Using methylcholanthrene-induced fibrosarcomas to study tumor immunology.

Mouse models of cancer are essential in furthering our understanding both of the mechanisms that drive tumor development and the immune response that develops in parallel, and also in providing a platform for testing novel anti-cancer therapies. The majority of solid tumor models available rely on the injection of existing cancer cell lines into naïve hosts which, while providing quick and reproducible model systems, typically lack the development of a tumor microenvironment that recapitulates those seen in human cancers. Administration of the carcinogen 3-methylcholanthrene (MCA), allows tumors to develop in situ, forming a tumor microenvironment with an established stroma and vasculature. This article provides a detailed set of protocols for the administration of MCA into mice and the subsequent monitoring of tumors. Protocols are also provided for some of the routinely used downstream applications that can be used for MCA tumors.

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.

Highly prevalent colorectal cancer-infiltrating LAP⁺ Foxp3⁻ T cells exhibit more potent immunosuppressive activity than Foxp3⁺ regulatory T cells.

Although elevated CD4⁺Foxp3⁺ regulatory T cell (Treg) frequencies within tumors are well documented, the functional and phenotypic characteristics of CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ T cell subsets from matched blood, healthy colon, and colorectal cancer require in-depth investigation. Flow cytometry revealed that the majority of intratumoral CD4⁺Foxp3⁺ T cells (Tregs) were Helios⁺ and expressed higher levels of cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD39 than Tregs from colon and blood. Moreover, ∼30% of intratumoral CD4⁺Foxp3⁻ T cells expressed markers associated with regulatory functions, including latency-associated peptide (LAP), lymphocyte activation gene-3 (LAG-3), and CD25. This unique population of cells produced interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß), and was ∼50-fold more suppressive than Foxp3⁺ Tregs. Thus, intratumoral Tregs are diverse, posing multiple obstacles to immunotherapeutic intervention in colorectal malignancies.

Herd characteristics and cow-level factors associated with Prototheca mastitis on dairy farms in Ontario, Canada.

Prototheca spp. are algae that cause incurable acute or chronic mastitis in dairy cows. The aim of this case-control study was the identification of cow- and herd-level risk factors for this unusual mastitis pathogen. Aseptically collected composite milk samples from 2,428 milking cows in 23 case and 23 control herds were collected between January and May 2011. A questionnaire was administered to the producers, and cow-level production and demographic data were gathered. In 58 of 64 isolates, Prototheca spp. and Prototheca zopfii genotypes were differentiated using PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. All isolates were identified as Prototheca zopfii genotype 2. The mean within-herd prevalence for Prototheca spp. was 5.1% (range 0.0-12.5%). Case herds had a significantly lower herd-level prevalence of Staphylococcus aureus and a higher prevalence of yeasts than did control herds. The final logistic regression model for herd-level risk factors included use of intramammary injections of a non-intramammary drug [odds ratio (OR) = 136.8], the number of different injectable antibiotic products being used (OR = 2.82), the use of any dry cow teat sealant (external OR = 80.0; internal OR = 34.2), and having treated 3 or more displaced abomasums in the last 12 mo OR = 44.7). The final logistic regression model for cow-level risk factors included second or greater lactation (OR = 4.40) and the logarithm of the lactation-average somatic cell count (OR = 2.99). Unsanitary or repeated intramammary infusions, antibiotic treatment, and off-label use of injectable drugs in the udder might promote Prototheca udder infection.

Milk production and somatic cell counts: a cow-level analysis.

The objectives of this study were to quantify the relationship between 24-h milk loss and lactation milk loss due to mastitis at the cow level. For the year 2009, individual cow test-day production records from 2,835 Ontario dairy herds were examined. Each record consisted of 24-h milk and component yields, stage of lactation (days in milk, DIM), somatic cell count (SCC, ×10(3) cells/mL) and parity. The modeling was completed in 2 stages. In stage 1, for each animal in the study, the estimated slope from a linear regression of 24-h milk yield (kg), adjusted for DIM, the quadratic effect of DIM, and the 24-h fat yield (kg) on ln(SCC) was determined. In stage 2, the estimated slope were modeled using a mixed model with a random component due to herd. The fixed effects included season (warm: May to September, cool: October to April), milk quartile class [MQ, determined by the rank of the 24-h average milk yield (kg) over a lactation within the herd] and parity. The estimated slopes from the mixed model analysis were used to estimate 24-h milk loss (kg) by comparing to a referent healthy animal with an SCC value of 100 (×10(3) cells/mL) or less. Lactation milk loss (kg) was then estimated by using estimated 24-h milk loss within lactation by means of a test-day interval method. Lactation average milk loss (kg) and SCC were also estimated. Lastly, lactation milk loss (kg) was modeled on the log scale using a mixed model, which included the random effect of herd and fixed effects, parity, and the linear and quadratic effect of the number of 24-h test days within a lactation where SCC exceeded 100 (×10(3) cells/mL; S100). The effect of SCC was significant with respect to 24-h milk loss (kg), increasing across parity and MQ. In general, first-parity animals in the first MQ (lower milk yield animals) were estimated to have 45% less milk loss than later parity animals. Milk losses were estimated to be 33% less for animals in first parity and MQ 2 through 4 than later parity animals in comparable MQ. Therefore, the relative level of milk production was found to be a significant risk factor for milk loss due to mastitis. For animals with 24-h SCC, values of 200 (×10(3) cells/mL), 24-h milk loss ranged from 0.35 to 1.09 kg; with 24-h SCC values of 2,000 (×10(3) cells/mL), milk loss ranged from 1.49 to 4.70 kg. Lactation milk loss (kg) increased significantly as lactation average SCC increased, ranging from 165 to 919 kg. The linear and quadratic effect of S100 was a significant risk factor for lactation milk loss (kg), where greatest losses occurred in lactations with 5 or more 24-h test days where SCC exceeded 100 (×10(3) cells/mL).

Changes in management practices and apparent prevalence on Canadian dairy farms participating in a voluntary risk assessment-based Johne's disease control program.

The objectives of this study were (1) to describe the change in Mycobacterium avium ssp. paratuberculosis (MAP) antibody milk ELISA-positive prevalence in Canadian dairy herds that participated in a risk assessment (RA)-based Johne's disease (JD) control program; (2) to describe the distribution of so-called high-risk management practices on Canadian dairy farms; and (3) to assess if compliance with selected recommendations translated into changes in the scores of associated RA questions. In Ontario and western Canada, 226 herds voluntarily participated in a RA-based JD control program for several years. In 2005-2007, a previsit survey, RA, and MAP-antibody milk ELISA of the entire milking herd were conducted. Therefore, the interpretation of the results of this study is strictly for the MAP-antibody milk ELISA status of cows or herds, because no culture of MAP (of fecal or environmental samples) was conducted due to economic restrictions. In early 2008, a telephone interview was used to determine compliance with recommended management changes after the first RA. In 2008-2009, a second RA and another whole-herd MAP antibody milk ELISA were performed. At both herd tests, about 35% of the farms had at least one MAP-antibody milk ELISA-positive cow, classifying them as a MAP-antibody milk ELISA-positive herd. However, 28.8% of herds had changed their MAP-antibody milk ELISA status between the 2 tests, demonstrating that a single herd test was insufficient to determine the long-term MAP-antibody ELISA status of a herd. The average within-herd MAP-antibody milk ELISA-positive prevalence changed from 5.4 to 4.2% over the study period, but management practices did not change much throughout the 2- to 3-yr period and were similar to those reported in other parts of North America. The overall RA scores decreased at the second RA, in particular for management practices in the calving and preweaned calf area, and when herds were test-positive at the first test. This was not surprising, because many of the recommendations at the first RA focused on these management areas and compliance with some recommended farm-specific management practices in this area might be linked to reduced scores for associated RA questions. In conclusion, the participating farms did, on average, decrease their within-herd MAP-antibody milk ELISA positive-prevalence and RA total scores. Changes in RA scores could be linked to improved management practices, indicating that the RA questions appropriately reflected management practices. Some herds changed their MAP-antibody milk ELISA status between tests, which underlines that a current test of the entire milking herd is necessary to determine the present MAP-antibody milk ELISA status of a dairy herd.

Associations between paratuberculosis milk ELISA result, milk production, and breed in Canadian dairy cows.

The 3 objectives of this study were (1) to quantify milk production differences among cows with different paratuberculosis (ParaTB) milk ELISA results; (2) to determine if production differences existed in lactations preceding the test among cows with different ParaTB milk ELISA results; and (3) to assess whether Channel Island breeds were more likely to test positive with the ParaTB milk ELISA than other dairy breeds. Current and completed lactation records from 35,591 dairy cows in Ontario and western Canada that had been tested with a commercial ParaTB milk ELISA were included in the analysis. The first occurrence of the highest categorical test result was used to classify the cow. Cows were then grouped by the lactation in which the first high-positive (HTP), low-positive, or negative milk ELISA occurred, and comparisons were made within lactation groups. High test-positive cows were defined as those that had an optical density ≥ 1.0 on at least 1 ParaTB milk ELISA. The associations between ParaTB milk ELISA status and milk production, as measured by the 305-d milk yield, were assessed with a series of linear mixed models. The effect of breed on the likelihood of testing positive with the milk ELISA was assessed using a logistic mixed model for the lactation in which the first negative or positive ParaTB milk ELISA occurred. Test-positive cows produced on average 2.9 to 6.8% less milk than negative herdmates in the lactation in which they were tested. The HTP cows produced on average 466, 514, and 598 kg less milk than low-positive herdmates in lactations 1, 2, and 4, respectively. Cows testing low-positive in their second lactation had, on average, a 218-kg higher milk yield in their first lactation than their test-negative herdmates. Otherwise, no association was found between test result and milk production in preceding lactations. Differences in milk production among negative, test-positive, and HTP cows increased with increasing parity. Cows of the Channel Island breeds had 1.4 to 8.3 times the odds to test positive compared with other dairy breeds. The findings of this study are consistent with previous studies that have reported that milk production is lower in test-positive animals. The differences in milk production increased with increasing ELISA optical density scores and parity in which the animal tested positive. However, with the exception of second-lactation cows, no differences in milk production were observed in tests preceding lactations. The differences in milk ELISA status among dairy breeds support the need for further studies investigating the genetic component of ParaTB susceptibility.

Attitudes of Canadian dairy farmers toward a voluntary Johne's disease control program.

The success of Johne's disease (JD) control programs based on risk assessment (RA) depends on producers' compliance with suggested management practices. One objective of this study was to describe the perception of participating Canadian dairy farmers of the impact of JD, the RA process, and suggested management strategies. The second objective was to describe the cost of changes in management practices following the RA. A telephone survey was conducted with 238 dairy farmers in Ontario, Manitoba, Saskatchewan, Alberta, and British Columbia. The producers agreed to participate in this follow-up study after they had been enrolled in an RA-based voluntary JD control program and had tested their herd with the JD milk ELISA test in 2005 to 2007. The majority of farms had no JD test-positive cows and, although some producers thought they had experienced the economic impact of JD, many did not see JD as a current problem for their herd. The majority of producers enrolled in this program because they were concerned that Mycobacterium avium ssp. paratuberculosis could be perceived by consumers as a cause for Crohn's disease in humans, which could lead to altered purchasing behavior of milk and milk products. Fifty-two farm-specific recommendations had been made after the initial RA. Although the producers generally liked the program and found the recommendations reasonable and feasible, on average only 2 of 6 suggestions made specifically to them were implemented. The recommendation with the highest compliance was culling of JD test-positive cows. The main reasons for noncompliance were that the dairy producer did not believe a change of management practices was necessary or the available barn setting or space did not allow the change. Producers were generally uncomfortable estimating time and monetary expenses for management changes, but found that several suggested management practices actually saved time and money. In addition, 39% of the producers that implemented at least 1 recommendation thought their calf and herd health had improved subsequently. This indicates that the communication of associated benefits needs to be improved to increase the compliance of producers with recommended management practices.

The impact of regulatory T cells on carcinogen-induced sarcogenesis.

Burnet proposed in the 1950's that the immune system is engaged in identifying and destroying abnormal cancerous cells. This process, termed immune surveillance, has been at the centre of intense debate for decades. Results using immunodeficient mice lend support to the immune surveillance hypothesis. We surmised that immune surveillance would be hampered by the inhibitory effect of naturally occurring FoxP3(+) regulatory T cells, a population of T cells shown to be present at an increased frequency in a variety of human tumours. The carcinogen, methylcholanthrene was injected subcutaneously into mice and the steady development of fibrosarcomas was observed over approximately 200 days. These fibrosarcomas were strikingly infiltrated with FoxP3(+) regulatory T cells implying that these cells impinge upon immune-mediated rejection of the tumour. This was confirmed by partial ablation of FoxP3(+) regulatory T-cell activity, which resulted in a marked reduction in tumour incidence. The reduction of tumour incidence was ablated in mice that lacked interferon gamma. These data offer strong support for the concept of immune surveillance and indicate that this process is limited by the inhibitory effect of FoxP3(+) regulatory T cells.

Immune tolerance to hepatitis C virus acquired during engraftment of bone marrow transplant.

The CD4+ T-cell response appears to be important for clearance of hepatitis C virus (HCV) in the majority of individuals. We have recently described a series of human leucocyte antigen (HLA)-DR11-restricted T-cell epitopes derived from HCV proteins which enables distinct populations of memory CD4+ T cells to be detected and counted in all nonviraemic HCV subjects. We examined the case of an HLA-DR11+ recipient of a haematopoietic stem-cell transplant who was concurrently infected with HCV from an HLA-DR11+ donor sibling. An acute HCV hepatitis developed and was treated with type I interferon. After successful viral clearance, the recipient demonstrated a selective lack of HCV epitope-specific CD4+ T cells and absence of serological responses compared with the treated donor. The recipient had no evidence of any nonspecific immunosuppression. The subsequent effects of concurrent infection during immune reconstitution are not known in adult humans, but data from murine models suggest this can lead to a skewing of the T-cell repertoire because of thymic selection. From the above observations, it is plausible that the introduction of foreign viral antigen into the thymus may lead to subsequent acquired central tolerance.

Evidence for lack of cross-genotype protection of CD4+ T cell responses during chronic hepatitis C virus infection.

CD4+ T lymphocyte responses are thought to play a major role in control of the hepatitis C virus (HCV). Few, however, have been mapped down to the level of peptide and HLA restriction. Furthermore, the ability of such T cells to respond to viruses which differ in genotype has not been addressed in detail. In most cases of persistent infection with HCV, CD4 proliferative responses are weak or absent. From a large cohort of persistently infected patients, we identified an individual with unusually robust and persistent responses in the face of chronic infection. We firstly mapped two peptide epitopes to regions of the nonstructural protein NS4 (aa1686-1705 and aa 1746-1765). However, in contrast to the genotype 1a derived antigens used for mapping, the infecting virus was identified as genotype 3a. Strikingly, the patient's CD4 response to these epitopes were specific only for the genotype 1a sequence, and did not recognize genotype 3a synthetic peptides. Serologic assays indicated that prior exposure to HCV of genotype 1 had occurred. This patient therefore maintains strong CD4 proliferative responses which are genotype specific and not cross-reactive. The apparent 'misdirection' of these nonprotective responses has important implications for the role of natural and vaccine induced CD4 responses in the face of variable viruses.

Characterization of novel HLA-DR11-restricted HCV epitopes reveals both qualitative and quantitative differences in HCV-specific CD4+ T cell responses in chronically infected and non-viremic patients.

The CD4+ T cell response is of critical importance in determining the fate of many viral infections. Clearance of HCV has a strong association with the MHC class II antigen HLA-DR11 suggesting a key role for CD4+ T cells. We used an epitope-prediction program to identify multiple novel HLA-DR11-restricted epitopes derived from several HCV proteins. These epitopes then allowed us to explore the qualitative and quantitative aspects of specific CD4+ T cell responses in HLA-DR11+ patients. Irrespective of the time since viral clearance, all the non-viremic patients recognized four epitopes with a high frequency of IFN-gamma-producing memory CD4+ T cells. There appeared to be two subpopulations of memory cells, immediate "effector" memory cells (Th1 phenotype) and resting "central" memory cells (Th1/0). Chronically infected patients revealed an almost complete absence of HCV epitope-specific IFN-gamma-producing T cells. However, three of these epitopes induced IL-10 production (down-regulatory) raising the question as to whether these cells play a role in viral persistence. The frequency and phenotype of memory cells is likely to reflect the magnitude of the initial immune response, and suggests that a high frequency of IFN-gamma-secreting CD4+ T cells to multiple epitopes are important in clearance of HCV.

Naturally processed HLA class II peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-MHC interactions.

MHC class II heterodimers bind peptides 12-20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRS: We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.

In vivo antigen challenge in celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T-cell epitope.

Celiac disease (CD) is an increasingly diagnosed enteropathy (prevalence, 1:200-1:300) that is induced by dietary exposure to wheat gliadins (as well as related proteins in rye and barley) and is strongly associated with HLA-DQ2 (alpha1*0501, beta1*0201), which is present in over 90% of CD patients. Because a variety of gliadin peptides have been identified as epitopes for gliadin-specific T-cell clones and as bioactive sequences in feeding studies and in ex vivo CD intestinal biopsy challenge, it has been unclear whether a 'dominant' T-cell epitope is associated with CD. Here, we used fresh peripheral blood lymphocytes from individual subjects undergoing short-term antigen challenge and tissue transglutaminase-treated, overlapping synthetic peptides spanning A-gliadin to demonstrate a transient, disease-specific, DQ2-restricted, CD4 T-cell response to a single dominant epitope. Optimal gamma interferon release in an ELISPOT assay was elicited by a 17-amino-acid peptide corresponding to the partially deamidated peptide of A-gliadin amino acids 57-73 (Q65E). Consistent with earlier reports indicating that host tissue transglutaminase modification of gliadin enhances gliadin-specific CD T-cell responses, tissue transglutaminase specifically deamidated Q65 in the peptide of A-gliadin amino acids 56-75. Discovery of this dominant epitope may allow development of antigen-specific immunotherapy for CD.