Mouse Dendritic Cells Pulsed with Capsular Polysaccharide Induce Resistance to Lethal Pneumococcal Challenge: Roles of T Cells and B Cells

Mice are exceedingly sensitive to intra-peritoneal (IP) challenge with some virulent pneumococci (LD50 = 1 bacterium). To investigate how peripheral contact with bacterial capsular polysaccharide (PS) antigen can induce resistance, we pulsed bone marrow dendritic cells (BMDC) of C57BL/6 mice with type 4 or type 3 PS, injected the BMDC intra-foot pad (IFP) and challenged the mice IP with supra-lethal doses of pneumococci. We examined the responses of T cells and B cells in the draining popliteal lymph node and measured the effects on the bacteria in the peritoneum and blood. We now report that: 1) The PS co-localized with MHC molecules on the BMDC surface; 2) PS-specific T and B cell proliferation and IFNγ secretion was detected in the draining popliteal lymph nodes on day 4; 3) Type-specific resistance to lethal IP challenge was manifested only after day 5; 4) Type-specific IgM and IgG antibodies were detected in the sera of only some of the mice, but B cells were essential for resistance; 5) Control mice vaccinated with a single injection of soluble PS did not develop a response in the draining popliteal lymph node and were not protected; 6) Mice injected with unpulsed BMDC also did not resist challenge: In unprotected mice, pneumococci entered the blood shortly after IP inoculation and multiplied exponentially in both blood and peritoneum killing the mice within 20 hours. Mice vaccinated with PS-pulsed BMDC trapped the bacteria in the peritoneum. The trapped bacteria proliferated exponentially IP, but died suddenly at 18–20 hours. Thus, a single injection of PS antigen associated with intact BMDC is a more effective vaccine than the soluble PS alone. This model system provides a platform for studying novel aspects of PS-targeted vaccination

A Systems Immunology Approach to the Host-Tumor Interaction: Large-Scale Patterns of Natural Autoantibodies Distinguish Healthy and Tumor-Bearing Mice

Traditionally, immunology has considered a meaningful antibody response to be marked by large amounts of high-affinity antibodies reactive with the specific inciting antigen; the detection of small amounts of low-affinity antibodies binding to seemingly unrelated antigens has been considered to be beneath the threshold of immunological meaning. A systems-biology approach to immunology, however, suggests that large-scale patterns in the antibody repertoire might also reflect the functional state of the immune system. To investigate such global patterns of antibodies, we have used an antigen-microarray device combined with informatic analysis. Here we asked whether antibody-repertoire patterns might reflect the state of an implanted tumor. We studied the serum antibodies of inbred C57BL/6 mice before and after implantation of syngeneic 3LL tumor cells of either metastatic or non-metastatic clones. We analyzed patterns of IgG and IgM autoantibodies binding to over 300 self-antigens arrayed on slides using support vector machines and genetic algorithm techniques. We now report that antibody patterns, but not single antibodies, were informative: 1) mice, even before tumor implantation, manifest both individual and common patterns of low-titer natural autoantibodies; 2) the patterns of these autoantibodies respond to the growth of the tumor cells, and can distinguish between metastatic and non-metastatic tumor clones; and 3) curative tumor resection induces dynamic changes in these low-titer autoantibody patterns. The informative patterns included autoantibodies binding to self-molecules not known to be tumor-associated antigens (including insulin, DNA, myosin, fibrinogen) as well as to known tumor-associated antigens (including p53, cytokeratin, carbonic anhydrases, tyrosinase). Thus, low-titer autoantibodies that are not the direct products of tumor-specific immunization can still generate an immune biomarker of the body-tumor interaction. System-wide profiling of autoantibody repertoires can be informative

Trogocytosis of MHC-I/Peptide Complexes Derived from Tumors and Infected Cells Enhances Dendritic Cell Cross-Priming and Promotes Adaptive T Cell Responses

The transporter associated with antigen processing (TAP) and the major histocompatibility complex class I (MHC-I), two important components of the MHC-I antigen presentation pathway, are often deficient in tumor cells. The restoration of their expression has been shown to restore the antigenicity and immunogenicity of tumor cells. However, it is unclear whether TAP and MHC-I expression in tumor cells can affect the induction phase of the T cell response. To address this issue, we expressed viral antigens in tumors that are either deficient or proficient in TAP and MHC-I expression. The relative efficiency of direct immunization or immunization through cross-presentation in promoting adaptive T cell responses was compared. The results demonstrated that stimulation of animals with TAP and MHC-I proficient tumor cells generated antigen specific T cells with greater killing activities than those of TAP and MHC-I deficient tumor cells. This discrepancy was traced to differences in the ability of dendritic cells (DCs) to access and sample different antigen reservoirs in TAP and MHC-I proficient versus deficient cells and thereby stimulate adaptive immune responses through the process of cross-presentation. In addition, our data suggest that the increased activity of T cells is caused by the enhanced DC uptake and utilization of MHC-I/peptide complexes from the proficient cells as an additional source of processed antigen. Furthermore, we demonstrate that immune-escape and metastasis are promoted in the absence of this DC ‘arming’ mechanism. Physiologically, this novel form of DC antigen sampling resembles trogocytosis, and acts to enhance T cell priming and increase the efficacy of adaptive immune responses against tumors and infectious pathogens

Production of LacZ inducible T cell hybridoma specific for human and mouse gp100₂₅₋₃₃ peptides.

Identification and quantification of immunogenic peptides and tumor-derived epitopes presented on MHC-I molecules are essential for basic studies and vaccines generation. Although lymphocytes derived from transgenic mice can serve as sensitive detectors of processes of antigen presentation and recognition, they are not always available. The use of cell lines might be extremely useful. In this study, we generated a lacZ inducible CD8⁺ hybridoma (BUSA14) capable of recognizing both human and mouse gp100₂₅₋₃₃ melanoma antigens presented on dendritic and tumor cell lines. This hybridoma expresses a variety of membranal T cell markers and secretes IL-2 and TNFα. Thus, BUSA14 offers a quantifiable, cheap and straightforward tool for studying peptide presentation by MHC-I molecules on the cell surface

Anti‐metastatic vaccination of tumor‐bearing mice with il‐2‐gene‐inserted tumor cells

IL‐2 gene was introduced through retroviral vectors into the highly malignant and poorly immunogenic 3LL‐D122 clone. Both high and low D122‐11‐2 secretors showed elimination of tumorigenicity in syngeneic immune‐competent mice; however, in nude mice only the high IL‐2 secretor showed reduced tumorigenicity as compared with parental D 122 cells. Also, following intravenous inoculation, only the high IL‐2 secretor showed reduced generation of metastases, whereas the low IL‐2 secretors were as highly metastatic as parental cells. These results seem to indicate that low levels of IL‐2 secreted by tumor cells are sufficient to activate T cells, while higher levels are needed in order to activate non‐T‐cell effectors. D122‐IL‐2 secretors induced high levels of anti‐tumor CTL, while their sensitivity to the lytic activity of these CTL was similar to the sensitivity of D122 cells, thus indicating that the elevation of immunogenicity of IL‐2 secretors was essentially due to the secreted IL‐2. In accordance with CTL induction, pre‐immunization with IL‐2 secretors protected mice against subsequent challenge of parental cells. Moreover, immunization in an “immunotherapy protocol” i.e., vaccination of mice carrying an established tumor of parental D122 cells with inactivated D122‐IL‐2 infectants, almost completely eliminated the generation of lung metastases by D122 cells, thus providing a rationale for the use of IL‐2 gene transferred tumor cells as a modality for treatment of metastasis

BUSA14 are activated by hgp100_25–33 and mgp100_25–33 presented on melanoma cell lines.

<p><b>A.</b> B16-MO5, F10.9, D122 and EL4 tumor cell lines were analyzed by flow cytometry with monoclonal antibodies to H-2K^b and H-2D^b to analyze MHC-I membranal expression. MFI values are presented in the figure. <b>B.</b> Twenty thousand B16-MO5, F10.9 and D122 cells were loaded with 30 µg/ml hgp100_25–33 or SIINFEKL peptides. Cells were washed and co-incubated with 6×10⁴ BUSA14 and BWZ.36/CD8α for 12 hours. Cells were then lysed and β-Gal enzymatic activity was monitored with CPRG. Cultures with D122 served as reference for CPRG background levels. Representative results (1 of 3 experiments) are presented as ΔOD (sample OD-background OD) measured after 12 hours. <b>C.</b> Sixty thousand BUSA14 and BWZ.36/CD8α cells/well were incubated overnight, in triplicates, with 2×10⁴ B16-MO5, F10.9 or D122 tumor cell lines. Representative results (1 of 2 experiments) are presented as ΔOD (sample OD-background OD) measured after 24 hours. Statistical analysis was done using student T test (*p<0.05, **p<0.01, ***p<0.001).</p

Detection of cytokines produced by BUSA14 cells.

<p>BUSA14 or BWZ.36/CD8α were co-incubated with DC2.4 cells loaded with hgp100_25–33 or SIINFEKL. Cells alone or co-cultured with unloaded DC2.4 or with PMA and ionomycin served as negative and positive controls, respectively. All cells were intracellulary stained with antibodies to CD8, IL-2/TNFα (<b>A</b>), IL-4/IFNγ (<b>B</b>) and analyzed by flow cytometry. Cells were gated for CD8 to exclude DC2.4 cells.</p