Background In Denmark and many other European countries, harvest records suggest a marked decline in European brown hare numbers, a decline often attributed to the agricultural practice. In the present study, we analyse the association between agricultural land-use, predator abundance and winter severity on the number of European brown hares harvested in Denmark in the years 1955 through 2000.
Background
In Denmark and many other European countries, harvest records suggest a marked decline in European brown hare numbers, a decline often attributed to the agricultural practice. In the present study, we analyse the association between agricultural land-use, predator abundance and winter severity on the number of European brown hares harvested in Denmark in the years 1955 through 2000.Results Winter cereals had a significant negative association with European brown hare numbers. In contrast to this, root crop area was positively related to their numbers. Remaining crop categories were not significantly associated with the European brown hare numbers, though grass out of rotation tended to be positively related. The areas of root crop production and of grass out of rotation have been reduced by approximately 80% and 50%, respectively, while the area of winter cereals has increased markedly (>70%). However, European brown hare numbers were primarily negatively associated with the number of red fox. Finally, we also found a positive association between mild winters and European brown hare numbers.
Results
Winter cereals had a significant negative association with European brown hare numbers. In contrast to this, root crop area was positively related to their numbers. Remaining crop categories were not significantly associated with the European brown hare numbers, though grass out of rotation tended to be positively related. The areas of root crop production and of grass out of rotation have been reduced by approximately 80% and 50%, respectively, while the area of winter cereals has increased markedly (>70%). However, European brown hare numbers were primarily negatively associated with the number of red fox. Finally, we also found a positive association between mild winters and European brown hare numbers.Conclusion The decline of Danish European brown hare populations can mainly be attributed to predation by red fox, but the development in agricultural land-use during the last 45 years have also affected the European brown hare numbers negatively. Additionally, though mild winters were beneficial to European brown hares, the increasing frequency of mild winters during the study period was insufficient to reverse the negative population trend.
Conclusion
The decline of Danish European brown hare populations can mainly be attributed to predation by red fox, but the development in agricultural land-use during the last 45 years have also affected the European brown hare numbers negatively. Additionally, though mild winters were beneficial to European brown hares, the increasing frequency of mild winters during the study period was insufficient to reverse the negative population trend.

Background In most countries in Western Europe, the landscape has undergone dramatic changes during the last century due to changes in the agricultural practices. In Denmark, both the uncultivated land and the semi-cultivated land, such as permanent grass areas, have been reduced dramatically, reflecting the general intensification of agriculture [ 1 ]. Additionally, fields have become larger, which has resulted in widespread fragmentation of remaining habitats, and today the landscape appears as a mosaic of natural habitats surrounded by cultivated land [ 1 , 2 ]. These changes in agriculture have affected a number of wildlife species living in this man-made landscape. For instance, the shift in agricultural practice has severely influenced the diversity and abundance of insects with concomitant consequences for the dynamics of a wide range of farmland birds [ 3 , 4 ], including the grey partridge ( Perdix perdix ) [ 5 ]. Among mammals, the European brown hare ( Lepus europaeus ) in particular has experienced a dramatic decline in many European countries [reviewed by [ 6 ]], including Denmark [ 7 , 8 ]. Despite its currently declining numbers, the European brown hare is still common and one of the most important game species throughout the country [ 8 ]. The dynamics of European brown hares seem resilient to even heavy hunting pressure [ 9 ], though local population dynamic data may be needed to ensure sustainable harvest [ 10 ]. In Denmark hunting of European brown hares is generally assumed to be without regulating effect [ 8 ]. The European brown hare is a typical grass steppe herbivore, and inhabits primarily open landscapes, including cultivated farmland [ 11 ], which is the predominant landform in Denmark [ 1 ]. The species is rather sedentary, and has generally small home ranges [e.g. [ 12 ]]. This site fidelity makes European brown hares highly susceptible to changes in their surrounding habitats, and the general decline in the European brown hare populations in Europe is mainly being attributed to changes in agriculture practice and land-use [reviewed by [ 6 , 12 ]]. European brown hares are important prey primarily for mammalian predators. In Northern Europe, the red fox ( Vulpes vulpes ) is the main predator on European brown hares, and foxes have been reported to influence the dynamics of several European brown hare populations substantially [ 13 - 17 ]. Also, infectious diseases such as the European brown hare syndrome virus, pseudotuberculosis, pasteurellosis and coccidiosis are present in many European brown hare populations [ 18 - 20 ]. Haerer et al. [ 19 ], however, concluded that diseases were not responsible for the decline of brown hare populations in Switzerland. Similarly, Frölich et al. [ 20 ] found that compared to red foxes, infectious diseases seemed to play a minor role in the dynamics of European brown hare populations in Germany. An increasing number of papers have documented the importance of climate for a number of life history traits and abundance of many terrestrial vertebrates [ 21 ], and European brown hare populations are affected negatively by cold winters and cold springs [ 12 , 22 - 24 ]. Factors regulating vertebrate populations may, however, exhibit large spatial variation, and even among proximate populations spatial variation and gradients in vertebrate population dynamics may exist [e.g. [ 25 ]]. In this study, we analyse and contrast the simultaneous associations between agricultural land-use, the number of red fox harvested, winter severity and the population dynamics of European brown hares across 11 Danish districts during 45 years.
Background
In most countries in Western Europe, the landscape has undergone dramatic changes during the last century due to changes in the agricultural practices. In Denmark, both the uncultivated land and the semi-cultivated land, such as permanent grass areas, have been reduced dramatically, reflecting the general intensification of agriculture [ 1 ]. Additionally, fields have become larger, which has resulted in widespread fragmentation of remaining habitats, and today the landscape appears as a mosaic of natural habitats surrounded by cultivated land [ 1 , 2 ]. These changes in agriculture have affected a number of wildlife species living in this man-made landscape. For instance, the shift in agricultural practice has severely influenced the diversity and abundance of insects with concomitant consequences for the dynamics of a wide range of farmland birds [ 3 , 4 ], including the grey partridge ( Perdix perdix ) [ 5 ]. Among mammals, the European brown hare ( Lepus europaeus ) in particular has experienced a dramatic decline in many European countries [reviewed by [ 6 ]], including Denmark [ 7 , 8 ].Perdix perdixLepus europaeusDespite its currently declining numbers, the European brown hare is still common and one of the most important game species throughout the country [ 8 ]. The dynamics of European brown hares seem resilient to even heavy hunting pressure [ 9 ], though local population dynamic data may be needed to ensure sustainable harvest [ 10 ]. In Denmark hunting of European brown hares is generally assumed to be without regulating effect [ 8 ]. The European brown hare is a typical grass steppe herbivore, and inhabits primarily open landscapes, including cultivated farmland [ 11 ], which is the predominant landform in Denmark [ 1 ]. The species is rather sedentary, and has generally small home ranges [e.g. [ 12 ]]. This site fidelity makes European brown hares highly susceptible to changes in their surrounding habitats, and the general decline in the European brown hare populations in Europe is mainly being attributed to changes in agriculture practice and land-use [reviewed by [ 6 , 12 ]].European brown hares are important prey primarily for mammalian predators. In Northern Europe, the red fox ( Vulpes vulpes ) is the main predator on European brown hares, and foxes have been reported to influence the dynamics of several European brown hare populations substantially [ 13 - 17 ]. Also, infectious diseases such as the European brown hare syndrome virus, pseudotuberculosis, pasteurellosis and coccidiosis are present in many European brown hare populations [ 18 - 20 ]. Haerer et al. [ 19 ], however, concluded that diseases were not responsible for the decline of brown hare populations in Switzerland. Similarly, Frölich et al. [ 20 ] found that compared to red foxes, infectious diseases seemed to play a minor role in the dynamics of European brown hare populations in Germany.Vulpes vulpesAn increasing number of papers have documented the importance of climate for a number of life history traits and abundance of many terrestrial vertebrates [ 21 ], and European brown hare populations are affected negatively by cold winters and cold springs [ 12 , 22 - 24 ]. Factors regulating vertebrate populations may, however, exhibit large spatial variation, and even among proximate populations spatial variation and gradients in vertebrate population dynamics may exist [e.g. [ 25 ]].In this study, we analyse and contrast the simultaneous associations between agricultural land-use, the number of red fox harvested, winter severity and the population dynamics of European brown hares across 11 Danish districts during 45 years.Results In the period 1955–2000 the European brown hare harvest record declined steadily in all the Danish districts but one: On the island Bornholm the European brown hare population declined until the late 1980s, but has increased markedly since then, and has now reached a level higher than that of 1955 (Fig.  1 ). Figure 1 Harvest records of European brown hare (bags per 1000 ha) from 11 Danish districts, 1955–2000. We found statistical significant direct density-dependence ( X t-1 ) in the European brown hare time series (G = 281.4, df = 2, P < 0.0001). Additionally, the effect of district was also significant (G = 769.0, df = 1, P < 0.0001). From 1955 to 2000, agricultural land-use changed markedly in Denmark, resulting in large temporal shifts in the areas covered by the different crop categories (Fig.  2 ). The areas covered with grass and green fodder in rotation and in particular the areas out of rotation have been reduced, the latter by approximately 50% since the mid 1950s. An even more dramatic decline has been observed for the root crops in the same period, a decline by more than 80% (Fig.  2 ). In Storstrøm, however, the area covered with root crops has been relatively stable over the years. In general, cereals were the dominant crop category in 1955 through 2000, with a shift from a predominance of spring cereals to winter cereals in the 1980s (Fig.  2 ). Figure 2 The total areas of the seven crop categories in Denmark 1955–2000. Note that prior to 1960 no data on winter cereals exist. The areas covered with winter cereals had a marked negative association with the number of European brown hares (Table  2 ), whereas root crops had a marked positive relation. Neither spring cereals, nor winter and spring rape seemed to be associated with European brown hare numbers (Table  2 ). Similarly, neither grass areas in or out of rotation were significantly related to European brown hare numbers, though the latter tended to have a positive association (Table  2 ). Table 2 Summarised results from the analysis of the impact of agricultural land-use, red fox and winter climate on European brown hare harvest records from 11 Danish counties, 1955–2000. Also given is the change in model deviance (Δ deviance) explained by the variable when fitted last. Type 3 sums of squares. Variable Coefficient SE F value P value Δdeviance Winter cereals (t) -0.08322 0.03778 4.85 0.0282 37.7 Spring cereals (t) -0.06243 0.04675 1.78 0.1825 2.5 Grass in rotation (t) 0.03217 0.03562 0.82 0.3669 4.1 Grass out of rotation (t) 0.05766 0.03070 3.53 0.0612 1.7 Winter rape (t) -0.03272 0.03094 1.12 0.2909 4.0 Spring rape (t) -0.02704 0.03948 0.47 0.4938 4.2 Root crops (t) 0.10370 0.04753 4.76 0.0297 0.4 Fox (t) -0.01791 0.02024 0.78 0.3767 5.2 Fox (t-1) -0.15720 0.01971 63.62 <.0001 52.8 NAO (t) 0.03732 0.01110 11.31 0.0008 4.0 Note: Due to the standardization of model variables, regression coefficients and Δ deviance values are directly comparable. The number of red foxes harvested in year t-1  had a marked negative effect on the number of European brown hares harvested the following year (Table  2 ), whereas red fox number in year t  seemed unimportant for the European brown hare numbers. Mild winters, i.e. high winter state of the large-scale climatic phenomenon the North Atlantic Oscillation [NAO; [ 26 ]], had a significant positive effect on the European brown hare numbers (Table  2 ).
Results
In the period 1955–2000 the European brown hare harvest record declined steadily in all the Danish districts but one: On the island Bornholm the European brown hare population declined until the late 1980s, but has increased markedly since then, and has now reached a level higher than that of 1955 (Fig.  1 ).We found statistical significant direct density-dependence ( X t-1 ) in the European brown hare time series (G = 281.4, df = 2, P < 0.0001). Additionally, the effect of district was also significant (G = 769.0, df = 1, P < 0.0001).Xt-1From 1955 to 2000, agricultural land-use changed markedly in Denmark, resulting in large temporal shifts in the areas covered by the different crop categories (Fig.  2 ). The areas covered with grass and green fodder in rotation and in particular the areas out of rotation have been reduced, the latter by approximately 50% since the mid 1950s. An even more dramatic decline has been observed for the root crops in the same period, a decline by more than 80% (Fig.  2 ). In Storstrøm, however, the area covered with root crops has been relatively stable over the years. In general, cereals were the dominant crop category in 1955 through 2000, with a shift from a predominance of spring cereals to winter cereals in the 1980s (Fig.  2 ).The areas covered with winter cereals had a marked negative association with the number of European brown hares (Table  2 ), whereas root crops had a marked positive relation. Neither spring cereals, nor winter and spring rape seemed to be associated with European brown hare numbers (Table  2 ). Similarly, neither grass areas in or out of rotation were significantly related to European brown hare numbers, though the latter tended to have a positive association (Table  2 ).Summarised results from the analysis of the impact of agricultural land-use, red fox and winter climate on European brown hare harvest records from 11 Danish counties, 1955–2000. Also given is the change in model deviance (Δ deviance) explained by the variable when fitted last. Type 3 sums of squares.(t)(t)(t)(t)(t)(t)(t)(t)(t-1)(t)Note: Due to the standardization of model variables, regression coefficients and Δ deviance values are directly comparable.The number of red foxes harvested in year t-1  had a marked negative effect on the number of European brown hares harvested the following year (Table  2 ), whereas red fox number in year t  seemed unimportant for the European brown hare numbers.t-1tMild winters, i.e. high winter state of the large-scale climatic phenomenon the North Atlantic Oscillation [NAO; [ 26 ]], had a significant positive effect on the European brown hare numbers (Table  2 ).Discussion Despite its high reproductive potential [e.g. [ 27 ]], the Danish European brown hare has, according to annual harvest records, declined dramatically since 1955. Studies of Danish European brown hare populations indicate that its reproductive success is low compared to that of con-specifics in other countries [ 27 ], and has, in turn, declined from the 1950s to the 1990s [ 28 ]. Hansen [ 27 ] suggested that the low reproductive success observed in Danish European brown hares might be attributed to the agricultural practice. Using data covering almost half a century, our analyses suggest that the dramatic decline in the Danish European brown hares can be attributed mainly to the negative effect of red foxes, but also to the agricultural land-use. The area of winter cereal production has increased dramatically during the last century, and became the dominating crop in the early 1990s. We also found a significant positive association between root crops, a crop type that has declined dramatically in the second half of the last century, and European brown hare numbers. European brown hares mainly forage on grasses and herbs [ 12 ], and cereals such as wheat are preferred during winter [ 29 , 30 ], which seems to contradict the results of our analyses. However, as European brown hares choose to feed on specific crops according to plant phenology [ 12 , 31 ], cereals, although important in winter, still occupy large areas when no longer of nutritional value to European brown hares, which may result in low availability of food especially during summer. Similarly, rape is avoided in the diet [ 29 ], but European brown hares may spend a substantial fraction of their time in rape fields during winter [ 30 ] prior to the development of glucosinulates [see [ 29 ]]. Apart from winter cereals and root crops, the crop categories did not affect the European brown hare numbers significantly. The lacking effect of grass and green fodder areas, especially those out of rotation, was unexpected as other studies have shown that hares prefer grass areas year-round [ 12 , 30 ]. This, however, may be attributed to the fact that we were unable to separate grass areas into those e.g. with and without cattle, factors that might affect European brown hare use of grass areas [ 12 ]. In a recent study, Fox [ 32 ] showed that farmland birds seemed to benefit from the reduced application of pesticides and inorganic fertilisers seen in Denmark since the early 1980s. The continuing decline in European brown hare numbers in that period therefore suggests that hares are not directly affected by the use of such substances, but rather respond to the loss of suitable habitat and space. European brown hares mainly move along field margins [ 12 , 33 ], and the increasing field size [ 34 ] combined with the general loss of suitable habitats possibly force hares to aggregate in the remaining patches of non-agricultural and non-urbanized land. Here, density-dependence may perpetuate the negative population development as European brown hares aggregate in the few, remaining pockets of suitable habitat. In line with this, Frylestam [ 23 ] reported an inverse relationship between fertility and density in European brown hares, which he attributed to shortage of food at least in some parts of the year, which also has been suggested in a number of other studies [ 12 , 24 , 27 ]. Hence, agricultural land-use, especially the increasing use of winter cereals and the marked reduction in root crops, but probably also grass areas out of rotation, seem to have contributed to the European brown hare decline in Denmark. However, the single-most important parameter for the number of European brown hares was the number of red foxes. Hence, our results are consistent with other studies reporting that red foxes may affect European brown hare populations negatively through predation [ 13 - 17 ]. This relationship is also particular evident from the positive development in the European brown hare population on the island Bornholm following the outbreak of sarcoptic mange there [ 35 ]. To elaborate the fox-hare interaction further, we reran the analyses including all variables but the red fox variables, and added the delayed AR term (i.e.  X t-2 ). In seven of the 11 districts the delayed density dependence was significant (P < 0.05), suggesting important inter-specific interactions [ 36 , 37 ]. The significant association with red fox numbers in year t-1  (and not year t ) most likely reflects that compared to adults, juvenile European brown hares suffer more from predation [e.g. [ 19 ]]. Hence, high red fox numbers in year t  result in low harvest of European brown hares in year t , which affects the reproductive potential of the populations, and, hence, the number of European brown hares the following year (year t+1 ). Also, the significant effect of district may point to differences in habitat quality, but also differences in the history of the sarcoptic mange, i.e. time since the eruption of the mange, among districts [see [ 35 ]]. Both European brown hare over-winter survival [ 24 ] and reproductive rate [ 23 ] increases with temperature, which may be attributed to improved forage availability following mild winters [see [ 38 , 39 ]]. Our analyses revealed similar results as mild winters affected the European brown hares positively. There may, however, also be a negative effect of mild winters, namely through transmission of diseases and parasites, which may be enhanced under mild climatic conditions [ 18 ]. Nevertheless, the overall effect of mild winters seemed positive. The upward trend in the NAO since the 1960s [ 40 ], however, was not sufficient to reverse the European brown hare decline, but may have decelerated it.
Discussion
Despite its high reproductive potential [e.g. [ 27 ]], the Danish European brown hare has, according to annual harvest records, declined dramatically since 1955. Studies of Danish European brown hare populations indicate that its reproductive success is low compared to that of con-specifics in other countries [ 27 ], and has, in turn, declined from the 1950s to the 1990s [ 28 ]. Hansen [ 27 ] suggested that the low reproductive success observed in Danish European brown hares might be attributed to the agricultural practice. Using data covering almost half a century, our analyses suggest that the dramatic decline in the Danish European brown hares can be attributed mainly to the negative effect of red foxes, but also to the agricultural land-use. The area of winter cereal production has increased dramatically during the last century, and became the dominating crop in the early 1990s. We also found a significant positive association between root crops, a crop type that has declined dramatically in the second half of the last century, and European brown hare numbers.European brown hares mainly forage on grasses and herbs [ 12 ], and cereals such as wheat are preferred during winter [ 29 , 30 ], which seems to contradict the results of our analyses. However, as European brown hares choose to feed on specific crops according to plant phenology [ 12 , 31 ], cereals, although important in winter, still occupy large areas when no longer of nutritional value to European brown hares, which may result in low availability of food especially during summer. Similarly, rape is avoided in the diet [ 29 ], but European brown hares may spend a substantial fraction of their time in rape fields during winter [ 30 ] prior to the development of glucosinulates [see [ 29 ]]. Apart from winter cereals and root crops, the crop categories did not affect the European brown hare numbers significantly. The lacking effect of grass and green fodder areas, especially those out of rotation, was unexpected as other studies have shown that hares prefer grass areas year-round [ 12 , 30 ]. This, however, may be attributed to the fact that we were unable to separate grass areas into those e.g. with and without cattle, factors that might affect European brown hare use of grass areas [ 12 ].In a recent study, Fox [ 32 ] showed that farmland birds seemed to benefit from the reduced application of pesticides and inorganic fertilisers seen in Denmark since the early 1980s. The continuing decline in European brown hare numbers in that period therefore suggests that hares are not directly affected by the use of such substances, but rather respond to the loss of suitable habitat and space. European brown hares mainly move along field margins [ 12 , 33 ], and the increasing field size [ 34 ] combined with the general loss of suitable habitats possibly force hares to aggregate in the remaining patches of non-agricultural and non-urbanized land. Here, density-dependence may perpetuate the negative population development as European brown hares aggregate in the few, remaining pockets of suitable habitat. In line with this, Frylestam [ 23 ] reported an inverse relationship between fertility and density in European brown hares, which he attributed to shortage of food at least in some parts of the year, which also has been suggested in a number of other studies [ 12 , 24 , 27 ]. Hence, agricultural land-use, especially the increasing use of winter cereals and the marked reduction in root crops, but probably also grass areas out of rotation, seem to have contributed to the European brown hare decline in Denmark. However, the single-most important parameter for the number of European brown hares was the number of red foxes. Hence, our results are consistent with other studies reporting that red foxes may affect European brown hare populations negatively through predation [ 13 - 17 ]. This relationship is also particular evident from the positive development in the European brown hare population on the island Bornholm following the outbreak of sarcoptic mange there [ 35 ]. To elaborate the fox-hare interaction further, we reran the analyses including all variables but the red fox variables, and added the delayed AR term (i.e.  X t-2 ). In seven of the 11 districts the delayed density dependence was significant (P < 0.05), suggesting important inter-specific interactions [ 36 , 37 ]. The significant association with red fox numbers in year t-1  (and not year t ) most likely reflects that compared to adults, juvenile European brown hares suffer more from predation [e.g. [ 19 ]]. Hence, high red fox numbers in year t  result in low harvest of European brown hares in year t , which affects the reproductive potential of the populations, and, hence, the number of European brown hares the following year (year t+1 ). Also, the significant effect of district may point to differences in habitat quality, but also differences in the history of the sarcoptic mange, i.e. time since the eruption of the mange, among districts [see [ 35 ]].Xt-2t-1tttt+1Both European brown hare over-winter survival [ 24 ] and reproductive rate [ 23 ] increases with temperature, which may be attributed to improved forage availability following mild winters [see [ 38 , 39 ]]. Our analyses revealed similar results as mild winters affected the European brown hares positively. There may, however, also be a negative effect of mild winters, namely through transmission of diseases and parasites, which may be enhanced under mild climatic conditions [ 18 ]. Nevertheless, the overall effect of mild winters seemed positive. The upward trend in the NAO since the 1960s [ 40 ], however, was not sufficient to reverse the European brown hare decline, but may have decelerated it.Conclusion Our analyses have provided important insight into the structure of the European brown hare dynamics in Denmark, and documented important patterns within the mechanisms regulating European brown hares. Hence, we have documented that the decline of European brown hares in Denmark mainly can be attributed to predation by red fox, but also to changes in agricultural land-use. Additionally, though mild winters were beneficial to European brown hares, the increasing frequency of mild winters during the study period was insufficient to reverse the negative population trend.
Conclusion
Our analyses have provided important insight into the structure of the European brown hare dynamics in Denmark, and documented important patterns within the mechanisms regulating European brown hares. Hence, we have documented that the decline of European brown hares in Denmark mainly can be attributed to predation by red fox, but also to changes in agricultural land-use. Additionally, though mild winters were beneficial to European brown hares, the increasing frequency of mild winters during the study period was insufficient to reverse the negative population trend.Methods European brown hare and red fox density indices As indices of European brown hare and red fox density, we used the harvest records from 14 Danish counties from 1955–2000. In 1970, the geographical borders of some of the counties were changed, which in two cases lead to substantial changes in area [ 7 ]. This, together with inconsistency in crop registration, forced us to lump together some counties, and we therefore present analyses of European brown hare harvest records from 11 districts (Fig.  1 ). European brown hare and red fox harvest records were expressed as the number of animals shot per hectare per year. Harvest records may seem rather crude indicators of density compared to e.g. spotlight surveys and line transects [see [ 41 ]]. Unfortunately, no alternative indices of European brown hare density in Denmark are available. Harvest records, however, may still be useful indicators of the long-term trends in European brown hare numbers [ 41 ]. Sarcoptic mange was first encountered in Denmark in the early 1980s, and is now present in seven of the 11 districts [ 35 ]. In one district (Bornholm; Fig.  1 ), the disease has practically eliminated the entire red fox population, and red fox hunting has been prohibited here since 1991. Consequently, we restrict our analyses of the Bornholm district to 1955 through 1990. In all other districts, red fox hunting is open from 1 September to 31 January, and for European brown hares from 1 October to 31 December. Neither red fox nor European brown hare hunting is quota-based. Agricultural land-use Time series quantifying agricultural land-use data, i.e. annual crop areas in hectares, covering the period 1955–81 were obtained from Statistics Denmark. Data for the period 1982–2000 were obtained from the official website of Statistics Denmark  . The annual crop data were grouped into seven categories (Table  1 ). Table 1 Description of model variables used in the analyses of European brown hare harvest records from Denmark, 1955–2000. Variable Description Winter cereals Winter cereals, including winter wheat, winter barley, and winter rye Spring cereals Spring cereals, including spring wheat, spring barley, spring rye, and oat Grass in rotation Grass and green fodder in rotation, including grass, clover, maize, and Lucerne Grass out of rotation Grass areas permanently out of rotation Winter rape Winter rape Spring rape Spring rape Root crops Sugar beets for sugar production and fodder, mangolds, turnips, and carrots for feed Fox The number of red fox harvested NAO The winter state of the North Atlantic Oscillation No data on the fraction of winter and spring cereals, respectively, were available for 1955–61, and these data points were omitted in the analyses. Similarly, prior to 1982 no data on the fraction of winter barley and spring barley were available. However up until 1968, winter barley was only sown on a few thousand hectares in Denmark, when it was forbidden due to its function as reservoir for mildew attacking spring barley [ 42 ]. The use of mildew resistant winter barley was permitted in 1982. Hence, from 1955–81 barley was regarded as spring barley. Finally, no data on the fraction of winter and spring rye are available from 1955–61 and 1979–98, but in Denmark spring rye is generally sown on very few hectares only [ 42 ], and we regarded rye as winter rye unless otherwise specified [see also [ 32 ]]. Oat is sown as spring crop only. No data on the areas covered with grass and green fodder in or out of rotation were available for 1994, and the data point was interpolated, i.e. 1994 equalled the average of 1993 and 1995. Climatic variability As an index of winter severity, we used the winter state of the North Atlantic Oscillation [NAO; [ 26 ]]. The climatic phenomenon NAO is defined as the difference in sea-level pressure between Portugal and Iceland, affecting the temperature, precipitation and wind across the Northern Hemisphere [ 26 , 43 , 44 ]. The NAO, thus, integrates the effects of a number of abiotic factors, and therefore seems particularly useful when analysing the dynamics of larger, endothermic animals with relatively large buffer-capacity against climatic variability. The relationship between the winter NAO (December t-1  through March t ) and temperature, precipitation and wind is particularly strong in Northern Europe [ 45 ]. When NAO is in its high state, the winter climate in Northern Europe is warm, wet, and windy, while when in its low state winters are cold and dry [ 44 ]. The NAO winter index has shown an upward trend since the 1960s, which accounts for a substantial fraction of the general warming in the Northern Hemisphere [ 40 ]. The NAO index data are available from the Climate Analysis Section, National Center for Atmospheric Research, USA  . Data analyses To remove heteroscedasticity in the system, data were log e -transformed, i.e.  X (t)  = ln( N (t)  + 1), where  N (t)  is the number of European brown hares harvested in year (t) . To allow the direct comparison of regression coefficients, variables were standardized prior to the analyses (i.e., [ X (t) - µ (1955–2000) ]/ s (1955–2000) ). Regression coefficients therefore express the rate of change in standard deviation units of the independent variable per one standard deviation unit of the dependent variable [ 46 ]. To obtain stationarity in the time series [see [ 47 ] for details], data were detrended following [ 48 ] by including year as covariate in all models. For each district, we tested for the presence of multi-collinearity among parameters prior to the analyses by means of condition indices and variance proportions [ 49 ], but multi-collinearity was not observed in any districts (condition index < 12.47; [see [ 49 ] for details]). The European brown hare time series were tested for non-linearity in  X (t)  vs.  X (t-1)  by applying the likelihood ratio test [ 50 ]. In all districts, linearity in  X (t)  vs.  X (t-1)  was not rejected λ< 7.76, P > 0.05). We analysed the data using a first-order autoregressive (AR(1)) mixed model approach with district as fixed factor: X (t) ~( µ , V ), where and where LAND_USE represents the seven crop categories (see Table  1 ). Because of the inclusion of red fox numbers, we did not include a delayed AR term (i.e.  X t-2 ), as inclusion of both delayed density dependence and predator abundance can be seen as redundant [ 37 ]. All analyses were performed in SAS 8.2, using the PROC MIXED procedure with restricted maximum-likelihood estimation of regression coefficients [ 49 ]. Model reduction was conducted using log-likelihood ratio tests [ 51 ].
Methods
European brown hare and red fox density indices As indices of European brown hare and red fox density, we used the harvest records from 14 Danish counties from 1955–2000. In 1970, the geographical borders of some of the counties were changed, which in two cases lead to substantial changes in area [ 7 ]. This, together with inconsistency in crop registration, forced us to lump together some counties, and we therefore present analyses of European brown hare harvest records from 11 districts (Fig.  1 ). European brown hare and red fox harvest records were expressed as the number of animals shot per hectare per year. Harvest records may seem rather crude indicators of density compared to e.g. spotlight surveys and line transects [see [ 41 ]]. Unfortunately, no alternative indices of European brown hare density in Denmark are available. Harvest records, however, may still be useful indicators of the long-term trends in European brown hare numbers [ 41 ]. Sarcoptic mange was first encountered in Denmark in the early 1980s, and is now present in seven of the 11 districts [ 35 ]. In one district (Bornholm; Fig.  1 ), the disease has practically eliminated the entire red fox population, and red fox hunting has been prohibited here since 1991. Consequently, we restrict our analyses of the Bornholm district to 1955 through 1990. In all other districts, red fox hunting is open from 1 September to 31 January, and for European brown hares from 1 October to 31 December. Neither red fox nor European brown hare hunting is quota-based.
European brown hare and red fox density indices
As indices of European brown hare and red fox density, we used the harvest records from 14 Danish counties from 1955–2000. In 1970, the geographical borders of some of the counties were changed, which in two cases lead to substantial changes in area [ 7 ]. This, together with inconsistency in crop registration, forced us to lump together some counties, and we therefore present analyses of European brown hare harvest records from 11 districts (Fig.  1 ). European brown hare and red fox harvest records were expressed as the number of animals shot per hectare per year. Harvest records may seem rather crude indicators of density compared to e.g. spotlight surveys and line transects [see [ 41 ]]. Unfortunately, no alternative indices of European brown hare density in Denmark are available. Harvest records, however, may still be useful indicators of the long-term trends in European brown hare numbers [ 41 ].Sarcoptic mange was first encountered in Denmark in the early 1980s, and is now present in seven of the 11 districts [ 35 ]. In one district (Bornholm; Fig.  1 ), the disease has practically eliminated the entire red fox population, and red fox hunting has been prohibited here since 1991. Consequently, we restrict our analyses of the Bornholm district to 1955 through 1990. In all other districts, red fox hunting is open from 1 September to 31 January, and for European brown hares from 1 October to 31 December. Neither red fox nor European brown hare hunting is quota-based.Agricultural land-use Time series quantifying agricultural land-use data, i.e. annual crop areas in hectares, covering the period 1955–81 were obtained from Statistics Denmark. Data for the period 1982–2000 were obtained from the official website of Statistics Denmark  . The annual crop data were grouped into seven categories (Table  1 ). Table 1 Description of model variables used in the analyses of European brown hare harvest records from Denmark, 1955–2000. Variable Description Winter cereals Winter cereals, including winter wheat, winter barley, and winter rye Spring cereals Spring cereals, including spring wheat, spring barley, spring rye, and oat Grass in rotation Grass and green fodder in rotation, including grass, clover, maize, and Lucerne Grass out of rotation Grass areas permanently out of rotation Winter rape Winter rape Spring rape Spring rape Root crops Sugar beets for sugar production and fodder, mangolds, turnips, and carrots for feed Fox The number of red fox harvested NAO The winter state of the North Atlantic Oscillation No data on the fraction of winter and spring cereals, respectively, were available for 1955–61, and these data points were omitted in the analyses. Similarly, prior to 1982 no data on the fraction of winter barley and spring barley were available. However up until 1968, winter barley was only sown on a few thousand hectares in Denmark, when it was forbidden due to its function as reservoir for mildew attacking spring barley [ 42 ]. The use of mildew resistant winter barley was permitted in 1982. Hence, from 1955–81 barley was regarded as spring barley. Finally, no data on the fraction of winter and spring rye are available from 1955–61 and 1979–98, but in Denmark spring rye is generally sown on very few hectares only [ 42 ], and we regarded rye as winter rye unless otherwise specified [see also [ 32 ]]. Oat is sown as spring crop only. No data on the areas covered with grass and green fodder in or out of rotation were available for 1994, and the data point was interpolated, i.e. 1994 equalled the average of 1993 and 1995.
Agricultural land-use
Time series quantifying agricultural land-use data, i.e. annual crop areas in hectares, covering the period 1955–81 were obtained from Statistics Denmark. Data for the period 1982–2000 were obtained from the official website of Statistics Denmark  . The annual crop data were grouped into seven categories (Table  1 ).Description of model variables used in the analyses of European brown hare harvest records from Denmark, 1955–2000.No data on the fraction of winter and spring cereals, respectively, were available for 1955–61, and these data points were omitted in the analyses. Similarly, prior to 1982 no data on the fraction of winter barley and spring barley were available. However up until 1968, winter barley was only sown on a few thousand hectares in Denmark, when it was forbidden due to its function as reservoir for mildew attacking spring barley [ 42 ]. The use of mildew resistant winter barley was permitted in 1982. Hence, from 1955–81 barley was regarded as spring barley. Finally, no data on the fraction of winter and spring rye are available from 1955–61 and 1979–98, but in Denmark spring rye is generally sown on very few hectares only [ 42 ], and we regarded rye as winter rye unless otherwise specified [see also [ 32 ]]. Oat is sown as spring crop only. No data on the areas covered with grass and green fodder in or out of rotation were available for 1994, and the data point was interpolated, i.e. 1994 equalled the average of 1993 and 1995.Climatic variability As an index of winter severity, we used the winter state of the North Atlantic Oscillation [NAO; [ 26 ]]. The climatic phenomenon NAO is defined as the difference in sea-level pressure between Portugal and Iceland, affecting the temperature, precipitation and wind across the Northern Hemisphere [ 26 , 43 , 44 ]. The NAO, thus, integrates the effects of a number of abiotic factors, and therefore seems particularly useful when analysing the dynamics of larger, endothermic animals with relatively large buffer-capacity against climatic variability. The relationship between the winter NAO (December t-1  through March t ) and temperature, precipitation and wind is particularly strong in Northern Europe [ 45 ]. When NAO is in its high state, the winter climate in Northern Europe is warm, wet, and windy, while when in its low state winters are cold and dry [ 44 ]. The NAO winter index has shown an upward trend since the 1960s, which accounts for a substantial fraction of the general warming in the Northern Hemisphere [ 40 ]. The NAO index data are available from the Climate Analysis Section, National Center for Atmospheric Research, USA  .
Climatic variability
As an index of winter severity, we used the winter state of the North Atlantic Oscillation [NAO; [ 26 ]]. The climatic phenomenon NAO is defined as the difference in sea-level pressure between Portugal and Iceland, affecting the temperature, precipitation and wind across the Northern Hemisphere [ 26 , 43 , 44 ]. The NAO, thus, integrates the effects of a number of abiotic factors, and therefore seems particularly useful when analysing the dynamics of larger, endothermic animals with relatively large buffer-capacity against climatic variability.The relationship between the winter NAO (December t-1  through March t ) and temperature, precipitation and wind is particularly strong in Northern Europe [ 45 ]. When NAO is in its high state, the winter climate in Northern Europe is warm, wet, and windy, while when in its low state winters are cold and dry [ 44 ]. The NAO winter index has shown an upward trend since the 1960s, which accounts for a substantial fraction of the general warming in the Northern Hemisphere [ 40 ]. The NAO index data are available from the Climate Analysis Section, National Center for Atmospheric Research, USA  .t-1tData analyses To remove heteroscedasticity in the system, data were log e -transformed, i.e.  X (t)  = ln( N (t)  + 1), where  N (t)  is the number of European brown hares harvested in year (t) . To allow the direct comparison of regression coefficients, variables were standardized prior to the analyses (i.e., [ X (t) - µ (1955–2000) ]/ s (1955–2000) ). Regression coefficients therefore express the rate of change in standard deviation units of the independent variable per one standard deviation unit of the dependent variable [ 46 ]. To obtain stationarity in the time series [see [ 47 ] for details], data were detrended following [ 48 ] by including year as covariate in all models. For each district, we tested for the presence of multi-collinearity among parameters prior to the analyses by means of condition indices and variance proportions [ 49 ], but multi-collinearity was not observed in any districts (condition index < 12.47; [see [ 49 ] for details]). The European brown hare time series were tested for non-linearity in  X (t)  vs.  X (t-1)  by applying the likelihood ratio test [ 50 ]. In all districts, linearity in  X (t)  vs.  X (t-1)  was not rejected λ< 7.76, P > 0.05). We analysed the data using a first-order autoregressive (AR(1)) mixed model approach with district as fixed factor: X (t) ~( µ , V ), where and where LAND_USE represents the seven crop categories (see Table  1 ). Because of the inclusion of red fox numbers, we did not include a delayed AR term (i.e.  X t-2 ), as inclusion of both delayed density dependence and predator abundance can be seen as redundant [ 37 ]. All analyses were performed in SAS 8.2, using the PROC MIXED procedure with restricted maximum-likelihood estimation of regression coefficients [ 49 ]. Model reduction was conducted using log-likelihood ratio tests [ 51 ].
Data analyses
To remove heteroscedasticity in the system, data were log e -transformed, i.e.  X (t)  = ln( N (t)  + 1), where  N (t)  is the number of European brown hares harvested in year (t) . To allow the direct comparison of regression coefficients, variables were standardized prior to the analyses (i.e., [ X (t) - µ (1955–2000) ]/ s (1955–2000) ). Regression coefficients therefore express the rate of change in standard deviation units of the independent variable per one standard deviation unit of the dependent variable [ 46 ]. To obtain stationarity in the time series [see [ 47 ] for details], data were detrended following [ 48 ] by including year as covariate in all models.X(t)N(t)N(t)(t)X(t)µsFor each district, we tested for the presence of multi-collinearity among parameters prior to the analyses by means of condition indices and variance proportions [ 49 ], but multi-collinearity was not observed in any districts (condition index < 12.47; [see [ 49 ] for details]).The European brown hare time series were tested for non-linearity in  X (t)  vs.  X (t-1)  by applying the likelihood ratio test [ 50 ]. In all districts, linearity in  X (t)  vs.  X (t-1)  was not rejected λ< 7.76, P > 0.05). We analysed the data using a first-order autoregressive (AR(1)) mixed model approach with district as fixed factor:X(t)X(t-1)X(t)X(t-1)X (t) ~( µ , V ),X(t)µVwhereandwhere LAND_USE represents the seven crop categories (see Table  1 ). Because of the inclusion of red fox numbers, we did not include a delayed AR term (i.e.  X t-2 ), as inclusion of both delayed density dependence and predator abundance can be seen as redundant [ 37 ]. All analyses were performed in SAS 8.2, using the PROC MIXED procedure with restricted maximum-likelihood estimation of regression coefficients [ 49 ]. Model reduction was conducted using log-likelihood ratio tests [ 51 ].Xt-2Authors' contributions NMS designed the study, participated in data preparation, carried out the statistical analyses, and drafted the manuscript. TA participated in data preparation. MCF supported the data analyses and contributed to the writing. All authors read and approved the final manuscript.
Authors' contributions
NMS designed the study, participated in data preparation, carried out the statistical analyses, and drafted the manuscript. TA participated in data preparation. MCF supported the data analyses and contributed to the writing. All authors read and approved the final manuscript.


Introduction Spontaneous T cell responses against specific tumor-associated antigens (TAA) are frequently detected in peripheral blood of tumor patients of various histiotypes. However, little is known about whether these circulating, spontaneously occurring, TAA-reactive T cells influence the clinical course of disease.
Introduction
Spontaneous T cell responses against specific tumor-associated antigens (TAA) are frequently detected in peripheral blood of tumor patients of various histiotypes. However, little is known about whether these circulating, spontaneously occurring, TAA-reactive T cells influence the clinical course of disease.Methods Fifty-four HLA-A2 positive colorectal cancer patients had been analyzed for the presence of T cell responses against epitopes derived from the TAA Ep-CAM, her-2/ neu , and CEA either by ELISPOT assay or by intracellular cytokine staining. Then, Kaplan-Meier survival analysis was performed comparing T-cell-responders and T-cell-non-responders. For comparison, a group of T-cell-non-responders was compiled stringently matched to T-cell-responders based on clinical criteria and also analyzed for survival.
Methods
Fifty-four HLA-A2 positive colorectal cancer patients had been analyzed for the presence of T cell responses against epitopes derived from the TAA Ep-CAM, her-2/ neu , and CEA either by ELISPOT assay or by intracellular cytokine staining. Then, Kaplan-Meier survival analysis was performed comparing T-cell-responders and T-cell-non-responders. For comparison, a group of T-cell-non-responders was compiled stringently matched to T-cell-responders based on clinical criteria and also analyzed for survival.neuResults Sixteen out of 54 patients had a detectable T cell response against at least one of the three tested TAA. Two out of 21 patients (9.5%) with limited stage of disease (UICC I and II) and 14 out of 33 patients (42.4%) with advanced disease (UICC III and IV) were T cell response positive. Comparing all T-cell-responders (n = 16) and all T-cell-non-responders (n = 38), no survival difference was found. In an attempt to reduce the influence of confounding clinical factors, we then compared 16 responders and 16 non-responders in a matched group survival analysis; and again no survival difference was found (p = 0.7).
Results
Sixteen out of 54 patients had a detectable T cell response against at least one of the three tested TAA. Two out of 21 patients (9.5%) with limited stage of disease (UICC I and II) and 14 out of 33 patients (42.4%) with advanced disease (UICC III and IV) were T cell response positive. Comparing all T-cell-responders (n = 16) and all T-cell-non-responders (n = 38), no survival difference was found. In an attempt to reduce the influence of confounding clinical factors, we then compared 16 responders and 16 non-responders in a matched group survival analysis; and again no survival difference was found (p = 0.7).Conclusion In summary, we found no evidence that spontaneous peripheral T cell responses against HLA-A2-binding epitopes of CEA, her-2/neu and Ep-CAM are a strong prognostic factor for survival.
Conclusion
In summary, we found no evidence that spontaneous peripheral T cell responses against HLA-A2-binding epitopes of CEA, her-2/neu and Ep-CAM are a strong prognostic factor for survival.

Introduction The importance of the immune system in containing tumor growth is supported by animal studies and various observations in humans [ 1 , 2 ]. These include increased prevalence of certain tumors following immunosuppression as well as the demonstration, that the presence of intralesional T cells is correlated with improved clinical outcome in various solid tumors [ 1 , 3 - 6 ]. In particular in CRC, the presence of CD8+ T cells within the tumor microenvironment was significantly associated with a better survival in several studies [ 3 , 7 - 9 ]. However, the antigen-specificity of these cells was not determined. T cell responses against specific tumor-associated antigens (TAA) are frequently detected in the peripheral blood of tumor patients [reviewed in [ 10 ]] of various histiotypes including colorectal cancer [ 11 ], melanoma [ 12 , 13 ], acute myeloid leukemia [ 14 ], breast cancer [ 15 ], neuroblastoma [ 16 ], and head and neck cancer [ 17 ]. Data from selected single patients suggest a favorable clinical course in patients with peripheral, spontaneous TAA-directed T cells [ 18 , 19 ]. However, this type of analysis does not allow firm conclusions. The only study comparing clinical outcome of patients with presence of antigen-specific immune responses including natural as well as vaccine-induced antibodies against melanoma antigen GM2 showed an improved survival in favor of immune responders [ 20 ]. TAA-directed T cell responses can reliably be induced using various vaccination approaches [reviewed in [ 21 ]]. Several recent reports have found a correlation between induction of a TAA-directed T cell response by vaccination and clinical response [ 22 - 25 ]. Preliminary data also suggest a possibly favorable clinical effect of vaccine-induced T cells in adjuvant vaccination [ 26 - 29 ]. Taken together, these data lead to the question, whether the presence of spontaneous TAA-specific T cells might be a positive prognostic factor. So far, however, no study has systematically compared survival data of patients with and without presence of a spontaneous TAA-directed T cell response. In previous studies, we have demonstrated spontaneous T cell responses against the TAA CEA, Ep-CAM, or her-2/neu in peripheral blood of approximately 25% of colorectal cancer patients [ 11 , 30 ]. These cells were identified in functional T cell assays by antigen-induced IFNγ production. More detailed analyses in some samples revealed a CD3+ CD8+ IFNγ+ CD69+ CD45RA+ phenotype [ 11 ], indicative of an effector T cell subset that is able to directly mediate tumor cell lysis [ 19 ]. Spontaneous TAA-specific T cells with the potential of effector cells should, theoretically, be capable of destroying tumor cells and thereby lead to elimination of residual disease or prevent tumor progression. To investigate whether a peripheral, spontaneous T cell response has an effect on the clinical outcome of tumor patients, we analyzed survival data of CRC patients with a TAA-directed T cell response and compared these data with the clinical course of CRC patients without detectable T cell response.
Introduction
The importance of the immune system in containing tumor growth is supported by animal studies and various observations in humans [ 1 , 2 ]. These include increased prevalence of certain tumors following immunosuppression as well as the demonstration, that the presence of intralesional T cells is correlated with improved clinical outcome in various solid tumors [ 1 , 3 - 6 ]. In particular in CRC, the presence of CD8+ T cells within the tumor microenvironment was significantly associated with a better survival in several studies [ 3 , 7 - 9 ]. However, the antigen-specificity of these cells was not determined. T cell responses against specific tumor-associated antigens (TAA) are frequently detected in the peripheral blood of tumor patients [reviewed in [ 10 ]] of various histiotypes including colorectal cancer [ 11 ], melanoma [ 12 , 13 ], acute myeloid leukemia [ 14 ], breast cancer [ 15 ], neuroblastoma [ 16 ], and head and neck cancer [ 17 ]. Data from selected single patients suggest a favorable clinical course in patients with peripheral, spontaneous TAA-directed T cells [ 18 , 19 ]. However, this type of analysis does not allow firm conclusions. The only study comparing clinical outcome of patients with presence of antigen-specific immune responses including natural as well as vaccine-induced antibodies against melanoma antigen GM2 showed an improved survival in favor of immune responders [ 20 ]. TAA-directed T cell responses can reliably be induced using various vaccination approaches [reviewed in [ 21 ]]. Several recent reports have found a correlation between induction of a TAA-directed T cell response by vaccination and clinical response [ 22 - 25 ]. Preliminary data also suggest a possibly favorable clinical effect of vaccine-induced T cells in adjuvant vaccination [ 26 - 29 ]. Taken together, these data lead to the question, whether the presence of spontaneous TAA-specific T cells might be a positive prognostic factor. So far, however, no study has systematically compared survival data of patients with and without presence of a spontaneous TAA-directed T cell response.In previous studies, we have demonstrated spontaneous T cell responses against the TAA CEA, Ep-CAM, or her-2/neu in peripheral blood of approximately 25% of colorectal cancer patients [ 11 , 30 ]. These cells were identified in functional T cell assays by antigen-induced IFNγ production. More detailed analyses in some samples revealed a CD3+ CD8+ IFNγ+ CD69+ CD45RA+ phenotype [ 11 ], indicative of an effector T cell subset that is able to directly mediate tumor cell lysis [ 19 ]. Spontaneous TAA-specific T cells with the potential of effector cells should, theoretically, be capable of destroying tumor cells and thereby lead to elimination of residual disease or prevent tumor progression. To investigate whether a peripheral, spontaneous T cell response has an effect on the clinical outcome of tumor patients, we analyzed survival data of CRC patients with a TAA-directed T cell response and compared these data with the clinical course of CRC patients without detectable T cell response.Patients, materials, and methods Patient selection and T cell assays After institutional review board approval and informed consent, peripheral blood mononuclear cells from 132 patients with CRC in all stages of disease had been prospectively collected and frozen for T cell analysis. All analyses have been performed in compliance with the Helsinki Declaration. Fifty-four patients were tested positive for HLA-A2 and were subsequently analyzed for the presence of T cell responses against the HLA-A*0201 presented T cell epitopes Ep-CAM p263–271 [ 31 ], her-2/ neu  p654–662 [ 32 , 33 ], and CEA p571–579 [ 34 ] either by ELISPOT assay or by intracellular cytokine staining. HLA analysis, ELISPOT, and intracellular cytokine staining were performed as previously described [ 11 , 30 ]. Positive responses were defined as previously described [ 11 , 30 ]. Survival analysis First, we performed a Kaplan-Meier survival analysis comparing all T-cell-responders and all T-cell-non-responders. Additionally, a two-sided log rank test was used to test statistical significance. Then, in an attempt to reduce the influence of external factors, we compiled a patient group from non-responders matching them to responders according to the following criteria: UICC stage of disease, gender, presence of clinically detectable tumor at time of blood draw, duration of disease until blood draw, age at first diagnosis, and previous therapy. Survival in both groups was compared using Kaplan-Meier analysis and by a two-sided log rank test. A level of p < 0.05 was considered significant. SPSS (11.5) software was used.
Patients, materials, and methods
Patient selection and T cell assays After institutional review board approval and informed consent, peripheral blood mononuclear cells from 132 patients with CRC in all stages of disease had been prospectively collected and frozen for T cell analysis. All analyses have been performed in compliance with the Helsinki Declaration. Fifty-four patients were tested positive for HLA-A2 and were subsequently analyzed for the presence of T cell responses against the HLA-A*0201 presented T cell epitopes Ep-CAM p263–271 [ 31 ], her-2/ neu  p654–662 [ 32 , 33 ], and CEA p571–579 [ 34 ] either by ELISPOT assay or by intracellular cytokine staining. HLA analysis, ELISPOT, and intracellular cytokine staining were performed as previously described [ 11 , 30 ]. Positive responses were defined as previously described [ 11 , 30 ].
Patient selection and T cell assays
After institutional review board approval and informed consent, peripheral blood mononuclear cells from 132 patients with CRC in all stages of disease had been prospectively collected and frozen for T cell analysis. All analyses have been performed in compliance with the Helsinki Declaration. Fifty-four patients were tested positive for HLA-A2 and were subsequently analyzed for the presence of T cell responses against the HLA-A*0201 presented T cell epitopes Ep-CAM p263–271 [ 31 ], her-2/ neu  p654–662 [ 32 , 33 ], and CEA p571–579 [ 34 ] either by ELISPOT assay or by intracellular cytokine staining. HLA analysis, ELISPOT, and intracellular cytokine staining were performed as previously described [ 11 , 30 ]. Positive responses were defined as previously described [ 11 , 30 ].neuSurvival analysis First, we performed a Kaplan-Meier survival analysis comparing all T-cell-responders and all T-cell-non-responders. Additionally, a two-sided log rank test was used to test statistical significance. Then, in an attempt to reduce the influence of external factors, we compiled a patient group from non-responders matching them to responders according to the following criteria: UICC stage of disease, gender, presence of clinically detectable tumor at time of blood draw, duration of disease until blood draw, age at first diagnosis, and previous therapy. Survival in both groups was compared using Kaplan-Meier analysis and by a two-sided log rank test. A level of p < 0.05 was considered significant. SPSS (11.5) software was used.
Survival analysis
First, we performed a Kaplan-Meier survival analysis comparing all T-cell-responders and all T-cell-non-responders. Additionally, a two-sided log rank test was used to test statistical significance. Then, in an attempt to reduce the influence of external factors, we compiled a patient group from non-responders matching them to responders according to the following criteria: UICC stage of disease, gender, presence of clinically detectable tumor at time of blood draw, duration of disease until blood draw, age at first diagnosis, and previous therapy. Survival in both groups was compared using Kaplan-Meier analysis and by a two-sided log rank test. A level of p < 0.05 was considered significant. SPSS (11.5) software was used.Results Patients, T cell response, survival of responders and non-responders Fifty-four HLA-A2 positive CRC patients who had been analyzed for T cell responses were included in this study [ 11 , 30 ] and retrospectively analyzed for survival. The overall survival rate was 66.7% at a median of 27.5 months follow-up after blood draw for T cell analysis. In 16 out of 54 patients a total number of 26 T cell responses (between 10 and 1110 specific T cell per 10 6  PBMC) against one of the three TAA Ep-CAM, her-2/ neu , and CEA had been detected. Comparing all responders (n = 16) and all non-responders (n = 38), no survival difference was found in a two-sided log rank test (p = 0.4; Fig.  1A ). A very slight trend toward better survival was found in favor of non-responders, which is, however, far from being statistically significant. Since we have previously observed that T cell responses occur more frequently in patients with advanced stages of disease, we compared clinical data for all patients; and found that among non-responders only 50% (19 out of 38) had clinical stage III or IV disease while 87.5% (14 out of 16) responding patients had stage III or IV disease. Thus, the data on survival are possibly based on a strong stage-related bias. Therefore, we subsequently used an approach matching non-responders to responders. Figure 1 Kaplan-Meier survival analyses of colorectal cancer patients based on their T cell response state. Dashed lines refer to T-cell-responders, full lines refer to T-cell-non-responders, crosses mark censored cases. Time point 0 refers to the time of blood draw for T cell analysis.  A . Two groups of CRC patients (total n = 54) were analyzed for survival after T cell analysis. One group had a spontaneous T cell response against the tumor associated antigens CEA, Ep-CAM, or her-2/neu (n = 16, dashed line). The other group had no T cell response against these antigens (n = 38, full line). No survival difference between the groups was found (log rank, p = 0.4).  B . Two matched groups of CRC patients (total n = 32) were analyzed for survival after T cell analysis. One group had a spontaneous T cell response against tumor antigens CEA, Ep-CAM, or her-2/neu (n = 16, dashed line). The other group had no T cell response against these antigens and was selected by having similar clinical characteristics for stage, gender, presence of clinically detectable tumor at time of blood draw, duration of disease, age, and prior therapy (n = 16, full line). No survival difference was found (log rank, p = 0.7). Survival in matched patient groups Sixteen patients from the above group of 38 non-responders were matched with 16 responders to obtain two groups comparable for potentially confounding clinical factors, in particular stage of disease (see table  1 ). At the time of the survival analysis 13 of the total of 32 (40.6%) patients had died: seven T-cell-responders (n = 1 stage III, n = 6 stage IV) and six T-cell-non-responders (n = 1 stage III, n = 5 stage IV). All deaths were CRC-related. Median time to death among T-cell-responders was 11 months, among T-cell-non-responders 14.5 months. The calculated mean survival time after blood draw for patients without T cell response was 37.0 months (± 4.8 SEM) with a 95% confidence interval 27.5–46.5. Mean survival of T-cell-responders was 40.2 months (± 6.5 SEM) with 95% confidence interval of 27.5–52.9 (Fig.  1B ). In a two-sided log rank test, survival did not show a statistically significant difference between responders and non-responders (p = 0.7). Of note, one to two years after blood draw, patients without T cell response had an up to 20% higher survival rate (approx. 80% vs. approx 60%). These results were, however, not significant. In a two-sided test with β = 0.2, a survival difference of 70% could have been considered significant at a level of α = 0.05 in a population of this size. Table 1 Patient characteristics Peripheral spontaneous T cell response against TAA Positive Negative (matched) Negative (total) Number of patients 16 16 38 Age at first diagnosis, mean ± SD 61.2 ± 12.1 59.9 ± 9.1 60.8 ± 11.1 Male/Female 11/5 11/5 19/19 Duration of disease until T cell analysis (mean) 31.8 30.2 27.4 Stage (UICC) I 0 0 1 (2.6%) II 2 (12.5%) 2 (12.5%) 18 (47.4%) III 3 (18.8%) 3 (18.8%) 5 (13.2%) IV 11 (68.8%) 11 (68.8%) 14 (36.8%) Previous therapy CT 9 (56.3%) 12 (75%) 18 (47.4%) RT 3 (18.8%) 4 (25%) 7 (18.4%) Surgery 12 (75%) 14 (87.5%) 30 (78.9%) Patients without evidence of tumor at time of blood draw 4 (25%, 2 II, 2 III) 4 (25%, 2 II, 2 III) 17 (45%, 13 II, 3 III, 1 IV) Time from blood draw until death or last contact, mean ± SD 28.1 ± 19.2 27.4 ± 15.0 25.7 ± 14.0 Abbreviations: SD standard deviation, CT chemotherapy (5-FU + folic acid, in few cases plus Irinotecan), RT radiotherapy
Results
Patients, T cell response, survival of responders and non-responders Fifty-four HLA-A2 positive CRC patients who had been analyzed for T cell responses were included in this study [ 11 , 30 ] and retrospectively analyzed for survival. The overall survival rate was 66.7% at a median of 27.5 months follow-up after blood draw for T cell analysis. In 16 out of 54 patients a total number of 26 T cell responses (between 10 and 1110 specific T cell per 10 6  PBMC) against one of the three TAA Ep-CAM, her-2/ neu , and CEA had been detected. Comparing all responders (n = 16) and all non-responders (n = 38), no survival difference was found in a two-sided log rank test (p = 0.4; Fig.  1A ). A very slight trend toward better survival was found in favor of non-responders, which is, however, far from being statistically significant. Since we have previously observed that T cell responses occur more frequently in patients with advanced stages of disease, we compared clinical data for all patients; and found that among non-responders only 50% (19 out of 38) had clinical stage III or IV disease while 87.5% (14 out of 16) responding patients had stage III or IV disease. Thus, the data on survival are possibly based on a strong stage-related bias. Therefore, we subsequently used an approach matching non-responders to responders. Figure 1 Kaplan-Meier survival analyses of colorectal cancer patients based on their T cell response state. Dashed lines refer to T-cell-responders, full lines refer to T-cell-non-responders, crosses mark censored cases. Time point 0 refers to the time of blood draw for T cell analysis.  A . Two groups of CRC patients (total n = 54) were analyzed for survival after T cell analysis. One group had a spontaneous T cell response against the tumor associated antigens CEA, Ep-CAM, or her-2/neu (n = 16, dashed line). The other group had no T cell response against these antigens (n = 38, full line). No survival difference between the groups was found (log rank, p = 0.4).  B . Two matched groups of CRC patients (total n = 32) were analyzed for survival after T cell analysis. One group had a spontaneous T cell response against tumor antigens CEA, Ep-CAM, or her-2/neu (n = 16, dashed line). The other group had no T cell response against these antigens and was selected by having similar clinical characteristics for stage, gender, presence of clinically detectable tumor at time of blood draw, duration of disease, age, and prior therapy (n = 16, full line). No survival difference was found (log rank, p = 0.7).
Patients, T cell response, survival of responders and non-responders
Fifty-four HLA-A2 positive CRC patients who had been analyzed for T cell responses were included in this study [ 11 , 30 ] and retrospectively analyzed for survival. The overall survival rate was 66.7% at a median of 27.5 months follow-up after blood draw for T cell analysis. In 16 out of 54 patients a total number of 26 T cell responses (between 10 and 1110 specific T cell per 10 6  PBMC) against one of the three TAA Ep-CAM, her-2/ neu , and CEA had been detected. Comparing all responders (n = 16) and all non-responders (n = 38), no survival difference was found in a two-sided log rank test (p = 0.4; Fig.  1A ). A very slight trend toward better survival was found in favor of non-responders, which is, however, far from being statistically significant. Since we have previously observed that T cell responses occur more frequently in patients with advanced stages of disease, we compared clinical data for all patients; and found that among non-responders only 50% (19 out of 38) had clinical stage III or IV disease while 87.5% (14 out of 16) responding patients had stage III or IV disease. Thus, the data on survival are possibly based on a strong stage-related bias. Therefore, we subsequently used an approach matching non-responders to responders.neuSurvival in matched patient groups Sixteen patients from the above group of 38 non-responders were matched with 16 responders to obtain two groups comparable for potentially confounding clinical factors, in particular stage of disease (see table  1 ). At the time of the survival analysis 13 of the total of 32 (40.6%) patients had died: seven T-cell-responders (n = 1 stage III, n = 6 stage IV) and six T-cell-non-responders (n = 1 stage III, n = 5 stage IV). All deaths were CRC-related. Median time to death among T-cell-responders was 11 months, among T-cell-non-responders 14.5 months. The calculated mean survival time after blood draw for patients without T cell response was 37.0 months (± 4.8 SEM) with a 95% confidence interval 27.5–46.5. Mean survival of T-cell-responders was 40.2 months (± 6.5 SEM) with 95% confidence interval of 27.5–52.9 (Fig.  1B ). In a two-sided log rank test, survival did not show a statistically significant difference between responders and non-responders (p = 0.7). Of note, one to two years after blood draw, patients without T cell response had an up to 20% higher survival rate (approx. 80% vs. approx 60%). These results were, however, not significant. In a two-sided test with β = 0.2, a survival difference of 70% could have been considered significant at a level of α = 0.05 in a population of this size. Table 1 Patient characteristics Peripheral spontaneous T cell response against TAA Positive Negative (matched) Negative (total) Number of patients 16 16 38 Age at first diagnosis, mean ± SD 61.2 ± 12.1 59.9 ± 9.1 60.8 ± 11.1 Male/Female 11/5 11/5 19/19 Duration of disease until T cell analysis (mean) 31.8 30.2 27.4 Stage (UICC) I 0 0 1 (2.6%) II 2 (12.5%) 2 (12.5%) 18 (47.4%) III 3 (18.8%) 3 (18.8%) 5 (13.2%) IV 11 (68.8%) 11 (68.8%) 14 (36.8%) Previous therapy CT 9 (56.3%) 12 (75%) 18 (47.4%) RT 3 (18.8%) 4 (25%) 7 (18.4%) Surgery 12 (75%) 14 (87.5%) 30 (78.9%) Patients without evidence of tumor at time of blood draw 4 (25%, 2 II, 2 III) 4 (25%, 2 II, 2 III) 17 (45%, 13 II, 3 III, 1 IV) Time from blood draw until death or last contact, mean ± SD 28.1 ± 19.2 27.4 ± 15.0 25.7 ± 14.0 Abbreviations: SD standard deviation, CT chemotherapy (5-FU + folic acid, in few cases plus Irinotecan), RT radiotherapy
Survival in matched patient groups
Sixteen patients from the above group of 38 non-responders were matched with 16 responders to obtain two groups comparable for potentially confounding clinical factors, in particular stage of disease (see table  1 ). At the time of the survival analysis 13 of the total of 32 (40.6%) patients had died: seven T-cell-responders (n = 1 stage III, n = 6 stage IV) and six T-cell-non-responders (n = 1 stage III, n = 5 stage IV). All deaths were CRC-related. Median time to death among T-cell-responders was 11 months, among T-cell-non-responders 14.5 months. The calculated mean survival time after blood draw for patients without T cell response was 37.0 months (± 4.8 SEM) with a 95% confidence interval 27.5–46.5. Mean survival of T-cell-responders was 40.2 months (± 6.5 SEM) with 95% confidence interval of 27.5–52.9 (Fig.  1B ). In a two-sided log rank test, survival did not show a statistically significant difference between responders and non-responders (p = 0.7). Of note, one to two years after blood draw, patients without T cell response had an up to 20% higher survival rate (approx. 80% vs. approx 60%). These results were, however, not significant. In a two-sided test with β = 0.2, a survival difference of 70% could have been considered significant at a level of α = 0.05 in a population of this size.Patient characteristicsPeripheral spontaneous T cell response against TAAPositiveNegative (matched)Negative (total)Abbreviations: SD standard deviation, CT chemotherapy (5-FU + folic acid, in few cases plus Irinotecan), RT radiotherapyDiscussion In the present study, we analyzed the clinical course of colorectal cancer patients with or without T cells reactive against HLA-A2-binding epitopes of Ep-Cam, her-2/neu, and CEA. No survival difference between T-cell-responders and T-cell-non-responders was found. This result has to be interpreted cautiously due to the small number of HLA-A2+ patients responding to the above antigens. Obviously, these small numbers cause a high beta error. Thus, this study is only a first indication that the tested spontaneous T cell responses are not important prognostic factors for survival. The second limitation of our study is that the known repertoire of TAA as potentially important T cell targets in CRC grows every year; and our T cell analysis included only a fraction of potential epitopes. Various other CRC-associated antigens, such as MUC1 or p53, and additional MHC class I antigenic epitopes of CEA and her2-neu have been described [summarized in [ 35 ]]. T cell responses against antigens in addition to the ones tested here could potentially play a role in immune surveillance of CRC. Immune surveillance is understood as a complex process in which T cells and tumor cells influence each other in several ways ["immunoediting", [ 1 ]]. There are various potential factors related to tumor cells as well as T cells which may explain a lack of survival effect by TAA-specific T cells. The frequency of TAA-specific T cells detected in most patients was quite low in the range of 10 to 100 T cells per 10 6  PBMC. These numbers may be too low to control tumor growth especially in patients with a higher tumor burden. Furthermore, a general T cell dysfunction including anergic T cells and T cells with downregulated CD3-zeta chains has been described in CRC patients [ 36 , 37 ]. It is possible that the specific T cells detected in the present study are functionally unable to destroy tumor cells. This assumption, however, is not supported by our previous finding that TAA-specific T cells have an effector potential as analyzed in selected patients [ 11 ]. Since we have analyzed peripheral blood, we do not know if the circulating T cells have the potential to migrate to the tumor site or compartments where CRC cells frequently migrate to including lymph node, liver and bone marrow. Furthermore, tumor cells may not be recognizable by TAA-specific T cells. It has been shown that CRC cell lines secrete immunosuppressive cytokines and that development of T cell responses is impeded due to low HLA expression and lack of intercellular adhesion molecule-1 (ICAM-1) and HLA-DR [ 38 , 39 ]. This is especially relevant considering the fact that TAA-specific T cell responses in peripheral blood are more frequently detectable in advanced stages of CRC [ 11 , 40 ], as well as other tumors [ 40 , 41 ]. These data led to the hypothesis that metastasizing of tumor cells to lymph nodes is a prerequisite for the development of circulating T cell responses [ 11 , 42 ]. Furthermore, the presence of TAA-directed T cell responses may have selected immune escape tumor variants. A broad variety of tumor escape mechanisms, such as antigen loss or loss of HLA expression, is described in various clinical conditions [ 43 ]. It is possible that we encounter similar mechanisms in the present study since malignant cells had grown  in vivo  during the presence of specific T cell responses. Finally, the role of suppressor and regulatory T cells in this specific context is unknown. Taken together, no evidence was found that peripheral, spontaneous T cell responses against HLA-A*0201-binding epitopes of CEA, Ep-CAM, or her-2/neu influence survival of CRC patients. Since the low patient number limits the conclusion, further studies should investigate more patients, more detailed function and migratory pattern of spontaneous T cell responses as well as the genetic profile of the tumor; and consider a broader antigen and epitope repertoire. These studies could have implications for vaccination therapy as we learn more about why immune surveillance may fail to control tumors and if the presence of a natural T cell response may impact on the efficacy of a vaccine.
Discussion
In the present study, we analyzed the clinical course of colorectal cancer patients with or without T cells reactive against HLA-A2-binding epitopes of Ep-Cam, her-2/neu, and CEA. No survival difference between T-cell-responders and T-cell-non-responders was found. This result has to be interpreted cautiously due to the small number of HLA-A2+ patients responding to the above antigens. Obviously, these small numbers cause a high beta error. Thus, this study is only a first indication that the tested spontaneous T cell responses are not important prognostic factors for survival.The second limitation of our study is that the known repertoire of TAA as potentially important T cell targets in CRC grows every year; and our T cell analysis included only a fraction of potential epitopes. Various other CRC-associated antigens, such as MUC1 or p53, and additional MHC class I antigenic epitopes of CEA and her2-neu have been described [summarized in [ 35 ]]. T cell responses against antigens in addition to the ones tested here could potentially play a role in immune surveillance of CRC.Immune surveillance is understood as a complex process in which T cells and tumor cells influence each other in several ways ["immunoediting", [ 1 ]]. There are various potential factors related to tumor cells as well as T cells which may explain a lack of survival effect by TAA-specific T cells. The frequency of TAA-specific T cells detected in most patients was quite low in the range of 10 to 100 T cells per 10 6  PBMC. These numbers may be too low to control tumor growth especially in patients with a higher tumor burden. Furthermore, a general T cell dysfunction including anergic T cells and T cells with downregulated CD3-zeta chains has been described in CRC patients [ 36 , 37 ]. It is possible that the specific T cells detected in the present study are functionally unable to destroy tumor cells. This assumption, however, is not supported by our previous finding that TAA-specific T cells have an effector potential as analyzed in selected patients [ 11 ]. Since we have analyzed peripheral blood, we do not know if the circulating T cells have the potential to migrate to the tumor site or compartments where CRC cells frequently migrate to including lymph node, liver and bone marrow.Furthermore, tumor cells may not be recognizable by TAA-specific T cells. It has been shown that CRC cell lines secrete immunosuppressive cytokines and that development of T cell responses is impeded due to low HLA expression and lack of intercellular adhesion molecule-1 (ICAM-1) and HLA-DR [ 38 , 39 ]. This is especially relevant considering the fact that TAA-specific T cell responses in peripheral blood are more frequently detectable in advanced stages of CRC [ 11 , 40 ], as well as other tumors [ 40 , 41 ]. These data led to the hypothesis that metastasizing of tumor cells to lymph nodes is a prerequisite for the development of circulating T cell responses [ 11 , 42 ]. Furthermore, the presence of TAA-directed T cell responses may have selected immune escape tumor variants. A broad variety of tumor escape mechanisms, such as antigen loss or loss of HLA expression, is described in various clinical conditions [ 43 ]. It is possible that we encounter similar mechanisms in the present study since malignant cells had grown  in vivo  during the presence of specific T cell responses. Finally, the role of suppressor and regulatory T cells in this specific context is unknown.in vivoTaken together, no evidence was found that peripheral, spontaneous T cell responses against HLA-A*0201-binding epitopes of CEA, Ep-CAM, or her-2/neu influence survival of CRC patients. Since the low patient number limits the conclusion, further studies should investigate more patients, more detailed function and migratory pattern of spontaneous T cell responses as well as the genetic profile of the tumor; and consider a broader antigen and epitope repertoire. These studies could have implications for vaccination therapy as we learn more about why immune surveillance may fail to control tumors and if the presence of a natural T cell response may impact on the efficacy of a vaccine.Abbreviations CEA carcinoembryonic antigen, CRC, colorectal carcinoma; ELISPOT, enzyme-linked immunospot; IFNγ, Interferon-γ; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cells, TAA, tumor associated antigen.
Abbreviations
CEA carcinoembryonic antigen, CRC, colorectal carcinoma; ELISPOT, enzyme-linked immunospot; IFNγ, Interferon-γ; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cells, TAA, tumor associated antigen.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.