992. Briefly, cells from the bone marrow (from the tibias and femurs of C57BL/6 mice 8-20 weeks) were grown in the presence of GM-CSF, at day 10 cells were tested by flow cytometry for CD11c, IA/IE and Gr1 to ensure that they had differentiated properly. They were matured with LPS day before use.

[0084] Primary CD4.sup.+ T from OT-II mice and CD8.sup.+ T from OT-I mice were obtained from lymph nodes and spleen by negative magnetic selection, as is described in Cruz-Adalia et al., 2014.

Cell Lines

[0085] The EL-4 lymphoma line was maintained in RPMI 1640 (Fisher Scientific), supplemented with 10% FCS, 0.1 U/ml penicillin, 0.1 mg/ml streptomycin (Lonza) and 0.05 mM 2-mercaptoethanol.

[0086] Melanoma B16 OVA line was maintained in RPMI 1640 with 0.4 mg/ml geneticin. Antibiotics were washed 48h before inoculation.

Antibodies:

[0087] Antibodies used were anti-CD69, -CD25, -CD4, -CD8, -CD11c, -IA/IE, -GO (BD and Immunostep), biotinylated antibodies against CD45.1, CD3, CD4, CD8, CD28, IgM, B220, CD19, MHC class II (1-Ab), CD11b, CD11c DX5, CD25 and CD16/CD32 (BD and Immunostep), and anti-tubulin FITC-conjugated (Santa Cruz). The monoclonal antibody 25-D1.16 specific for SIINFEKL/H-2K.sup.b (SEQ ID NO: 1/H-2K.sup.b) and labeled with allophycocyanin (APC) was purchased from eBioscience. Anti-TAP-1 (M-18), -ERK-2 and damin B were purchased from Santa Cruz. Secondary antibody duck anti-mouse and goat anti-hamster conjugated with AlexaFluor488, 647, or 568 were purchased from Life Technologies; secondary antibodies peroxidase conjugated with goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Thermo Scientific.

Reagents

[0088] The OVAp/OT-II peptide (OVA 323-339; ISQAVHAAHAEINEAGR, SEQ ID NO: 3) and OVAp/OT-I (OVA 257-264, SIINFEKL (SEQ ID NO: 1)) were generated at the Center for Molecular Biology Severo Ochoa (CBMSO-CSIC). Mouse GM-CSF was from Peprotech. The peptide (MCCp) 88-103 (ANERADLIAYLKQATK; SEQ ID NO: 2) was purchased from GenScript. LPS (Sigma-Aldrich), microparticles coupled to streptavidin (MiltenyiBiotec), streptavidin-PerCP (BD) and Alexa Fluor 568-Phalloidin (Life Technologies). Poly-L-Lysine (Sigma-Aldrich), CellTrace? Violet (Life Technologies) and 7-AAD Viability staining solution of eBiosciences.

Transinfection of CD4.SUP.+ T Cells

[0089] CD4.sup.+ T cells from mice OT-II were transinfected with Listeria-OVA or Listeria-WT as described in Cruz-Adalia et al. 2014. Briefly, BM-DC infected and loaded with OVAp (OT-II) (to increase transinfection) formed conjugates with CD4.sup.+ T from OT-II mice. In some experiments the BM-DC and CD4.sup.+ T cells were isolated from AND mice expressing MHC-I haplotypes: H-2K.sup.b/k, or H-2K.sup.b negative, H-2K.sup.k/k. In these experiments the BM-DC were loaded with MCCp to enhance transinfection. After 3h of conjugates DC/T it was added gentamicin (100 ?g/ml) to the medium to remove extracellular bacteria. 24h later (in some experiments 48h) the tiCD4.sup.+ cells purified by cell sorting (FACS Synergy; iCyt).

Proliferation Assays of CD8.SUP.+ T

[0090] The purified population of tiCD4.sup.+ cells was incubated with na?ve CD8.sup.+ T cells isolated from OT-I mice. These CD8.sup.+ T cells previously stained with CellTrace? Violet to quantify their proliferation by flow cytometry (FACSAria; BD). In each cell division fluorescence is lost in the proliferating population, and it was observed as a leftward shift in the histogram. Only the living cells, which do not capture the dye 7AAD, were analyzed.

[0091] To analyze the proliferation of CD8.sup.+ T cells in vivo, 5?10.sup.6 na?ve CD8.sup.+ T cells (from mice CD45.1.sup.+ OT-I), stained with CellTrace? Violet, were injected intravenously into recipient mice (CD45.2.sup.+ C57BL/6). After 24h, the tiCD4.sup.+ cells were transferred to mice. Spleens were isolated after three days to analyze the proliferation of CD8.sup.+ T cells by flow cytometry.

Fluorescence Microscopy:

[0092] tiCD4.sup.+ and na?ve CD8.sup.+ T cells were allowed to form conjugates for 1 h, then fixed with 4% paraformaldehyde in PBS. CD8+ T cells were previously stained with CellTrace? Violet. Samples were permeabilized with 0.1% Triton? X-100 in PBS prior to staining with the indicated antibodies. F-actin was detected using phalloidin coupled to a fluorophore. Samples were visualized by confocal microscopy using a Leica TCS SP5 equipped with 63?-lenses and controlled by Leica LAS AF. Images were analyzed using ImageJ (NIH Bethesda, Md.).

Cytotoxicity Assay

[0093] CD8.sup.+ T cells both effector and cytotoxic (CTLs) were prepared from na?ve CD8.sup.+ cells cells (OT-I) activated with tiCD4.sup.+ transinfected with Listeria-OVA. As positive controls were used CD8.sup.+ T cells from OT-I activated with splenocytes loaded with OVAp.

[0094] EL-4 cells were incubated with 0.5 ?M OVAp/OT-I for 1 h (or not, as control). After PBS washes, EL-4 cells loaded with OVAp were stained with CellTrace? Violet (5 ?M). On the other hand, EL-4 cells without loading OVAp were stained with CellTrace? Violet (0.5 ?M). After washing with culture medium both populations were mixed and incubated with different ratios of CTLs (5:1, 2:1, 1:1, 0.5:1) (EL4/CTLs). After 4h, samples were analyzed by flow cytometry. To calculate the specific cytotoxicity it was employed the following formula: 1?(% of EL-4 Cell Violet.sup.high/% of EL-4 Cell Violet.sup.low)?100 (Lang et al., 2005). Cytotoxicity is calculated relative subtracting the negative control (EL-4 cells not incubated with CTLs).

Protection Assay Against Tumors (B16-OVA Melanoma)

[0095] CD4.sup.+ T cells transinfected with Listeria-WT (as negative control) or Listeria-OVA were prepared as described in previous sections. 24h after transinfection, tiTCD4.sup.+ are re-isolated cell sorting and resuspended in PBS. na?ve CD8.sup.+ T cells from OT-I were purified by magnetic columns as previously described (Cruz-Adalia et al., 2014) and resuspended in PBS. 5?10.sup.5 B16-OVA cells were injected subcutaneously into the right flank of recipient mice (C57BL/6). After that, mice were divided into three groups and transferred intravenously (iv) in a single injection: Group 1: PBS; Group 2:5?10.sup.5 tiCD4.sup.+ cells transinfected with Listeria-WT; and Group 3: tiCD4.sup.+ cells transinfected with Listeria-OVA. All mice in all groups were also injected, simultaneously to tiCD4.sup.+ or PBS vehicle, with 10.sup.3 CD8.sup.+ T cells from OT-I (also i.v.). Tumor growth was monitored every 2-3 days using a caliper-dial, and the areas were determined by multiplying the length and width. The experimental groups were assigned randomly and the experiment was conducted in a double blind (people who measured the tumors did not know to which group each mouse belonged). Mice were sacrificed when tumors reached 300 mm.sup.2 according to the criteria endpoint as directed by the European Union and FELASA for experimental animals, and in accordance with the current legislation in Spain.

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