Lymphadenectomy exacerbates tumor growth while lymphadenectomy plus the adoptive transfer of autologous cytotoxic cells and low-dose cyclophosphamide induces regression of an established murine fibrosarcoma.

Tumor-draining lymph node (TDLN) ablation is routinely performed in the management of cancer; nevertheless, its usefulness is at present a matter of debate. TDLN are central sites where T cell priming to tumor antigens and onset of the antitumor immune response occur. However, tumor-induced immunosuppression has been demonstrated at TDLN, leading to downregulation of antitumor reaction and tolerance induction. Tolerance in turn is a main impairment for immunotherapy trials. We used a murine immunogenic fibrosarcoma that evolves to a tolerogenic state, to study the cellular and molecular mechanisms underlying tolerance induction at the level of TDLN and to design an appropriate immunotherapy. We determined that following a transient activation, the established tumor induces signs of immunosuppression at TDLN that coexist with local and systemic evidences of antitumor response. Therefore, we evaluated the feasibility of removing TDLN in order to eliminate a focus of immunosuppression and favor tumor rejection; but instead, a marked exacerbation of tumor growth was induced. Combining TDLN ablation with the in vivo depletion of regulatory cells by low-dose cyclophosphamide and the restoring of the TDLN-derived cells into the donor mouse by adoptive transference, resulted in lowered tumor growth, enhanced survival and a considerable degree of tumor regression. Our results demonstrate that important antitumor elements can be eliminated by lymphadenectomy and proved that the concurrent administration of low-dose chemotherapy along with the reinoculation of autologous cytotoxic cells provides protection. We suggest that this protocol may be useful, especially in the cases where lymphadenectomy is mandatory.

[Regulatory T cell depletion increases the number of CD8 cells during mouse mammary tumor virus infection]. / La depleción de las células T regulatorias aumenta el número de las células CD8 durante la infección con el virus del tumor mamario murino.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. We have shown in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (Sag)-specific CD4+ CD25+ Foxp3+ regulatory T cells (Treg) in Peyer's patches. Herein, we evaluated whether the depletion of Treg cells affects the CD8+ population during milk-borne MMTV infection. At day 6 of infection, the depletion of Treg cells increased the percentage and absolute number of CD8+ cells in lymph nodes as well as the mean intensity fluorescence of the CD44 activation marker. The absolute number of CD8+ cells was increased in cells bearing both Sag reactive and non-reactive TCR Vß chains. We have previously shown that regulatory T cell depletion at day 6 of infection decrease viral load. Results reported herein suggest that at least after day 6 of MMTV infection Treg cells play an inhibiting role on CD8 antiviral response.

Increases in IgA(+) B cells in Peyer's patches during milk-borne mouse mammary tumor virus infection are influenced by Toll-like receptor 4 and are completely dependent on the superantigen response.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Although mammary glands are the final target of infection, Peyer's patches (PP) are the entry site of the virus. Herein, we show that the infection induces increases in the number of PP IgA(+) B cells and higher expression of the α circular transcript, which is a specific marker of the switch to IgA. In addition, IgA(+) B-cell increases correlated with higher levels of cytokines related to IgA class switching, such as interleukin (IL)-5 and IL-6. Of interest, the increases in IgA(+) B cells were lower in Toll-like receptor 4-deficient mice and were completely dependent on the presence of superantigen-reactive T cells. Our results point to a novel mechanism involved in MMTV infection and suggest that IgA(+) B cells may play an important role in carrying the virus to the mammary glands.

Early increases in superantigen-specific Foxp3+ regulatory T cells during mouse mammary tumor virus infection.

Mouse mammary tumor virus (MMTV) is a milk-borne betaretrovirus that has developed strategies to exploit and subvert the host immune system. Here, we show in a natural model of MMTV infection that the virus causes early and progressive increases in superantigen (SAg)-specific Foxp3(+) regulatory T cells (T(reg)) in Peyer's patches (PP). These increases were shown to be dependent on the presence of dendritic cells. CD4(+) CD25(+) T cells from the PP of infected mice preferentially suppress the proliferative response of T cells to SAg-expressing antigen-presenting cells ex vivo. We investigated the influence of the depletion of CD25(+) cells at different stages of the infection. When CD25(+) cells were depleted before MMTV infection, an increase in the number of PP SAg-cognate Foxp3(-) T cells was found at day 6 of infection. Since the SAg response is associated with viral amplification, the possibility exists that T(reg) cells attenuate the increase in viral load at the beginning of the infection. In contrast, depletion of CD25(+) cells once the initial SAg response has developed caused a lower viral load, suggesting that at later stages T(reg) cells may favor viral persistence. Thus, our results indicated that T(reg) cells play an important and complex role during MMTV infection.

A B-Cell Superantigen Induces the Apoptosis of Murine and Human Malignant B Cells.

B-cell superantigens (Sags) bind to conserved sites of the VH or VL regions of immunoglobulin molecules outside their complementarity-determining regions causing the apoptosis of normal cognate B cells. No attempts to investigate whether B-cell Sags are able to induce the apoptosis of cognate malignant B cells were reported. In the present study we show that protein L (PpL), secreted by Finegoldia magna, a B-cell Sag which interacts with κ+ bearing cells, induces the apoptosis of murine and human κ+ lymphoma B cells both in vitro and in vivo. Apoptosis was not altered by caspase-8 inhibitor. No alterations in the levels of Bid, Fas and Fas-L were found suggesting that PpL does not activate the extrinsic pathway of apoptosis. The involvement of the intrinsic pathway was clearly indicated by: i) alterations in mitochondrial membrane potential (ΔΨm) both in murine and human lymphoma cells exposed to PpL; ii) decreased levels of apoptosis in the presence of caspase-9 inhibitor; iii) significant increases of Bim and Bax protein levels and downregulation of Bcl-2; iv) the translocation from the cytoplasm to the mitochondria of Bax and Bim pro-apoptotic proteins and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor and v) the translocation of Bcl-2 protein from the mitochondria to the cytosol and its inhibition by caspase-9 inhibitor but not by caspase-8 inhibitor. The possibility of a therapeutic use of Sags in lymphoma/leukemia B cell malignancies is discussed.

A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation.

Superantigens increase the survival of mice bearing T cell lymphomas by inducing apoptosis of neoplastic cells.

Superantigens bind to major histocompatibility complex class II molecules and interact with T cells expressing a particular T cell receptor Vß inducing a strong proliferation/deletion response of the superantigen-reactive T cells. However, there have been no attempts to investigate the ability of Sags to induce apoptosis in neoplastic T cells by signaling through the Vß region of their TCR. In the present study we show that bacterial and MMTV-encoded superantigens induce the apoptosis of AKR/J cognate lymphoma T cells both in vitro and in vivo. The Fas-Fas-L pathway was shown to be involved in the apoptosis of lymphoma T cells induced by bacterial superantigens. In vivo exposure to bacterial superantigens was able to improve the survival of lymphoma bearing mice. Moreover, the permanent expression of a retroviral encoded superantigen induced the complete remission of an aggressive lymphoma in a high percentage of mice. The possibility of a therapeutic use of superantigens in lymphoma/leukemia T cell malignancies is discussed.

A polymeric bacterial protein activates dendritic cells via TLR4.

The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.

Cathepsin-L influences the expression of extracellular matrix in lymphoid organs and plays a role in the regulation of thymic output and of peripheral T cell number.

Nackt mice, which are deficient in cathepsin-L (CTSL), show an early impairment during positive selection in the context of class II MHC molecules and as a consequence, the percentage and absolute number of CD4(+) thymocytes are significantly decreased. In this study, we show that lymph nodes from nackt mice are hypertrophied, showing normal absolute numbers of CD4(+) T cells and marked increases in the number of CD8(+) T lymphocytes. Basal proliferative levels are increased in the CD4(+) but not in the CD8(+) population. Lymph node T cells show increases in the expression of alpha(5), alpha(6), and beta(1) integrin chains. These alterations correlate with increases in the expression of extracellular matrix (ECM) components in lymph nodes. Interestingly, laminin, fibronectin, and collagen I and IV are markedly decreased in nackt thymus which shows an augmented output of CD8(+) cells. These results demonstrate that a mutation in the Ctsl gene influences the levels of ECM components in lymphoid organs, the thymic output, and the number of T cells in the periphery. They further raise the possibility that, by regulating the level of expression of ECM components in lymphoid organs, CTSL is able to broadly affect the immune system.