Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/-) retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK) cell-mediated cytotoxicity: i) B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/-) and FcγRIIIa deficient (CD16-/-) C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii) administration of NK1.1 specific IgG2a (mAb PK136) but not irrelevant IgG2a (myeloma M9144) prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii) splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv) purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v) adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi) degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei infected mice kill B cells, suppress humoral immunity and expedite early mortality.

Exclusion of T Cells From Pancreatic Carcinomas in Mice Is Regulated by Ly6C(low) F4/80(+) Extratumoral Macrophages.

BACKGROUND & AIMS: Immunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal adenocarcinoma (PDAC). We investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumor's immunosuppressive mechanisms, or both. METHODS: Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry. RESULTS: Gemcitabine in combination with a CD40 agonist induced T-cell-dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4(+) and CD8(+) T cells infiltrated subcutaneous tumors, but only CD4(+) T cells infiltrated spontaneous pancreatic tumors (not CD8(+) T cells). In mice depleted of Ly6C(low) F4/80(+) extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8(+) T cells and induced tumor regression, mediated by CD8(+) T cells. CONCLUSIONS: Ly6C(low) F4/80(+) macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.

Listeria and Salmonella bacterial vectors of tumor-associated antigens for cancer immunotherapy.

This review covers the use of the facultative intracellular bacteria, Listeriamonocytogenes and Salmonella enterica serovar typhimurium as delivery systems for tumor-associated antigens in tumor immunotherapy. Because of their ability to infect and survive in antigen presenting cells, these bacteria have been harnessed to deliver tumor antigens to the immune system both as bacterially expressed proteins and encoded on eukaryotic plasmids. They do this in the context of strong innate immunity, which provides the required stimulus to the immune response to break tolerance against those tumor-associated antigens that bear homology to self. Here we describe differences in the properties of these bacteria as vaccine vectors, a summary of the major therapies they have been applied to and their advancement towards the clinic.

T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis.

African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG) coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB) and Follicular B (FoB) cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves.

B Lymphocytes provide an infection niche for intracellular bacterium Brucella abortus.

BACKGROUND: Brucella spp. are intracellular bacteria that establish lifelong infections whose mechanisms of chronicity are poorly understood. Notably, B cells facilitate the establishment of the high infection plateau that persists for months. METHODS: We evaluated the contribution of murine B cells toward providing infection niches for Brucella by using flow cytometry and microscopy and by determining live bacterial counts associated with B cells both in vivo and in vitro. RESULTS: Herein we demonstrate that immunoglobulin M and complement-opsonized Brucella abortus infects and survives inside primary murine B cells protected from bactericidal effects of gentamicin. The entry was dependent on microfilaments for internalization and subsequently brucellae reside in a late endosomal/lysosomal compartment. Throughout the infection, 10% of colony-forming units from infected mice was associated with B cells, and these cells transferred disease to naive hosts. Furthermore, Brucella-positive cells were positive for transforming growth factor (TGF) ß1, and about 10% of such cells were B cells, similar to rates found for other intracellular pathogens that induce their hosts cells to produce TGF-ß1. CONCLUSIONS: To conclude, infected B cells contribute to chronic bacterial infections by providing an intracellular niche that may exert an immunoregulatory role. Although professional phagocytic cells harbor intracellular bacteria including Brucella, infection of lymphocytes by bacteria has not been previously appreciated.

Targeting tumor vasculature with novel Listeria-based vaccines directed against CD105.

The FDA approval of bevacizumab (Avastin®, Genentech/Roche), a monoclonal antibody raised against human VEGF-A, as second-line therapy for colon and lung carcinoma validated the approach of targeting human tumors with angiogenesis inhibitors. While the VEGF/VEGFR pathway is a viable target for anti-angiogenesis tumor therapy, additional targets involved in tumor neovascularization have been identified. One promising target present specifically on tumor vasculature is endoglin (CD105), a member of the TGF-ß receptor complex expressed on vascular endothelium and believed to play a role in angiogenesis. Monoclonal antibody therapy and preventive vaccination against CD105 has met with some success in controlling tumor growth. This report describes the in vivo proof-of-concept studies for two novel therapeutic vaccines, Lm-LLO-CD105A and Lm-LLO-CD105B, directed against CD105 as a strategy to target neovascularization of established tumors. Listeria-based vaccines directed against CD105 lead to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous Listeria-based vaccines targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Support is provided for epitope spreading to endogenous tumor antigens and reduction in tumor vascularity after vaccination with Listeria-based CD105 vaccines. Reported here, these CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

Trypanosomiasis-induced B cell apoptosis results in loss of protective anti-parasite antibody responses and abolishment of vaccine-induced memory responses.

African trypanosomes of the Trypanosoma brucei species are extra-cellular parasites that cause human African trypanosomiasis (HAT) as well as infections in game animals and livestock. Trypanosomes are known to evade the immune response of their mammalian host by continuous antigenic variation of their surface coat. Here, we aim to demonstrate that in addition, trypanosomes (i) cause the loss of various B cell populations, (ii) disable the hosts' capacity to raise a long-lasting specific protective anti-parasite antibody response, and (iii) abrogate vaccine-induced protective response to a non-related human pathogen such as Bordetella pertussis. Using a mouse model for T. brucei, various B cell populations were analyzed by FACS at different time points of infection. The results show that during early onset of a T. brucei infection, spleen remodeling results in the rapid loss of the IgM(+) marginal zone (IgM(+)MZ) B cell population characterized as B220(+)IgM(High)IgD(Int) CD21(High)CD23(Low)CD1d(+)CD138(-). These cells, when isolated during the first peak of infection, stained positive for Annexin V and had increased caspase-3 enzyme activity. Elevated caspase-3 mRNA levels coincided with decreased mRNA levels of the anti-apoptotic Bcl-2 protein and BAFF receptor (BAFF-R), indicating the onset of apoptosis. Moreover, affected B cells became unresponsive to stimulation by BCR cross-linking with anti-IgM Fab fragments. In vivo, infection-induced loss of IgM(+) B cells coincided with the disappearance of protective variant-specific T-independent IgM responses, rendering the host rapidly susceptible to re-challenge with previously encountered parasites. Finally, using the well-established human diphtheria, tetanus, and B. pertussis (DTPa) vaccination model in mice, we show that T. brucei infections abrogate vaccine-induced protective responses to a non-related pathogen such as B. pertussis. Infections with T. brucei parasites result in the rapid loss of T-cell independent IgM(+)MZ B cells that are normally functioning as the primary immune barrier against blood-borne pathogens. In addition, ongoing trypanosome infections results in the rapid loss of B cell responsiveness and prevent the induction of protective memory responses. Finally, trypanosome infections disable the host's capacity to recall vaccine-induced memory responses against non-related pathogens. In particular, these last results call for detailed studies of the effect of HAT on memory recall responses in humans, prior to the planning of any mass vaccination campaign in HAT endemic areas.

Cancer immunotherapy using Listeria monocytogenes and listerial virulence factors.

Our laboratory is interested in how immunogenicity may be modulated in vivo in order to better design more effective immunotherapeutics against cancer. Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic CTL epitopes. We have used this approach to target tumor antigens expressed on breast, melanoma and cervical cancer. We are also exploring the role of Listerial virulence factors in potentiating adaptive immune responses by activating innate immunity. Specifically, we are using these proteins as adjuvants for B cell lymphomas.

Rescue of CD8 T cell-mediated antimicrobial immunity with a nonspecific inflammatory stimulus.

Reconstitution of protective immunity by adoptive transfer of pathogen-specific T cells has been successful in patients with compromised cellular immunity. The in vivo effectiveness of in vitro-expanded CD8 CTLs is variable, however. For example, adoptively transferred Listeria monocytogenes-specific CD8 CTLs only confer protective immunity if challenge infection occurs within 48 hours of T cell infusion. Herein we show that transferred CTLs persist in lymphoid compartments for many weeks, but that their response to bacterial challenge decreases during the first week following transfer. While T cells transferred less than 48 hours before infection proliferate, those transferred 7 days before infection die. Remarkably, treatment of mice with anti-CD40 at the time of T cell infusion reprograms transferred T cells, allowing them to proliferate and confer protective immunity upon bacterial challenge 7 days later. Our study demonstrates, for the first time to our knowledge that CD40-mediated stimuli can influence CD8 T cell activation independent of concurrent antigen exposure. The ability to modulate long-term responsiveness of CD8 T cells with a transient, nonspecific inflammatory stimulus has importation implications for adoptive immunotherapy.

Anti-Trypanosoma brucei activity in Cape buffalo serum during the cryptic phase of parasitemia is mediated by antibodies.

Cape buffalo are reservoir hosts of African trypanosomes. They rapidly suppress population growth of the highly antigenically variable extracellular haemoprotozoa and subsequently maintain a cryptic infection. Here we use in vitro cultures of trypanosomes cloned from Cape buffalo blood during cryptic infection, as well as related and unrelated trypanosomes, to identify anti-trypanosome components present in cryptic-phase infection serum. Trypanosome clone-specific complement-dependent trypanolytic IgM and IgG arose after appearance of target trypanosomes during cryptic infection. Serum collected late in the cryptic phase of infection contained complement-independent growth-inhibitory IgG which varied in activity among target trypanosomes. Removal of protein A/G-binding IgG from the serum restored its capacity to support trypanosome growth in vitro. Recovered growth-inhibitory IgG reacted with the variable surface glycoprotein (VSG) of parasites most affected by it, and reacted with trypanosome common antigens, notably the endosome-restricted tomato lectin-binding glycoproteins (TL-antigens). The inclusion of purified TL-antigens in culture medium did not affect the trypanosome growth-inhibitory activity of immune Cape buffalo serum. In addition, hyperimmune rabbit IgG against TL-antigens showed little or no binding to intact trypanosomes and did not affect trypanosome growth in vitro although it did react strongly with TL-antigens and trypanosome endosomes. We conclude that antibodies, particularly clone-specific (putatively VSG-specific) antibodies are responsible for the anti-trypanosome activity of cryptic phase infection serum consistent with a dominant role in parasite control in Cape buffalo.

Interferon γ-induced intratumoral expression of CXCL9 alters the local distribution of T cells following immunotherapy with Listeria monocytogenes.

The ability of Listeria monocytogenes-based anticancer vaccines to induce tumor regression depends on the responsiveness of malignant cells to interferon γ (IFNγ). Inhibition of IFNγ limits the recruitment of T cells to the tumors of vaccinated mice. We hypothesized that vaccination with immunotherapeutic L. monocytogenes induces the IFNγ-dependent production of chemokines that regulate the migration of tumor-infiltrating T cells. To gain further insights into this issue, we examined the chemokine responses of a transplantable, human papillomavirus (HPV)-immortalized murine tumor model (TC-1) following the administration of a L. monocytogenes-based immunotherapeutic agent that expresses E7 from HPV-16. Here, we report that the administration of L. monocytogenes-based anticancer vaccines increases the secretion of chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL10 by tumors, hence favoring the recruitment of T cells bearing the cognate chemokine (C-X-C motif) receptor 3 (CXCR3). Furthermore, the expression of CXCL9, but not CXCL10, in TC-1 tumors was significantly reduced upon anti-IFNγ antibody treatment. CXCL9 was highly expressed by TC-1 cells following the administration of IFNγ and tumor necrosis factor α (TNFα), in vitro. Moreover, the inhibition of CXCL9 in TC-1 cells reduced the proportion of CD8+ T cells infiltrating tumors in vaccinated mice, while increasing that of CD4+ T cells, thus altering T-cell subset distribution. We conclude that the administration of L. monocytogenes-based anticancer vaccines regulates TH1 chemokine responses and that malignant cells are an important source of these chemokines.

Vaccination with immunotherapeutic Listeria monocytogenes induces IL-17(+) γδ T cells in a murine model for HPV associated cancer.

Interleukin 17 (IL-17) is produced during infection with Listeria monocytogenes and is also an important regulator of tumor development with both pro- and anti-tumorigenic effects. αß T cells and γδ T cells are among the principle producers of IL-17 in response to infection and other proinflammatory conditions. Listeria-based cancer immunotherapies induce IFNγ directed Th1 dependent tumor regression; however, the role of IL-17 in Listeria based immunotherapy has not been addressed. Therefore, we investigated the ability of attenuated Listeria-based immunotherapy to induce IL-17 producing cells in a model of cervical cancer and the potential impact that these cells have on anti-tumor vaccine efficacy. Here we show that vaccination of tumor bearing mice with Listeria vaccines resulted in elevated levels of intratumoral IL-17 and increased IL-17 production by γδ TCR+ cells, exclusively. IL-17 producing cells were lacking in tumors of γδ T-cell-deficient mice; however, the absence of γδ T cells, including IL-17+ γδ T cells, did not alter tumor progression or abrogate the efficacy of the Listeria-based vaccine indicating that αß T cells are key for clearance of the tumor. Th1 responses, known to be responsible for anti-tumor Listeria-based vaccine efficacy, appear to be sufficient for tumor regression in γδ T-cell-deficient mice. We conclude that the efficacy of Listeria-based vaccine does not rely on γδ T cells (or IL-17 produced by them) in a TC.1 tumor model; however, Listeria-based immunotherapy can be used to induce IL-17+ γδ T cells that are important for regression observed in alternative cancer models.

Listeria monocytogenes and its products as agents for cancer immunotherapy.

This review covers the use of Listeria monocytogenes and its virulence factors as cancer immunotherapeutics. We describe their development as vectors to carry protein tumor antigen and eukaryotic DNA plasmids to antigen-presenting cells and efforts to harness their tumor-homing properties. We also describe their use as vectors of angiogenic molecules to induce an immune response that will destroy tumor vasculature. The background knowledge necessary to understand the biology behind the rationale to develop Listeria as a vaccine vector for tumor immunotherapy is included as well as a brief summary of the major therapies that have used this approach thus far.

Distinct in vivo dendritic cell activation by live versus killed Listeria monocytogenes.

Immunization of mice with live or heat-killed Listeria monocytogenes (HKLM) efficiently primes pathogen-specific CD8(+) T cells. T lymphocytes primed by HKLM, however, undergo attenuated proliferation and do not fully differentiate. Thus, only infection with live bacteria induces long-term, CD8(+) T cell-mediated protective immunity. In this study we demonstrate that live and heat-killed bacteria, while both associating with Mac-3(+)CD11b(hi) cells, localize to distinct splenic areas following intravenous inoculation. While HKLM localize to the marginal zone and the splenic red pulp, live L. monocytogenes are carried to the T cell zone of splenic white pulp. Despite these differences, in vivo depletion of CD11c-expressing cells prevents priming of naive T cells by either HKLM or live L. monocytogenes. Analysis of CD11c(hi) dendritic cells (DC) reveals that infection with live L. monocytogenes induces higher levels of CD40, CD80 and CD86 expression than immunization with HKLM. Our results suggest that CD8(+) T cell priming following HKLM immunization or live infection is mediated by DC and that the disparate outcomes of priming can be attributed to suboptimal conditioning of DC in the absence of live, cytosol-invasive bacteria.

Promiscuity of MHC class Ib-restricted T cell responses.

Murine infection with the Gram-positive intracellular bacterium Listeria monocytogenes activates CD8(+) T cells that recognize bacterially derived N-formyl methionine peptides in the context of H2-M3 MHC class Ib molecules. Three peptides, fMIGWII, fMIVIL, and fMIVTLF, are targets of L. monocytogenes-specific CD8(+) T cells. To investigate epitope cross-recognition by H2-M3-restricted CD8(+) T cells, we deleted the sequence encoding fMIGWII from a virulent strain of L. monocytogenes. Infection with fMIGWII-deficient L. monocytogenes unexpectedly primed CD8(+) T cells that stain with fMIGWII/H2-M3 tetramers and lyse fMIGWII-coated target cells in vivo. Because the fMIGWII sequence is nonredundant, we speculated that other bacterially derived Ags are priming these responses. HPLC peptide fractionation of bacterial culture supernatants revealed several distinct L. monocytogenes-derived peptides that are recognized by fMIGWII-specific T cells. Our results demonstrate that the dominant H2-M3-restricted CD8(+) T cell population, although reactive with fMIGWII, is primed by other, non-fMIGWII peptides derived from L. monocytogenes. Although this degree of Ag receptor promiscuity is unusual for the adaptive immune system, it may be a more common feature of T cell responses restricted by nonpolymorphic MHC class Ib molecules.