Adoptive cellular immunotherapy utilizing tumor-reactive T cells has proven to be a promising strategy for cancer treatment. However, we hypothesize that successful treatment strategies will have to appropriately stimulate not only cellular immunity, but also humoral immunity. We previously reported that B cells in tumor-draining lymph nodes (TDLN) may function as antigen-presenting cells. In this study, we identified TDLN B cells as effector cells in an adoptive immunotherapy model. In vivo primed and in vitro activated TDLN B cells alone mediated effective (p<0.05) tumor regression after adoptive transfer into two histologically distinct murine pulmonary metastatic tumor models. Prior lymphodepletion of the host with either chemotherapy or whole-body irradiation augmented the therapeutic efficacy of the adoptively transferred TDLN B cells in the treatment of subcutaneous tumors as well as metastatic pulmonary tumors. Furthermore, B cell plus T cell transfers resulted in substantially more efficient antitumor responses than B cells or T cells alone (p<0.05). Activated TDLN B cells conferred strong humoral responses to tumor. This was evident by the production of IgM, IgG and IgG2b, which bound specifically to tumor cells and led to specific tumor cell lysis in the presence of complement. Collectively, these data indicate that in vivo primed and in vitro activated B cells can be employed as effector cells for cancer therapy. The synergistic antitumor efficacy of co-transferred activated B effector cells and T effector cells represents a novel approach for cancer adoptive immunotherapy.

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell-cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G-M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention. Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell-cell adhesion that may underlie progression to invasive SCC. .

To date, molecular targets chosen for Ab activation to generate antitumor effector cells have been confined on T cells, such as TCR/CD3, CD28, CD137 (4-1BB), CD134 (OX40), and inducible costimulator. In this report we investigated the immune function of murine tumor-draining lymph node (TDLN) cells after simultaneous Ab targeting of CD3 on T cells and CD40 on APCs. Anti-CD3 plus anti-CD40-activated TDLN cells secreted significantly higher amounts of IFN-γ, but less IL-10, compared with anti-CD3-activated cells. In adoptive immunotherapy, ligation of CD3 and CD40 resulted in the generation of more potent effector cells in mediating tumor regression. Freshly harvested TDLN cells were composed of ∼60% CD3+ T cells, 30–35% CD19+ B cells, 5% CD11c+ dendritic cells (DC), and few CD14+ or NK cells (each &lt;3%). CD40 was distributed predominantly on B cells and DCs. Cell depletion indicated that simultaneous targeting was toward CD3 on T cells and CD40 on APCs, respectively. Elimination of APCs completely abrogated the augmented antitumor responses induced by anti-CD40. Either B cell or DC removal partially, but significantly, reduced the therapeutic efficacy conferred by CD40 engagement. Furthermore, the immunomodulation function of anti-CD40 was associated with its capability to increase IL-12 secretion while inhibiting IL-4 production. Our study establishes a role for CD40 expressed on B cells or DCs in the costimulation of TDLN cells. Eliciting antitumor activity via simultaneous targeting of CD3 on T cells and CD40 on APCs is relevant for the design of effective T cell-based cancer immunotherapy.

We have previously described the antitumor reactivity of tumor-draining lymph node (TDLN) cells after secondary activation with antibodies. In this report, we examined the effects of interleukin (IL)-12 and IL-18 on modulating the immune function of antibody-activated murine TDLN cells. TDLN cells were activated with anti-CD3/anti-CD28 monoclonal antibody followed by stimulation with IL-12 and/or IL-18. IL-18 in combination with IL-12 showed a synergistic effect in augmenting IFNγ and granulocyte macrophage colony-stimulating factor secretion, whereas IL-18 alone had minimal effect. Concurrently, IL-18 prevented IL-12–stimulated TDLN cells from producing IL-10. The IL-12/IL-18–cultured TDLN cells therefore manifested cytokine responses skewed towards a Th1/Tc1 pattern. IL-12 and IL-18 stimulated CD4+ TDLN cells and enhanced IFNγ production by CD4+ cells to a greater extent than by CD8+ cells. Use of NF-κB p50−/− TDLN cells suggested the involvement of NF-κB in the IL-12/IL-18 polarization effect. Furthermore, a specific NF-κB inhibitor significantly suppressed IL-12/IL-18–induced IFNγ secretion, thus confirming the requirement for NF-κB activation in IL-12/IL-18 signaling. In adoptive immunotherapy, IL-12– and IL-18–cultured TDLN cells infiltrated pulmonary tumor nodules and eradicated established tumor metastases more efficiently than T cells generated with IL-12 or IL-18 alone. Antibody depletion revealed that both CD4+ and CD8+ cells were involved in the tumor rejection induced by IL-12/IL-18–cultured TDLN cells. These studies indicate that IL-12 and IL-18 can be used to generate potent CD4+ and CD8+ antitumor effector cells by synergistically polarizing antibody-activated TDLN cells towards a Th1 and Tc1 phenotype.

<p>Supplemental table S1 documents comparability between final groupings of baseline biopsies used for gene expression comparisons between persistent and regressive sites demonstrating comparability in all key histologic and clinical variables with the exception of higher inflammation in the regressive group. Supplemental table S2 describes the number of differentially expressed genes that distinguish the four initial groups of bronchial biopsies including persistent bronchial dysplasia (BD), regressive BD, progressive non-dysplasia (ND) and stable ND. The number of genes differentially expressed between the groups highlight the finding that differences are greater in respect to the outcome of the lesions at a given site versus the degree of dysplasia in the baseline biopsy. Most notably the difference between regressive BD and stable ND, two groups with histologically distinct baseline biopsies show fewer differentially expressed genes than all but one other group comparison including the histologically indistinguishable persistent and regressive BD groups. These findings are consistent with persistent and regressive sites representing biologically distinct lesions. Supplemental table S3 shows airway locations, temporal histologic features of full airways and clinical characteristics associated with each specimen used for gene expression analyses. The table is arranged by persistent versus regressive status and presents specimens in descending order of baseline histologic score within each group. The specimen numbers correspond to those notated in figure 1B of the manuscript. Supplemental figure S1 shows a heat map using differentially expressed genes to demonstrate the rationale for combining groups in the final comparison. The heatmap demonstrates that persistent BD and progressive ND sites, which are combined to represent persistent sites, show similar gene expression profiles, with only cases Group3_051, Group3_086, Group3_113 and Group4_070 showing profiles that demonstrate similarity to the regressive BD/stable ND group. The regressive BD and stable ND, which are combined to represent regressive sites, also show similar gene expression profiles that are opposite to those seen in the persistent site group with only cases Group2_037, Group2_085 and Group2_092 showing profiles that demonstrate similarity to the persistent BD/progressive ND group. Supplemental tables S4 - S6 present full data genelists for genes associated with pathways showing significantly altered activity in comparisons of persistent and regressive BD (supplemental table S3), genes associated with pathways that are altered in relation to increasing histologic score (supplemental table S4) and upstream regulators related to persistence and histologic score (supplemental table S5). Supplemental table S7 shows documents comparability between final groupings of baseline biopsies used for analyses of the composition of inflammatory infiltrates in comparisons between a validational set of persistent and regressive sites. Comparability is demonstrated in all key histologic and clinical variables including mean inflammation score and percent of high inflammation score biopsies between the two groups. A higher mean baseline histologic score was seen in the persistent group. Supplemental figure S2 shows subset analyses of the validational set of persistent and regressive sites with comparison of these groups by tissue compartment (epithelial and stromal) and overall degree of inflammation (supplemental figures S2A and S2B). Supplemental figure S2C shows the changes in expression of genes from the original gene expression array analysis data for those genes associated with polarization of subsets of inflammatory cells. The genes with significant or near significant change provide evidence that the activity of certain subsets of macrophages and T-lymphocytes correlate with persistence or regression of BD. These subsets are further studied in figure 5 of the main text.</p>