Testicular immune cell populations and macrophage polarisation in adult male mice and the influence of altered activin A levels.

Detailed morphological characterization of testicular leukocytes in the adult CX3CR1 gfp/+ transgenic mouse identified two distinct CX3CR1 + mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3+ (T lymphocytes) and NK1.1+ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206+MHCII+ sub-population (12% of total CD45+ cells). Rare testicular subsets of CX3CR1 +CD11c+F4/80+ (4.3%) mononuclear phagocytes and CD3+NK1.1+ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba+/-) or elevated activin A (Inha+/-) were assessed using flow cytometry. Although the proportion of F4/80+CD11b+ leukocytes (macrophages) was not affected, the frequency of CD206+MHCII+cells was significantly lower and CD206+MHCII- correspondingly higher in Inha+/- testes. This shift in expression of MHCII in CD206+ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.

An in vitro model demonstrates the potential of neoplastic human germ cells to influence the tumour microenvironment.

Testicular germ cell tumours (TGCT) typically contain high numbers of infiltrating immune cells, yet the functional nature and consequences of interactions between GCNIS (germ cell neoplasia in situ) or seminoma cells and immune cells remain unknown. A co-culture model using the seminoma-derived TCam-2 cell line and peripheral blood mononuclear cells (PBMC, n = 7 healthy donors) was established to investigate how tumour and immune cells each contribute to the cytokine microenvironment associated with TGCT. Three different co-culture approaches were employed: direct contact during culture to simulate in situ cellular interactions occurring within seminomas (n = 9); indirect contact using well inserts to mimic GCNIS, in which a basement membrane separates the neoplastic germ cells and immune cells (n = 3); and PBMC stimulation prior to direct contact during culture to overcome the potential lack of immune cell activation (n = 3). Transcript levels for key cytokines in PBMC and TCam-2 cell fractions were determined using RT-qPCR. TCam-2 cell fractions showed an immediate increase (within 24 h) in several cytokine mRNAs after direct contact with PBMC, whereas immune cell fractions did not. The high levels of interleukin-6 (IL6) mRNA and protein associated with TCam-2 cells implicate this cytokine as important to seminoma physiology. Use of PBMCs from different donors revealed a robust, repeatable pattern of changes in TCam-2 and PBMC cytokine mRNAs, independent of potential inter-donor variation in immune cell responsiveness. This in vitro model recapitulated previous data from clinical TGCT biopsies, revealing similar cytokine expression profiles and indicating its suitability for exploring the in vivo circumstances of TGCT. Despite the limitations of using a cell line to mimic in vivo events, these results indicate how neoplastic germ cells can directly shape the surrounding tumour microenvironment, including by influencing local immune responses. IL6 production by seminoma cells may be a practical target for early diagnosis and/or treatment of TGCT.

Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets.

Xenotransplantation of porcine islets into diabetic non-human primates is characterized by (i) an initial massive graft loss possibly due to the instant blood-mediated inflammatory reaction and (ii) the requirement of intensive, clinically unfriendly immunosuppressive therapy. We investigated whether the transgenic expression of a human complement-regulatory protein (hCD46) on porcine islets would improve the outcome of islet xenotransplantation in streptozotocin-induced diabetic Cynomolgus monkeys. Immunosuppression consisted of thymoglobulin, anti-CD154 mAb for costimulation blockade, and mycophenolate mofetil. Following the transplantation of islets from wild-type pigs (n = 2) or from 1,3-galactosyltransferase gene-knockout pigs (n = 2), islets survived for a maximum of only 46 days, as evidenced by return to hyperglycemia and the need for exogenous insulin therapy. The transplantation of islets from hCD46 pigs resulted in graft survival and insulin-independent normoglycemia in four of five monkeys for the 3 months follow-up of the experiment. One normalized recipient, selected at random, was followed for >12 months. Inhibition of complement activation by the expression of hCD46 on the pig islets did not substantially reduce the initial loss of islet mass, rather was effective in limiting antibody-mediated rejection. This resulted in a reduced need for immunosuppression to preserve a sufficient islet mass to maintain normoglycemia long-term.

Human dendritic cells pulsed with specific lipopeptides stimulate autologous antigen-specific T cells without the addition of exogenous maturation factors.

SUMMARY: Serum-free culture conditions to generate immature human monocyte-derived DC (Mo-DC) were optimized, and the parameters that influence their maturation after exposure to lipopeptides containing CD4(+) and CD8(+) T-cell epitopes were examined. The lipopeptides contained a single CD4(+) helper T-cell epitopes, one of a number of human leucocyte antigen (HLA)-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. To ensure complete maturation of the Mo-DC, we examined (i) the optimal lipopeptide concentration, (ii) the optimal Mo-DC density and (iii) the appropriate period of exposure of the Mo-DC to the lipopeptides. The results showed that a high dose of lipopeptide (30 microm) was no more efficient at upregulating maturation markers on Mo-DC than a low dose (6 microm). There was an inverse relationship between Mo-DC concentration and the mean fluorescence intensity of maturation markers. In addition, at the higher cell concentrations, the chemotactic capacity of the Mo-DC towards a cognate ligand, CCL21, was reduced. Thus, high cell concentrations during lipopeptide exposure were detrimental to Mo-DC maturation and function. The duration of exposure of Mo-DC to the lipopeptides had little effect on phenotype, although Mo-DC exposed to lipopeptides for 48 rather than 4 h showed an increased ability to stimulate autologous peripheral blood mononuclear cells to release interferon-gamma in the absence of exogenous maturation factors. These findings reveal conditions for generating mature antigen-loaded DC suitable for targeted immunotherapy.

Mechanism of measles virus-induced suppression of inflammatory immune responses.

Measles virus (MV) causes profound immunosuppression, resulting in high infant mortality. The mechanisms are poorly understood, largely due to the lack of a suitable animal model. Here, we report that particular MV proteins, in the absence of MV replication, could generate a systemic immunosuppression in mice through two pathways: (1) via MV-nucleoprotein and its receptor FcgammaR on dendritic cells; and (2) via virus envelope glycoproteins and the MV-hemagglutinin cellular receptor, CD46. The effects comprise reduced hypersensitivity responses associated with impaired function of dendritic cells, decreased production of IL-12, and the loss of antigen-specific T cell proliferation. These results introduce a novel model for testing the immunosuppressive potential of anti-measles vaccines and reveal a specific mechanism of MV-induced modulation of inflammatory reactions.

Genetic engineering for xenotransplantation.

Xenotransplantation is being pursued vigorously to solve the shortage of allogeneic donor organs. Experimental studies of the major xenoantigen (Gal) and of complement regulation enable model xenografts to survive hyperacute rejection. When the Gal antigen is removed or reduced and complement activation is controlled, the major barriers to xenograft survival include unregulated coagulation within the graft and cellular reactions involving macrophages, neutrophils, natural killer (NK) cells, and T lymphocytes. Unlike allografts, where specific immune responses are the sole barrier to graft survival, molecular differences between xenograft and recipient that affect normal receptor-ligand interactions (largely active at the cell surface and which may not be immunogenic), are also involved in xenograft failure. Transgenic strategies provide the best options to control antigen expression, complement activation, and coagulation. Although the Gal antigen can be eliminated by gene knockout in mice, that outcome has only become a possibility in pigs due to the recent cloning of pigs after nuclear transfer. Instead, the use of transgenic glycosyl transferase enzymes and glycosidases, which generate alternative terminal carbohydrates on glycolipids and glycoproteins, has reduced antigen in experimental models. As a result, novel strategies are being tested to seek the most effective solution. Transgenic pigs expressing human complement-regulating proteins (DAF/CD55, MCP/CD46, or CD59) have revealed that disordered regulation of the coagulation system requires attention. There will undoubtedly be other molecular incompatibilities that need addressing. Xenotransplantation, however, offers hope as a therapeutic solution and provides much information about homeostatic mechanisms.

A therapeutic human anti-idiotypic antibody mimics CD55 in three distinct regions.

The human anti-idiotypic antibody 105AD7 was isolated from a colorectal cancer patient receiving the anti-tumor antibody 791T/36 for radioimmuno-scintigraphy of liver metastases. We have mapped the binding site of 791T/36 to the first two small consensus repeat (SCR) domains of the complement regulatory protein (CD55) that is overexpressed by a wide range of solid tumors. Cloning of both antigen and anti-idiotype has identified the molecular basis of their mimicry. Amino acid homology has been identified between three complementarity-determining regions of 105AD7 and three regions of CD55 within the first two SCR domains. 791T/36 and anti-anti-idiotypic (Ab3) polyclonal antibodies raised against 105AD7 showed specific binding to these peptides. The antibodies were also found to bind synergistically to combinations of these peptides, indicating cooperativity between the peptides in stabilizing antibody binding. This also implies that the contact face on both CD55 antigen and 105AD7 is generated by the cooperation of several peptides positioned on two domains in each protein. Thus a human monoclonal anti-idiotypic antibody generated by a cancer patient is able to show both amino acid and structural homology with the complement regulatory protein CD55. These findings help identify the mechanism by which a human anti-idiotypic antibody is able to mimic a tumor-associated antigen and stimulate anti-tumor B and T cell responses.

Inhibition of hyperacute transplant rejection by soluble proteins with the functional domains of CD46 and FcgammaRII.

BACKGROUND: Recombinant soluble forms of complement regulatory molecules, including the human complement regulatory protein CD46 (rsCD46), have been shown to inhibit hyperacute transplant rejection (HAR) and protect against complement-mediated inflammatory tissue damage. Similarly, recombinant soluble forms of the immunoglobulin receptor FcgammaRII (rsFcgammaRII) can attenuate antibody-mediated inflammatory responses. We have produced and tested the function of novel recombinant chimeric proteins that incorporate the functional domains of both CD46 (membrane cofactor protein, MCP) and the low affinity human IgG receptor FcgammaRII (CD32). METHODS: Two recombinant soluble chimeric proteins (CD46:FcR and FcR:CD46) were designed and produced using a human cell expression system. Their ability to protect cells against complement-mediated lysis (through the CD46 domain) and bind human IgG (through the Fc receptor domain) was assessed in vitro. They were also tested in vivo in the rat reverse passive Arthus reaction and a murine model of hyperacute cardiac transplant rejection. RESULTS: In vitro, the functional domains of the chimeric proteins each retained their activity. In vivo, the serum half-life of the recombinant chimeric proteins in mice was more than either rsCD46 or rsFcgammaRII. In the rat reverse passive Arthus reaction, intradermal injection of each recombinant protein substantially reduced inflammatory skin edema (>50%) and polymorphonuclear neutrophil infiltration (>90%). In the hyperacute rejection model, i.v. treatment with FcR:CD46 prevented complement-mediated rejection, macroscopic bruising, edema, and thrombosis more effectively than rsCD46. CONCLUSIONS: CD46/FcgammaRII bifunctional proteins have an improved ability to control complement-mediated hyperacute graft rejection and have therapeutic potential in other conditions involving antibody-mediated inflammation.

Interaction of CD46 with measles virus: accessory role of CD46 short consensus repeat IV.

To define further the accessory role(s) of the CD46 (membrane cofactor protein) short consensus repeat (SCR) III and IV domains in the interaction of CD46 with measles virus (MV), chimeric proteins were generated by substituting domains from the structurally related protein decay accelerating factor (DAF, CD55): x3DAF (exchange of CD46 SCR III) and x4DAF (exchange of SCR IV). Transfected CHO cell lines that stably expressed these chimeric proteins were compared for MV binding and infection. Compared with wild-type CD46 (I-II-III-IV), a significant decrease in MV binding was observed with x4DAF. Despite this limited binding, these cells were still capable of supporting virus entry. In a quantitative fusion assay, no significant differences in fusion were observed as a result of the exchange of either CD46 SCR III or IV. However, the down-regulation of cell surface CD46 typically observed following MV infection was abolished with x4DAF, as was the redistribution of CD46 on the cell surface. Thus, CD46 SCR IV appears to be required for optimal virus binding and receptor down-regulation, although importantly, in spite of these functional limitations, x4DAF can still be used for MV entry.

Chimeric CD46/DAF molecules reveal a cryptic functional role for SCR1 of DAF in regulating complement activation.

Chimeric proteins using membrane cofactor (CD46) and decay accelerating factor (DAF or CD55) were generated to further investigate the functional domains involved in the regulation of human serum complement. Following activation of the classical pathway, the isolated substitution of CD46 SCR III (x3DAF) exhibited a modest regulatory activity comparable to that of CD46. The isolated substitution of CD46 SCR IV (x4DAF), and the combined CD46 SCR III+IV substitutions (x3/4DAF) were essentially as efficient as DAF. No regulation of C3b deposition was observed with the combined CD46 SCR I+II substitutions (x1/2DAF). When tested after activation of the alternative pathway, both the x3DAF and x3/4DAF chimeras failed to regulate C3b deposition, while the x4DAF chimera still displayed some activity. In contrast to that observed following classical pathway activation, the x1/2DAF chimera exhibited a similar efficiency to wild type CD46 and DAF in controlling C3b deposition. Using SCR specific antibodies, the regulatory activity of the x1/2DAF chimera against the alternative pathway was mapped to the first three distal SCR (i.e. DAF 1, DAF 2 and CD46 III). These data demonstrate that several combinations of SCR domains from two related complement regulators can result in functional molecules, and reveal a novel and cryptic functional role for DAF SCR1.

Autoreactive cytotoxic T cells in mice are induced by immunization with a conserved mitochondrial enzyme in Freund's complete adjuvant.

Standard methods to generate autoimmune reactions in mice, by immunization with antigens emulsified with adjuvants, stimulate strong helper (CD4) T-cell and antibody responses but are not reported to induce cytolytic CD8 T cells. The aim of this study was to assess whether specific autoreactive CD8 T cells could be readily generated after immunization with a 'weak' autoantigen in adjuvant. Mice were immunized intraperitoneally three times with the E3 subunit of the mitochondrial 2-oxoacid dehydrogenase enzyme complexes (dihydrolipoamide dehydrogenase) emulsified with Freund's complete adjuvant. Splenic and lymph node lymphocytes were harvested after 14 days for in vitro functional studies. T lymphocytes were tested for proliferative responses and cytotoxicity against antigen-loaded isogeneic target cells. An autoreactive cytolytic T lymphocyte (CTL) response was detectable only after the in vitro restimulation of lymphocytes with E3 antigen-loaded syngeneic splenocytes. These CTL were identified as H-2-restricted CD8+ T cells. A proliferative response to E3 was demonstrable against antigen-pulsed syngeneic splenocytes. Immunized mice also generated strong antibody responses to E3. Liver histology showed portal infiltrates interpreted as a response of the liver to a non-specific immunological stimulus. It is concluded that autoreactive cytolytic T cells can be generated experimentally upon appropriate stimulation of the immune system, and can be identified in vitro upon release from the controlling mechanisms that are likely to regulate them in vivo.

Current and future prospects for xenotransplantation.

The transplantation of organs and tissues between animal species, or xenotransplantation, is the focus of a growing field of research, owing primarily to the increasing shortage of allogeneic donor organs. The pig stands out as the most suitable donor animal for humans; however, xenografts (e.g. pig organs) used for human transplantation are normally destroyed by the host within minutes by hyperacute xenograft rejection. An improved understanding of the immune recognition and rejection of xenografts has resulted in new therapies that can partially overcome hyperacute rejection (HAR), delayed xenograft rejection (DXR) or acute vascular xenograft rejection. Strategies to diminish immunogenicity following xenotransplantation can be divided into two approaches: those directed at the recipient (e.g. antibodies or complement depletion or inhibition and tolerance induction) and those directed at the donor (e.g. transgenic modifications to express human complement-regulatory proteins or removal or displacement of alphaGal epitopes). DXR is likely to be controlled by transgenic inhibition of endothelial cell activation (e.g. inhibition of NF-kappaB). Transgenic pigs required for xenotransplantation will soon be generated at a greater efficiency and precision using nuclear transfer and cloning when compared to pronuclear injection. Of greater significance is that nuclear transfer offers the ability to target gene insertion selectively to specific gene loci and to delete specific genes in the pig. Experimental pig-to-primate organ xenotransplantation is currently under way, and results show increased transplant function from minutes to days and weeks. The final therapeutic regimen that allows survival of a discordant xenograft is likely to involve a combination of 'modified' functional genes in the donor organ, the development of immunological tolerance to pig antigens and administration of novel therapeutic agents, including immunosuppressants, that can control natural killer (NK) cell and monocyte mediated responses.

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.

Interactions between the ectodomains of haemagglutinin and CD46 as a primary step in measles virus entry.

Recombinant soluble forms of the ectodomains of measles virus haemagglutinin (sH) and of its receptor CD46 (sCD46) were obtained as a purified disulphide-bonded sH homodimer with an apparent molecular mass of 160 kDa and a purified sCD46 monomer with an apparent molecular mass of 60 kDa, without detectable contamination with moesin. Purified sH bound to purified and immobilized sCD46 and this binding was specifically inhibited by sCD46 in solution. sCD46 bound to wild-type H expressed on the cell surface and inhibited measles virus binding to CD46-expressing cells. Binding of sCD46 to cell surface H was increased about twofold when measles virus fusion protein was coexpressed with H. sH bound to wild-type cell surface CD46 and inhibited measles virus binding onto CD46-expressing cells. sCD46 also inhibited virus infection. Thus, the direct interaction between the ectodomains of H and CD46 is likely to be the primary event in measles virus infection.

Translation is enhanced after silent nucleotide substitutions in A+T- rich sequences of the coding region of CD46 cDNA.

Specific sequences in the coding region of CD46 (membrane cofactor protein) transcripts have been shown to have a marked effect on translation. Two A+T-rich regions of CD46 cDNA were altered by mutation without changing the CD46 amino acid sequence (silent nucleotide substitution). In one region, the A+T content was reduced from 78% to 55% and in the other a putative polyadenylation addition sequence was disrupted. In each example, mutated sequences transfected into COS-7 cells produced significantly more soluble or cell surface protein (up to a 20-fold increase) than wild-type sequences. The amount of cellular plasmid DNA and CD46 mRNA was not increased, suggesting that the effect was not due to increased transfection efficiency, or transcript synthesis or stability. Biosynthetically labelled transfected cells showed an increase in translation rate but cell-free in vitro translation studies demonstrated that wild-type and mutated transcripts were translated with similar efficiency. The data show that translation of CD46 is affected by specific mRNA coding sequences, 400-540 bases from the initiation codon, and suggest that these sequences require the structural integrity of the cell to exert their effect.

Engineering of recombinant soluble CD46: an inhibitor of complement activation.

Human CD46 (membrane cofactor protein) is a type 1 glycoprotein that functions to protect autologous cells from complement-mediated damage by binding C3b and C4b for their factor I-mediated cleavage. We now describe the production and function of recombinant soluble CD46 (rsCD46), which was produced as a truncated form by mutagenesis using the splice overlap extension polymerase chain reaction, by inserting a translational stop codon into the CD46 cDNA at the junction of the transmembrane and extracellular domains. After transfection of an expression construct into 293-EBNA (Epstein-Barr nuclear antigen)-transformed cells, secretion of rsCD46 protein was detected by immunoradiometric assay using monoclonal antibodies. Following a single-step immunoaffinity purification, the protein resolved as a single band of approximately 56,000 MW on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified rsCD46 (51 micrograms/ml) protected Chinese hamster ovary (CHO) cells from lysis initiated by a high titre rabbit anti-CHO antibody and complement from rabbit or human. The protection was specifically mediated by rsCD46 because the monoclonal antibody M177, which blocks interaction between CD46 and C3b/C4b, abrogated the protection. The results demonstrate that rsCD46 is effective as a fluid-phase regulator of complement activation on cell surfaces, even when initiated by the classical complement pathway. The in vivo efficacy of rsCD46 was investigated using a mouse heart to rat xenograft model. Administration of a bolus injection of rsCD46 was effective at delaying hyperacute graft rejection. These data suggest that rsCD46 may have a role as a therapeutic agent.

A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro.

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement-mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35 . More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase. In this report, the ability of rsCD46, rsD55 and rsCD35 to regulate human complement activation mediated by the classical pathway in vitro was clearly demonstrated by all three soluble proteins; however, rsCD35 was a more effective inhibitor than either rsCD46 or rsCD55. A combination of rsCD46+ rsCD55 was more potent than either of these proteins alone. Cell lysis via alternative pathway activation in vitro was efficiently regulated by rsCD46 and rsCD35 to a similar extent, whereas rsCD55 was not effective. Assays of rsCD46 in vivo have previously not been possible due to difficulties in expressing sufficient quantities of protein. This limitation has been overcome and now we report the ability of rsCD46 to inhibit immune complex-mediated inflammation in a rat using the reverse passive Arthus reaction model. Administration of rsCD46 significantly reduced the size of lesion, and histological examination showed a reduction in inflammatory infiltrate and edema. These data suggest that rsCD46, in addition to rsCd55 and rsCD35, may be useful a therapeutic agent.

CTL in mice immunized with human mucin 1 are MHC-restricted.

CTLs that recognize tumor Ags have been described in mice and humans, particularly for melanoma. These CTLs are CD8+, which is MHC-restricted. In contrast, in human carcinomas of the breast, pancreas, or ovary, and in multiple myeloma, CD8+ CTLs have been described that lyse targets expressing human MUC1 in a non-MHC-restricted manner. On the basis of these observations, we immunized mice with conjugates of mannan-human fusion protein, human mucin 1 (MUC1), which produced CD8+ CTLs. In contrast to the human anti-MUC1 CTLs found in cancer patients, the murine anti-MUC1 CTLs were clearly MHC-restricted, e.g., in inbred mice of the H-2-b, d, k, s, or z haplotypes; the H-2 restriction was also confirmed in H-2 congenic strains. Tests of H-2 recombinant strains demonstrated that MUC1 peptides were able to associate with D or K class I molecules of the b, d, or k haplotypes. Mice lacking MHC-class I molecules made weak CTL responses that were H-2Db-restricted, and in the class I H-2Kbm1 mutant strain, CTL restriction was also shown. Finally, cold target inhibition studies demonstrated that Kb and Db are recognized similarly, but Kk is less well recognized. Thus, anti-MUC1 CTLs induced by immunization of mice are different from those obtained from patients. The immunization of cancer patients with MUC1 peptides is now undergoing clinical trials and it will be of interest to observe whether the CTLs induced are HLA-restricted, not restricted, or whether both types of CTLs are produced.

Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses.

The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a T1-type response to such synthetic antigens. We report that T1-type (generating high CTL, low antibody) or T2-type (the reciprocal) responses can be induced by conjugation of the antigen to the carbohydrate polymer mannan: T1 responses are selected by using oxidizing conditions; T2 responses are selected by using reducing conditions for the conjugation. Using human MUC1 as a model antigen in mice, immunization with oxidized mannan-MUC1 fusion protein (ox-M-FP) led to complete tumor protection (challenge up to 5 x 10(7) MUC1+ tumor cells), CTLs, and a high CTL precursor (CTLp) frequency (1/6900), whereas immunization with reduced mannan-MUC1 FP (red-M-FP) led to poor protection after challenge with only 10(6) MUC1+ tumor cells, no CTLs, and a low CTLp frequency (1/87,800). Ox-M-FP selects for a T1 response (mediated here by CD8+ cells) with high interferon gamma (IFN-gamma) secretion, no interleukin 4 (IL-4), and a predominant IgG2a antibody response; red-M-FP selects for a T2-type response with IL-4 production and a high predominant IgG1 antibody response but no IFN-gamma.