Ian F. C. McKenzie

Size-Dependent Immunogenicity: Therapeutic and Protective Properties of Nano-Vaccines against Tumors

Infection can protect against subsequent disease by induction of both humoral and cellular immunity, but inert protein-based vaccines are not as effective. In this study, we present a new vaccine design, with Ag covalently conjugated to solid core nano-beads of narrowly defined size (0.04–0.05 μm) that localize to dendritic cells (DEC205+ CD40+, CD86+) in draining lymph nodes, inducing high levels of IFN-γ production (CD8 T cells: precursor frequencies 1/5000 to 1/1000) and high Ab titers in mice. Conjugation of Ag to these nano-beads induced responses that were significantly higher (2- to 10-fold) than those elicited by other bead sizes, and higher than a range of currently used adjuvants (alum, QuilA, monophosphoryl lipid A). Responses were comparable to CFA/IFA immunization for Abs and ex vivo peptide-pulsed dendritic cell immunization for CD8 T cells. A single dose of Ag-conjugated beads protected mice from tumors in two different model challenges and caused rapid clearance of established tumors in mice. Thus, a range of Ags conjugated to nano-beads was effective as immunogens in both therapeutic and prophylactic scenarios.

Galα(1,3)Gal, the Major Xenoantigen(s) Recognised in Pigs by Human Natural Antibodies

The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection--mediated by natural antibodies in humans to pig antigens, complement fixation to endothelial cells, and the rapid onset of intravascular coagulation. It is now clear that the major target of the natural IgM and IgG antibodies is the terminal carbohydrate epitope Gal alpha(1,3)Gal, formed by the alpha 1,3galactosyl transferase, which places a terminal galactose residue in an alpha-linkage to another galactose. The alpha 1,3galactosyl transferase in the pig gives rise to very high endothelial cell expression of Gal alpha(1,3)Gal, a ready explanation for the hyperacute rejection of vascularized organs. In addition the parenchuma of liver and kidneys have high levels of Gal alpha-(1,3)Gal. These tissues will all fail in a pig-to-human transplant in what can now be precisely defined in terms of antigen and antibody. We have already made some suggestions for removal of anti-Gal alpha(1,3)Gal antibodies and if the procedure were technically feasible xenotransplantation could be attempted now, especially in patients doomed to a certain death because of the absence of a donor (especially for liver where ex vivo perfusion could be performed). However, the immune system is far from simple, as is shown by the healthy status of mice lacking MHC Class I, Class II or both Class I & II molecules. Perhaps the curtain is about to go up to reveal a new scene! Islets differ from the other tissues and may well not undergo acute antibody-mediated hyperacute rejection--it will be of interest to see how these fare in xenotransplantation models or even in patients. Again, normal individuals do not have anti-islet antibodies; but a proportion of diabetic patients do have such antibodies--whether these will cause hyperacute or acute rejection or are markers of immunity of T-cell type, remains to be seen. Whatever, the area is exciting, is progressing rapidly and, as indicated elsewhere, within a few years we should know whether modified pig tissue can be grafted to some patients. The isolation of the cDNA clone encoding the pig alpha 1,3 galactosyl transferase is an essential first step in the production of a transgenic pig lacking the alpha 1,3Galactosyltransferase and therefore the Gal alpha(1,3)Gal epitope, and such animals could serve as donor for human transplantation.

Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG.

The low affinity receptor for IgG, FcγRII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. FcγRII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human FcγRII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by FcγRII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between FcγRII and the structurally related FcεRI α chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by FcγRII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of FcγRII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.

Distribution of the major xenoantigen (gal(α1–3)gal) for pig to human xenografts

We have previously demonstrated that the major epitope in pig tissues detected by naturally occurring human IgM antibodies is galactose (alpha 1-3)galactose. Subsequent biochemical studies demonstrated this epitope to be present on molecules (Mr40-220kDa) on both endothelial cells and lymphocytes. The objective of the present study was to define the distribution of gal(alpha 1-3)gal in different pig tissues, concentrating on those of relevance for the potential transplantation of pig organs or tissues to humans. Adult pig tissues were obtained fresh, fixed, and stained by the immunoperoxidase technique using biotinylated Griffonia simplicifolia lectin (IB4) which binds only to gal(alpha 1-3)gal, and examined histologically. Endothelial cells in all small vessels (capillaries, arterioles and venules) had a unifrom and dense expression of gal(alpha 1-3)gal; in larger vessels, like the aorta, they were less reactive. The highest concentrations were found in the liver parenchyma which stained uniformly, and in the kidney, where the highest amounts were found in the brush border of the proximal convoluted tubules. There was no staining of collecting ducts or glomeruli (except for endothelium) and moderate staining of the distal convoluted tubules. Heart muscle was nonreactive, although the high density of capillaries indicated a reasonable content of gal(alpha 1-3)gal. In contrast to these tissues was the distribution in the pancreas, which, apart from vessels and the lining of ducts, was nonreactive, i.e. islet cells were essentially lacking in gal(alpha 1-3)gal. Other tissues such as the lung contained moderate amounts of material lining the alveoli and bronchioles.(ABSTRACT TRUNCATED AT 250 WORDS)

Balb/c-h-2db. A new h-2 mutant in balb/ckh that identifies a locus associated with the d region.

A newH-2 mutant, BALB/c-H-2db, is described. This mutant originated in BALB/c, is inbred, and is coisogenic with the parental BALB/cKh strain. The mutation is of the loss type since BALB/c-H-db rejects BALB/c, but not vice versa. Complementation studies have localized the mutation to theD region of theH-2 complex. A cross between BALB/c-H-2db and B10.D2-H-2da failed to complement for either BALB/c or B10.D2 skin grafts, indicating that these are two separate mutations at the same locus (Z2). Direct serological analysis and absorption studies revealed that, with one exception, theH-2 andIa specificities of BALB/c and BALB/c-H-2db are identical. In particular,H-2.4, the H-2Dd private specificity, is quantitatively and qualitatively identical in the two strains. The exception is that of the specificities detected by antiserum D28b: (k×r)F1 anti-h, which contains anti-H-2.27, 28, and 29. These specificities appear to be absent from theH-2db mutant since they are not detected directly or by absorption. Other public specificities are present in normal amounts,e.g., the reaction with antisera to H-2.3, 8, 13, 35, and 36. The reaction with antiserum D28 (f×k)F1 anti-s, which contains antibodies to H-2.28, 36, and 42, is the same in both strains. Antiserum made between the two strains (H-2db anti-H-2d) reacts like an anti-H-2 serum, in that it reacts with both T and B cells by cytotoxicity, but is not a hemagglutinating antibody. The serum reacts as does the D28b serum in both strain distribution and in cross-absorption studies. We conclude that theH-2db mutation occurred at a locus in theD region, resulting in the loss of the H-2.28 public serological specificity and of a histocompatibility antigen. Whether these are one and the same antigen is not yet known. The data, in view of other evidence, imply that the public and private specificities are coded for by separate genes.

Ly-6.2: A New Lymphocyte Specificity of Peripheral T-Cells

A new cell-membrane alloantigen determining locus, Ly-6, has recently been described, and the single specificity Ly-6.2 has been defined by the serum (BALB/c× A)F1 anti-CXBD. Using both fluorescence and cytotoxicity, we found this specificity predominantly on peripheral (extrathymic) T cells, as tissues react thus: thymus, 0–5 percent; spleen, 25 percent; lymph nodes, 69 percent; bone marrow, 15 percent. These reactions agree with the proportion of (Thy+, Ig−) cells present in these tissues. Cortisone-resistant thymus cells were positive. Absorption studies with thymus cells demonstrated the sparse or absent representation of Ly-6.2 on intrathymic T cells. Examination of spleen and lymph node cells from T cell-depleted C57BL/6 mice (after in vitro treatment with anti-Thy-1 serum or examination of tissues of C57BL/6-nu/nu mice) also showed a depletion of Ly-6.2+ cells. Conversely, removal of Ig+ B cells, which caused a relative increase in the number of T cells in the residual population, also increased the number of Ly-6.2+ cells. Additive effects of anti-Thy-1.2 and anti-Ly-6.2 could not be demonstrated, which suggests that the same population was Thy-1.2+, Ly-6.2+. However, additive effects could be shown with an anti-Ia serum and anti-Ly-6.2. The Ly-6.2 specificity is not found on red cells, liver, brain, or antibody-forming cells, but has been identified on a T-cell (but not B-cell) tumor and on kidney. Ly-6.2 can therefore be considered to be a marker for peripheral T cells, and it differs from the Thy-1 and the Ly-1,2,3, and 5 specificities in its relative absence from the thymus.

Switching amino-terminal cytoplasmic domains of alpha(1,2)fucosyltransferase and alpha(1,3)galactosyltransferase alters the expression of H substance and Galalpha(1,3)Gal.

When α(1,2)fucosyltransferase cDNA is expressed in cells that normally express large amounts of the terminal carbohydrate Galα(1,3)Gal, and therefore the α(1,3)galactosyltransferase (GT), the Galα(1,3)Gal almost disappears, indicating that the presence of the α(1,2)fucosyltransferase (HT) gene/enzyme alters the synthesis of Galα(1,3)Gal. A possible mechanism to account for these findings is enzyme location within the Golgi apparatus. We examined the effect of Golgi localization by exchanging the cytoplasmic tails of HT and GT; if Golgi targeting signals are contained within the cytoplasmic tail sequences of these enzymes then a “tail switch” would permit GT first access to the substrate and thereby reverse the observed dominance of HT. Two chimeric glycosyltransferase proteins were constructed and compared with the normal glycosyltransferases after transfection into COS cells. The chimeric enzymes showed Km values and cell surface carbohydrate expression comparable with normal glycosyltransferases. Co-expression of the two chimeric glycosyltransferases resulted in cell surface expression of Galα(1,3)Gal, and virtually no HT product was expressed. Thus the cytoplasmic tail of HT determines the temporal order of action, and therefore dominance, of these two enzymes.

Low molecular weight ia antigens in normal mouse serum. I. Detection and production of a xenogeneic antiserum.

The observation that mouse serum can specifically inhibit cytotoxic Ia antisera indicates that substantial quantities of Ia antigen are present in normal mouse serum. The inhibitory substance in serum is dialyzable and is therefore probably of low molecular weight; it presumably represents a degraded form of cell-bound Ia antigen. A rabbit antiserum specific for murine Ia antigens was obtained by immunizing rabbits with mouse serum and then absorbing the resultant antiserum with dialyzed mouse serum. The binding of this antiserum to mouse leucocytes was detected by an indirect rosetting technique. The specificity of this antiserum was established as follows: (a) serum from mouse strains which possessed the corresponding Ia specificities absorbed out the antileucocyte antibodies; (b) binding of the rabbit antibody to leucocytes was inhibited by mouse anti-Ia sera but not by mouse antibodies against other regions of theH-2 complex; (c) conversely, binding of mouse cytotoxic Ia antibodies to leucocytes was specifically blocked by the rabbit antiserum; (d) antisera produced in rabbits against serum from recombinant mouse strains showed the correct Ia specificities. The rabbit anti-Ia serum was used to demonstrate that 90% of splenic B cells and 40 to 50% of splenic T cells are Ia+. in order of content of Ia+ cells, the different lymphoid populations were ranked spleen > thymus > bone marrow > peripheral blood > lymph node.

Serological and complementation studies in four C57BL/6H-2 mutants

C57BL/6 (H-2b) mice, and four mutants (B6.C-H-2ba, B6-H-2bg1, B6-H-2bg2, B6-H-2bh) derived from this strain after separate mutations had occurred at the same locus within theH-2 complex, were analyzed to determine whether the mutations had led to anyH-2 (or Ia) difference which could be detected serologically. The strains were typed directly with antisera specific for H-2K and H-2D public and private specificities and for the Ia specificities; quantitative absorption studies were also performed for the relevant H-2Kb, H-2Dd and Iab specificities. In no case was any quantitative or qualitative difference detected serologically between any of the strains. In addition, by using a variety of techniques to produce and assay for antibody, we failed to produce any antisera between the parental strains and the four mutants. TheH-2 mutations therefore appear to give rise to a type of antigenic specificity which is recognized byT cells and which generateT, but notB cell responses; nor are they recognized by H-2 or Ia alloantisera. The location of the mutating locus within theH-2 complex was shown by the complementation method to be within theK orIA region and not in theIB region, since crosses of the mutant strains with B10.A(4R) or D2.GD failed to complement for a subsequent C57BL/6 skin graft.

Expression of mucin 1 (MUC1) in esophageal squamous-cell carcinoma: its relationship with prognosis.

Using 2 anti‐mucin 1 (MUC1) monoclonal antibodies (MAbs), DF3 and BCP8, we examined MUC1 expression immunohistochemically in 192 esophageal squamous‐cell carcinomas (SCCs). In normal squamous epithelium of the esophagus, DF3 was not expressed, but BCP8 was expressed on the cell membrane, mainly in the surface layer. In esophageal SCCs, DF3 and BCP8 were expressed mainly on the cell membrane of SCC cells, but also in the cytoplasm in several cases. To analyze the correlation of MUC1 expression and the prognosis of the patients, the 192 cases were divided into 2 groups: high‐expression group (HEG, >50% of the neoplastic cells stained) and low‐expression group (LEG, <50% of neoplastic cells stained). DF3‐HEG (24 patients) showed a significantly poorer survival rate than DF3‐LEG (168 patients), whereas there was no significant difference in survival between BCP8‐HEG (43 patients) and BCP8‐LEG (149 patients). Also, in the analysis of 162 patients with advanced stage (submucosal or deeper invasion) to exclude the influence of low expression of DF3 and BCP8 in 30 patients with early stage (up to the level of muscularis mucosae), DF3‐HEG (24 patients) showed significantly poorer survival than DF3‐LEG (138 patients), whereas there was no significant difference in survival between BCP8‐HEG (42 patients) and BCP8‐LEG (120 patients). The results of our study on esophageal SCC suggest that the expression of sialyl oligosaccharides detected by DF3 is related to poor prognosis. Int. J. Cancer (Pred. Oncol.) 84:251–257, 1999.