Randall N. Knibbs

Mechanisms involved in radiation enhancement of intratumoral dendritic cell therapy.

We have previously reported that local tumor irradiation, without inducing cell death, can augment the therapeutic efficacy of intratumoral (IT) dendritic cell (DC) vaccination. This study examined potential mechanisms underlying radiation enhancement of IT DC therapy in this setting. Even though ionizing radiation did not mediate tumor cell killing, bone marrow-derived DCs acquired in vitro tumor antigens from irradiated D5 murine melanoma cells more efficiently than from untreated cells. This radiation-enhanced loading of DCs did not induce DC maturation, but was associated with improved cross-priming of T cells both in vitro and in vivo. Furthermore, in vivo pulsing of DCs with irradiated versus untreated tumor cells resulted in superior presentation of tumor antigens to T cells. In addition, tumor irradiation facilitated homing of IT administered DCs to the draining lymph node, possibly by down-regulating CCL21 expression within the tumor mass. Studies of the tumor microenvironment in irradiated versus untreated tumors did not reveal significant inflammatory changes. Moreover, radiation did not promote accumulation of CD4+ or CD8+ effector T cells within solid tumors. Our results indicate that, without inducing cytotoxicity, tumor irradiation can enhance the ability of DCs to capture tumor antigens, migrate to the draining lymph node, and present processed antigens to T cells. These findings may prove useful in designing future strategies for human cancer immunotherapy.

E- and P-selectins are essential for the development of cockroach allergen-induced airway responses.

Peribronchial inflammation contributes to the pathophysiology of allergic asthma. In many vascular beds, adhesive interactions between leukocytes and the endothelial surface initiate the recruitment of circulating cells. Previous studies using OVA-induced airway hyperreactivity indicated that P-selectin, a member of the selectin family expressed by activated platelets and endothelium, contributed to both inflammation and bronchoconstriction. The current study used cockroach allergen (CRA), an allergen that induces asthmatic responses in both humans and mice, to further investigate the role of selectins in the development of peribronchial inflammation and airway hyperreactivity. P- and E-selectin mRNAs were detected in extracts of CRA-sensitized animals beginning shortly after intratracheal challenge with CRA. The P-selectin mRNA was transiently induced at early time points while up-regulation of the E-selectin mRNA was more prolonged. Mice with targeted deletions in E-selectin (E−), P-selectin (P−), and both genes (E−/P−) showed 70–85% reductions in airway hyperreactivity, peribronchial inflammation, and eosinophil accumulation. The P− and E−/P− groups showed the most profound reductions. The transfer of splenic lymphocytes from CRA-primed E−/P− into naive wild-type (WT) mice produced the same level of airway hyperreactivity as transfers from CRA-primed WT into naive WT hosts, indicating that peripheral immunization was similar. The observed changes in the selectin-deficient animals were not related to inadequate sensitization, because CRA priming and challenge increased serum IgE levels. Furthermore, pulmonary Th2-type cytokines and chemokines in the E-selectin−/− and WT animals were similar. The findings indicate that both P- and E-selectin contribute to CRA-induced peribronchial inflammation and airway hyperreactivity.

Donor cell cycling, trafficking, and accumulation during adoptive immunotherapy for murine lung metastases.

Adoptive cellular immunotherapy treats metastatic cancer by infusing cultured T cells derived from resected tumors or primed lymph nodes. The infused cells must accumulate in metastatic lesions to suppress growth; however, this process and the resulting clinical response are dynamic and evolve during the days and weeks following cell infusion. This study used novel experimental techniques to determine the fate of infused, cultured tumor-draining lymph node (TDLN) cells during the treatment of murine pulmonary micrometastases. After infusion, the cultured TDLN cells accumulated in the pulmonary vasculature, systemic lymph nodes, and spleen. Donor cells were initially confined to alveolar capillaries with no movement into metastases. Within 4 h, TDLN cells began migrating across pulmonary postcapillary venules and first appeared within metastases. After 24 h, most donor cells in the lung were associated with tumor nodules. Donor cell proliferation within the lung and lymphoid organs was detected within 24 h of infusion and continued throughout the 5-day period of observation. Furthermore, those proliferating in lymphoid organs trafficked back to the tumor-bearing lungs, accounting for ∼50% of the donor cells recovered from these sites after 5 days. Finally, donor T cells entering metastases both early (within 1–2 days) and late (after 2 days) suppressed tumor growth, but the early recruits accounted for most of the therapeutic response. Thus, cultured TDLN cells migrate directly into tumor-bearing organs and seed the recirculating pool of lymphocytes after infusion. Small fractions of the later differentiate in lymphoid organs and migrate into the lungs but appear less effective than effector cells in the initial bolus.

Attachment, spreading and growth in vitro of highly malignant and low malignant murine fibrosarcoma cells

Highly malignant cell lines and low-malignant cell lines isolated from three different methylcholanthrene-induced murine fibrosarcomas were examined for their ability to attach to plastic dishes and collagen-coated dishes under serumfree conditions and in the presence of serum. Most of the cells from the three highly malignant lines attached and spread under all conditions. By 72h, there was a significant increase in the number of cells indicating that at least some of the cells had undergone division (even in the absence of serum). In contrast, fewer of the cells from the three low-malignant lines attached and spread on the plastic or collagen substrates in the absence of serum or in the presence of 0.1 per cent serum. However, when 15μg laminin per dish was added along with the lowmalignant cells, they then attached and spread on the plastic and collagen-coated dishes. Previous studies have indicated that the highly malignant lines express cell surface antigens that cross-react with laminin while the low-malignant cell lines do not. We speculate that the differences between the high- and low-malignant cells in the expression of cell surface laminin-like antigens contribute to the dissimilarities in attachment and spreading capacity. These differences may also contribute to the dissimilarity between these cells in malignant potential.

L-Selectin Serves as an E-Selectin Ligand on Cultured Human T Lymphoblasts

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by ∼60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, ∼60–70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.

Subset-Specific Reductions in Lung Lymphocyte Accumulation Following Intratracheal Antigen Challenge in Endothelial Selectin-Deficient Mice

We previously demonstrated induction and expression of CD62E and CD62P in the lungs of mice primed and then challenged with intratracheal (i.t.) SRBC. The current study examined accumulation of endogenous lymphocytes in the lungs of endothelial E- and P-selectin-deficient (E−P−) mice after i.t. SRBC challenge. Compared with syngeneic wild-type (wt) mice, E−P− mice showed an 85–95% decrease in CD8+ T cells and B cells in the lungs at both early and late time points. In contrast, CD4+ T cell accumulation was reduced by ∼60% early, but equivalent to wt levels later. Surprisingly, many γδ T cells were found in lungs and blood of E−P− mice but were undetectable in the lungs and blood of wt mice. Absolute numbers of peripheral blood CD4, CD8, and B lymphocytes in E−P− mice equaled or exceeded the levels in wt mice, particularly after challenge. Trafficking studies using αβ T lymphoblasts confirmed that the recruitment of circulating cells after challenge was markedly reduced in E−P− mice. Furthermore, Ag priming occurred normally in both the selectin-deficient and wt mice, because primed lymphocytes from both groups transferred Ag sensitivity into naive wt mice. Lung production of mRNA for six CC and two CXC chemokines after challenge was equivalent by RT-PCR analysis in wt and E−P− mice. Therefore, reduced lung accumulation of αβ T cells and B cells in E−P− mice did not result from reduced delivery of circulating lymphocytes to the lungs, unsuccessful Ag priming, or defective pulmonary chemokine production. Selectin-dependent lymphocyte recruitment into the lungs following i.t.-SRBC challenge is subset specific and time dependent.

Tumor-Specific Responses in Lymph Nodes Draining Murine Sarcomas Are Concentrated in Cells Expressing P-Selectin Binding Sites

Tumor-draining lymph node (TDLN) cells develop substantial antitumor activity after activation on immobilized αCD3 and culture in low-dose IL-2. This study found that the minor subset of TDLN T cells expressing binding sites for the adhesion receptor P-selectin (Plighigh T cells) produced T lymphoblasts with the most tumor-specific IFN-γ synthesis in vitro and antitumor activity following adoptive transfer in vivo. The Plighigh T cells constituted <25% of the cells with the phenotype of recently activated cells including high levels of CD69, CD44, or CD25, and low levels of CD62L. The cultured Plighigh TDLN were 10- to 20-fold more active against established pulmonary micrometastases than cultured unfractionated TDLN, and >30-fold more active than cultured TDLN cells depleted of the Plighigh fraction before expansion (Pliglow cells). Tumor-specific IFN-γ synthesis in vitro paralleled the antitumor activities of the cultured fractions in vivo, implying that increased Tc1 and Th1 effector functions contributed to the tumor suppression. Neither nonspecific interaction with the P-selectin chimera used for sorting nor endogenous costimulatory activity in the Plighigh fraction accounted for the marked increase in antitumor activities after culture. The cultured Plighigh fraction contained a variety of potential effector cells; however, the CD8 and CD4 subsets of αβ T cells accounted for 95–97% of its antitumor activity. The authors propose that P-selectin sorting increased antitumor activities by concentrating Tc1 and Th1 pre-effector/effector cells before culture.

Monoclonal antibodies that recognize the trisaccharide epitope Galα1-3Galβ1-4GlcNAc present on Ehrlich tumor cell membrane glycoproteins

Monoclonal antibodies were prepared against the trisaccharide Galα1-3Galβ1-4GlcNAc, a sequence which occurs on the surface of Ehrlich ascites tumor cells as well as in thyroglobulin, laminin and a variety of other proteins. This was accomplished by immunizing BALB/c mice with the fraction of Ehrlich cell membrane glycoproteins obtained by affinity chromatography on aGriffonia simplicifolia I (GS I) column which selectively binds α-d-galactosyl-terminated structures. Detection of Galα1-3Galβ1-4GlcNAc-specific antibodies was accomplished by employing glycoproteins containing the trisaccharide sequence; fusion with spleen cells from an immunized mouse was accomplished in the presence of polyethylene glycol (PEG1500). An enzyme-linked immunosorbent assay (ELISA) system was used to identify two clones (2.10G and 6.8E), which recognized the desired trisaccharide conjugate. These clones also recognized a thyroglobulin fraction isolated by GS I affinity chromatography and murine laminin, both of which possess the Galα1-3Galβ1-4GlcNAc sequence. Inhibition of antibody-trisaccharide reactivity, examined employing an ELISA assay, revealed that two trisaccharides, Galα1-3Galβ1-4GlcNAc/Glc, were the best inhibitory haptens; Galβ1-4GlcNAc (LacNAc), Galα1-3Gal and Galβ1-4Glc (lactose) were poor inhibitors. Indirect immunofluorescence staining of unfixed Ehrlich cells using the monoclonal antibody at 4° C revealed fluorescence over the entire cell surface. Indirect immunogold labeling of semithin and ultrathin sections of aldehyde fixed and Lowicryl K4M-embedded Ehrlich cells resulted in specific labeling of the cell surface and internal structure. Immunoblot analysis revealed that removal of the α-galactosyl residues of laminin by α-galactosidase abolished reactivity with the monoclonal antibodies. The availability of this antibody, which belongs to the IgM family of immunoglobulins, now makes possible the detection of this sugar sequence on cells and tissue sections, as well as on glycoproteins in solution.

Sustained High-Yield Production of Recombinant Proteins in Transiently Transfected COS-7 Cells Grown on Trimethylamine-Coated (Hillex) Microcarrier Beads

The present study shows that COS‐7 cells transiently transfected and maintained on positively charged (trimethylamine‐coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high‐level synthesis was observed with secreted chimeric proteins (murine E‐selectin– and P‐selectin‐human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte‐macrophage colony‐stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine‐coated microcarriers than to polystyrene flasks. After 10–14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5‐fold increase in protein production from a single transfection over two weeks. Thus, microcarrier‐based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.

Lymphocyte-endothelial cell adhesive interactions in lung immunity: lessons from the murine response to particulate antigen.

The adhesive interaction between lymphocytes and lung endothelial cells presents an attractive arena for the development of novel therapeutic agents to modify pathologic pulmonary immune responses. The conceptual basis for choosing molecular targets to modulate this adhesive interaction derives, in large part, from results of murine experimental model systems of the pulmonary immune response. This article reviews one such model, the response of primed C57BL/6 mice to the particulate antigen sheep erythrocytes. Novel data are presented on the effect of a blocking anti-alpha(4) integrin monoclonal antibody on lung leukocyte and lymphocyte subset accumulation after intratracheal (IT) antigen challenge. Results from this model system have indicated that lymphocytes may use either the endothelial selectins or alpha(4) integrin as independent pathways to initiate recruitment into the lungs.