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(Journal of Leukocyte Biology. 2001;69:271-279.)
© 2001 by Society for Leukocyte Biology

Differences in the induction of CD8⁺ T cell responses by subpopulations of dendritic cells from afferent lymph are related to IL-1 secretion

Jayne C. Hope, Paul Sopp, Robert A Collins and Chris J. Howard

Institute for Animal Health, Compton, Newbury, Berkshire, United Kingdom

Correspondence: Dr. J. C. Hope, Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK. E-mail: Jayne.Hope{at}BBSRC.ac.uk

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The major subset of dendritic cells (DC) from bovine afferent lymph expresses the SIRP MyD-1 antigen, but not CD11a or the antigen recognized by mAb CC81, and potently stimulates CD4⁺ and CD8⁺ T lymphocyte proliferation. The minor subpopulation, that is CD11a⁺CC81⁺MyD-1^-, effectively stimulates CD4⁺ but not CD8⁺ T lymphocyte proliferation. CD11a⁺CC81⁺MyD-1^- DC did not induce anergy or death or secrete an inhibitory factor. However, supernatant from cultures of CD8⁺ T cells with CD11a^-CC81^-MyD-1⁺ DC significantly enhanced proliferation of CD8⁺ T cells in response to CD11a⁺CC81⁺MyD-1^- DC, an effect that was blocked by interleukin (IL)-1, but not IL-1ß, specific mAb. The proliferation of CD8⁺ T cells with CD11a⁺CC81⁺MyD-1^- DC was also enhanced by adding IL-1. IL-1ß slightly enhanced proliferation, whereas IL-2, IL-6, IL-12, and IL-15 had no effect. We conclude that the failure to stimulate CD8⁺ T cell proliferation results from the lack of IL-1 synthesis by this population, which may have important consequences in vivo.

Key Words: afferent lymph veiled cells • CD8⁺ lymphocytes

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Dendritic cells (DC) are a system of antigen-presenting cells scattered sparsely throughout the body tissues, the function of which is to stimulate and control immune responses. DC are unique in their capacity to stimulate immune responses in naive T lymphocytes. Studies of murine Langerhans cells have provided a model in which cells in the periphery are effective at antigen uptake but poor stimulators of T lymphocyte responses. The process of migration is associated with functional and phenotypic changes such that, upon arrival within lymphoid tissues, DC are able to present processed antigen and stimulate the activation and proliferation of antigen-specific T lymphocytes [¹ ]. Dendritic cells from afferent lymph draining the skin or intestine are able to both take up antigen and to effectively stimulate proliferation of naive and memory T lymphocytes [² , ³ ].

Heterogeneity of DC biology has been reported in many studies. Differences in tissue location or maturation-induced differences in DC phenotype and function may account, in part, for this. However, there is also evidence that DC may derive from more than one lineage. Thus, the majority of DC in lymphoid organs are of myeloid origin. These cells can be isolated in vitro after culture of blood and bone marrow precursors in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and other cytokines, including tumor necrosis factor (TNF-), stem cell factor, and interleukin-4 (IL-4) [^{4 5 6} ]. Evidence from humans and mice indicates that cells of the lymphoid lineage, which are present in both bone marrow and thymus, can also differentiate into DC [⁷ , ⁸ ]. These DC express lymphoid markers, including CD8 in mice [⁹ ], and display different functional activities when compared to myeloid-derived DC. For example, mouse lymphoid DC (CD8⁺) were reported to display suppressive, rather than stimulatory, effects on both CD4⁺ and CD8⁺ T lymphocytes [¹⁰ , ¹¹ ]. However, recent evidence suggests that CD8⁺ DC may preferentially stimulate Th1 responses [¹² , ¹³ ] and secrete interferon- (IFN-) [¹⁴ ] and IL-12 [¹⁵ ]. In humans a putative lymphoid DC, the CD4⁺ CD11c^- CD3^- plasmacytoid cell, has recently been isolated from lymphoid tissue and blood [¹⁶ ]. It has been reported that these cells induce a Th2-biased response in contrast to the Th1 response induced by monocyte-derived DC [¹⁷ ].

Studies of rat and cattle afferent lymph veiled cells (ALVC) have also demonstrated heterogeneity of these cells in terms of surface phenotype and function [² , ³ ]. These cells represent a population of DC derived without requiring culture, allowing deductions to be made regarding their in vivo properties. DC isolated from bovine afferent lymph have previously been shown to comprise two subpopulations [² ]. The major subpopulation of bovine ALVC is characterized by expression of the MyD-1 antigen [¹⁸ ], a member of the SIRP family of proteins, and lack of expression of both CD11a and the antigen recognized by mAb CC81. The CD11a^- CC81^- MyD-1⁺ ALVC are potent stimulators of both CD4- and CD8-mediated proliferative responses. However, although the minor subpopulation of ALVC (CD11a⁺ CC81⁺ MyD-1^-), can stimulate allogeneic and antigen-specific proliferative responses of CD4⁺ lymphocytes, these cells are relatively inefficient at stimulating CD8 lymphocyte proliferation [² ]. The basis for the differential stimulatory capacity of these populations is not known, and both express similar levels of MHC class II, CD40, and CD80/86 [² ]. Two major subpopulations of rat ALVC have also been defined and can be distinguished on the basis of differential expression of CD4 and the SIRP protein homolog of bovine MyD-1 recognized by monoclonal antibody OX41 [³ , ¹⁹ ]. The subpopulations are also distinct in function: CD4⁺OX41⁺ rat ALVC stimulate vigorous T lymphocyte responses, whereas the CD4^-OX41^- population is less efficient and may be involved in the induction and maintenance of self tolerance [²⁰ ]. The basis for heterogeneity in function and phenotype of cattle and rat ALVC remains obscure; however, evidence suggests that their reported differences are unlikely to reflect different maturation stages of DC [² , ³ ]. It seems likely that differences in cytokine secretion or surface molecule expression may be central to the differential T cell stimulatory capacity of ALVC subsets.

We have studied the basis of the differential capacity of bovine ALVC populations to stimulate allogeneic CD8⁺ lymphocyte responses and demonstrate the potential importance of IL-1 as a factor that affects the differential induction of CD8⁺ T lymphocyte proliferation by ALVC subpopulations.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Monoclonal antibodies
The sources of mouse mAb and their isotypes, secondary reagents, and methods for flow cytometry have been described in detail elsewhere [² ]. The mAb used in this study that were specific for cattle antigens were as follows: CC63 (CD8; IgG2a), IL-A114 (WC6; IgG1), CC98 (WC6; IgG2b), CC149 (MyD-1; IgG2b), IL-A99 (CD11a, IgG2a), and CC81 (IgG1) that recognizes an undefined antigen expressed on a minor subpopulation of ALVC [² ]. The 210-kDa WC6 antigen is expressed at a high level on ALVC but not on monocytes [²¹ ]. The MyD-1 antigen is expressed on bovine monocytes, macrophages, and subsets of DC [¹⁸ ]. Bound mAb was detected with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) -labeled anti-mouse isotype-specific reagents (Southern Biotechnology Associates, Birmingham, AL).

Recombinant cytokines
Bovine rIL-2 [²² ] and human rIL-15 (Peprotech EC, London, UK) were titrated on freshly isolated CD8⁺ lymphocytes, and proliferation was assessed on day 5. Concentrations of IL-2 and IL-15 that induced suboptimal proliferation of purified CD8⁺ cells were selected for further experiments (2 U/mL and 0.2 ng/mL, respectively). Bovine IL-1 and IL-1ß, IL-6, and IL-12 were produced in a transfection COS-7 system [²³ ]. Stock solutions were titrated in functional assays. One unit was defined as the concentration giving half-maximal responses. A final concentration of 10 U/mL was used unless otherwise stated. mAb specific for bovine IL-6 (CC310) and IL-12 (CC301) [²⁴ ] were also prepared in this laboratory and were assessed for neutralizing capacity using biological assays. mAb 10.82 (anti-ovine IL-1 [²⁵ ], which also recognizes bovine IL-1) was provided by Dr. G. Barcham, School of Veterinary Science, University of Melbourne, Australia. The anti-bovine IL-1ß mAb was a kind gift from Dr. D. Werling, Institute of Animal Sciences, Schwerzenbach, Switzerland. Both monoclonal antibodies were validated for anti-IL-1 activity at the institutes from which they originated [²⁵ , ²⁶ ]. Isotype-matched controls were murine mAb against avian cell surface proteins: AV20 (Bu-1, IgG1) and AV37 (chicken spleen cell subset, IgG2a), both provided by F. Davidson, Institute for Animal Health, Compton. All mAbs were used at a final concentration of 25 µg IgG/mL [as assessed by enzyme-linked immunosorbent assay (ELISA)].

Preparation of CD8⁺ lymphocytes
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density gradient centrifugation (1.083 g/mL Histopaque, Sigma Chemical, Poole, UK). CD8⁺ cells were isolated from PBMC after staining with mAb CC63 [²⁷ ]. Thereafter cells were incubated with anti-mouse IgG2_a+b super-paramagnetic particles (Miltenyi-Biotech, Bergisch-Gladbach, Germany), and labeled cells were isolated from a Minimacs column (Miltenyi Biotech) according to the manufacturer’s instructions. The purity of the cells was evaluated by flow cytometry and shown to be >97%. Cells were adjusted to 10⁶/mL in RPMI 1640 medium containing Glutamax-1 (Life Technologies, Paisley, UK), 10% heat-inactivated fetal calf serum (FCS), 5 x 10^-5 M 2-mercaptoethanol (2-ME), 50 µg/mL gentamycin [tissue culture medium (TCM)].

Isolation of ALVC
Afferent lymph was obtained by cannulation of calves after surgical removal of the prescapular lymph node [²⁸ ]. After density gradient centrifugation (Histopaque, Sigma), cells were suspended in 10% dimethyl sulfoxide (DMSO)-FCS and frozen in liquid N₂. Immediately before use, afferent lymph samples were thawed and washed in Hanks’ balanced salt solution (Life Technologies) containing 1 mM EDTA. ALVC were identified on the basis of high forward scatter and expression of the WC6 antigen. Two distinct subpopulations of ALVC were further identified on the basis of expression of CD11a or differential staining with mAb CC81 [² ]. ALVC subsets were sorted by flow cytometry using a FACStar plus (Becton Dickinson, San Jose, CA) after staining with appropriate mAb to the WC6 antigen together with anti-CD11a mAb or mAb CC81 and isotype-specific secondary reagents [² ].

Proliferation assays
Allogeneic CD8⁺ lymphocytes (10⁵/well) were incubated in triplicate with 4 x 10³ irradiated ALVC (20 Gy from a ¹³⁷Cs source) in a total volume of 200 µL TCM. Supernatants harvested on day 4 from cultures of CD8⁺ cells that had been incubated with CD11a^- ALVC (SN), recombinant cytokines 4.2, or anti-cytokine mAb were also added in some experiments. Cultures were incubated for 5 days at 37°C. [³H]thymidine (³HTdR, 37 mBq; DuPont, Stevenage, UK) was added for the final 18 h of culture. Results are expressed as mean ± SD of triplicate wells.

Determination of CD8⁺ T lymphocyte cytolytic activity
The capacity of CD8⁺ cells to induce cytolysis in allogeneic target cells was assessed on day 5 after stimulation. Allogeneic targets were afferent lymph cells infected with Theileria annulata, a parasite that induces transformation of bovine cells [²² ]. Target cells were labeled with the membrane dye PKH-2 (Sigma) and incubated with CD8⁺ cells at effector-to-target ratios ranging from 50:1 to 0.39:1. After 4 h cells were stained with propidium iodide (PI) and the percentage of target cells expressing both PKH-2 and PI was assessed by flow cytometry as previously described [²⁹ ].

Assessment of apoptosis of stimulated CD8⁺ lymphocytes
CD8⁺ lymphocytes (10⁵/well) were incubated with allogeneic ALVC as described above. Apoptosis of CD8⁺ cells was assessed on consecutive days by staining with Annexin V-FITC (Boehringer Mannheim, Germany), which recognizes phosphatidylserine expression on early apoptotic cells. Necrotic cells were identified by incorporation of PI. Thus, early apoptotic cells were Annexin V-positive and did not incorporate PI, whereas necrotic cells were PI-positive and Annexin V-negative. Live cells expressed neither Annexin V nor incorporated PI.

Statistical analysis
Statistical analyses were performed using a paired Student’s t test. P values of less than 0.05 were considered significant.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CD11a^- CC81^-, but not CD11a⁺ CC81⁺, ALVC induce allogeneic proliferation of CD8⁺ T lymphocytes
CD11a^- and CD11a⁺ veiled cells were purified from afferent lymph by sorting with a FACStar plus, and their capacity to induce proliferation of allogeneic CD8⁺ T lymphocytes was investigated. The CD11a^- subpopulation induced a proliferative response in allogeneic CD8⁺ T cells that was significantly higher than that induced by the CD11a⁺ population (Fig. 1A ). Similar differences in the capacity to stimulate allogeneic CD8⁺ T cell proliferation were observed when the subpopulations of ALVC were purified on the basis of differential expression of the molecule recognized by mAb CC81 (Fig. 1B) .

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Figure 1. CD11a- CC81-, but not CD11a+ CC81+, ALVC stimulate allogeneic proliferation of CD8+ T lymphocytes. ALVC were identified on the basis of expression of WC6. Subsets were identified on the basis of expression of CD11a (A) or the undefined antigen recognized by mAb CC81 (B) and purified by sorting on a FACStar plus. ALVC were cultured with allogeneic CD8+ lymphocytes for 5 days. [3H]TdR was added for the final 18 h of culture. Results are expressed as mean cpm ± SD of triplicate wells.

CD8⁺ T lymphocytes stimulated by CD11a⁺ ALVC remain viable
To assess whether the CD11a⁺ subpopulation of ALVC was inducing death of CD8⁺ T cells, the level of apoptosis or necrosis of the responding CD8⁺ lymphocytes was assessed after stimulation with ALVC subsets. No significant difference in the percentage of dead cells was detected after stimulation of CD8⁺ T cells with either subpopulation of ALVC, with the number of live cells remaining relatively constant throughout the 5-day culture period (Table 1 ). Representative flow cytometric profiles of these cells are illustrated in Figure 2 .

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Table 1. CD8+ T Lymphocytes Stimulated by CD11a+ ALVC Remain Viable

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Figure 2. CD8+ T lymphocytes stimulated by CD11a+ ALVC remain viable. ALVC subsets were purified as for Figure 1 and cultured with purified CD8+ T lymphocytes. On each day of culture the percentage of live cells was assessed by flow cytometry. Apoptotic cells were identified by staining with Annexin V FITC, and necrotic cells by staining with PI. Live cells [in the lower left (LL) quadrant of each dot plot] expressed neither Annexin V nor incorporated PI. The flow cytometric data and the percentage of cells in each quadrant is shown for day 1 and day 5 of culture. The data for all 5 days of culture are summarized in Table 1 . Quadrants: UL, upper left; UR, upper right; LL, lower left; LR, lower right.

CD11a⁺ ALVC do not inhibit the response of CD8⁺ T lymphocytes through the secretion of an inhibitory cytokine nor through the induction of T cell anergy
The low proliferation of CD8⁺ T cells induced by CD11a⁺ ALVC could have reflected the secretion by the ALVC of an inhibitory cytokine or the induction of T cell anergy by these cells. CD8⁺ T lymphocytes were incubated with purified ALVC or a combination of the subsets. No significant down-regulation of the response to CD11a^- ALVC was observed when they were mixed with CD11a⁺ ALVC (Fig. 3 ). In addition, when CD8⁺ T lymphocytes were cultured with CD11a⁺ ALVC for 2 h before addition of CD11a^- ALVC (Fig. 3) , no significant inhibitory effect on the proliferative response of CD8⁺ T cells to CD11a^- ALVC was observed. These observations indicated that the CD8⁺ T cells were not anergized by contact with the CD11a⁺ subset, and that CD11a⁺ ALVC do not provide a down-regulatory signal.

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Figure 3. CD11a+ ALVC do not inhibit the response of CD8+ T lymphocytes. ALVC subsets were purified as for Figure 1 . Either both subsets were mixed at the same time with the allogeneic CD8+ T cells or CD11a+ ALVC were cultured with CD8+ T cells for 2 h before addition of the CD11a- subset. Proliferation was assessed on day 5 as [3H]TdR incorporation and results are expressed as mean cpm ± SD.

CD8⁺ T lymphocytes stimulated by both ALVC subsets are cytolytic
The ability of CD8⁺ T cells stimulated by ALVC subsets to lyse allogeneic target cells was investigated. Both CD11a^- and CD11a⁺ ALVC induced cytotoxic CD8⁺ T lymphocytes (Fig. 4 ). The cytotoxic capacity of CD8⁺ T cells stimulated by CD11a^- ALVC (Fig. 4 , filled symbols) was marginally higher than that of CD8⁺ T cells that had been stimulated by CD11a⁺ ALVC (Fig. 4 , open symbols).

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Figure 4. CD8+ T lymphocytes stimulated by both ALVC subsets are effective cytotoxic T cells. ALVC subsets were purified as described for Figure 1 and cultured with allogeneic CD8+ T cells for 5 days. The cytolytic activity of the recovered CD8+ T cells in response to allogeneic target cells was assessed by flow cytometry. The percentage of dead cells was calculated as the percentage of target cells that incorporated PI.

The differential capacity of CD11a^- and CD11a⁺ ALVC to induce CD8⁺ T lymphocyte proliferation is not due to a lack of IL-2 or IL-15 secretion
The reduced capacity of CD11a⁺ ALVC to induce allogeneic CD8⁺ T cell proliferation may have been due to an inability to induce IL-2 secretion by the responding lymphocytes. Production of IL-2 by CD8⁺ T cells was assessed in culture supernatants by biological assay. Low levels of IL-2 were detected after culture of CD8⁺ T cells alone, with no significant increase after culture with either subset of ALVC (data not shown). To further assess the potential role of IL-2, rIL-2 was added to cultures of CD8⁺ lymphocytes and CD11a⁺ ALVC. No enhancement of proliferation was observed upon addition of IL-2 (Fig. 5 ), and the difference in CD8⁺ T cell stimulation between CD11a^- and CD11a⁺ ALVC was maintained. Because IL-15 has similar properties to IL-2 we assessed the effect of addition of IL-15 to cultures of allogeneic CD8⁺ T lymphocytes and CD11a⁺ ALVC. As for IL-2, no enhancement of the proliferation of CD8⁺ T cells in response to CD11a⁺ ALVC was observed (Fig. 5) .

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Figure 5. The differential capacity of CD11a- and CD11a+ ALVC to induce CD8+ T lymphocyte proliferation is not due to a lack of IL-2 or IL-15. ALVC subsets were purified as described for Figure 1 and cultured with allogeneic CD8+ lymphocytes for 5 days, in the presence of either IL-2 (2 U/mL) or IL-15 (0.2 ng/mL). [3H]TdR was added for the final 18 h of culture. Results are expressed as mean cpm ± SD of triplicate wells.

Supernatants derived from cultures of CD11a^- ALVC and CD8⁺ lymphocytes enhance proliferation of CD8⁺ T cells in response to CD11a⁺ ALVC
The failure of CD11a⁺ ALVC to stimulate CD8⁺ T lymphocyte proliferation may have been due to the inability of these cells to secrete a costimulatory cytokine that is produced by the CD11a^- subset. Supernatants (SN) were derived from cultures of CD8⁺ lymphocytes and CD11a^- ALVC, and added to cultures of CD8⁺ T cells and CD11a⁺ ALVC. The addition of supernatant significantly enhanced the response to CD11a⁺ ALVC, such that the response of CD8⁺ T cells to both subsets of ALVC were similar in magnitude (P < 0.01; Fig. 6 ).

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Figure 6. Supernatants from cultures of CD11a- ALVC, and CD8+ lymphocytes enhance proliferation of CD8+ T cells in response to CD11a+ ALVC. CD11a+ ALVC were cultured with allogeneic CD8+ lymphocytes in the presence of the indicated dilutions of supernatant (SN) that had previously been derived from 4-day cultures of CD11a- ALVC with CD8+ lymphocytes. Results are expressed as mean cpm ± SD of triplicate wells. Purified CD11a- ALVC were cultured with CD8+ T cells as positive control.

Addition of IL-1 or IL-1ß, but not of IL-6 or IL-12, enhances the proliferative response of CD8⁺ T lymphocytes stimulated by CD11a⁺ ALVC
To determine whether lack of secretion of a costimulatory cytokine could account for the reduced allo-stimulatory capacity of CD11a⁺ ALVC, recombinant IL-1, IL-6, or IL-12 were added to cultures of CD8⁺ T cells and CD11a⁺ ALVC. Addition of 10 U/mL of IL-1ß (P=0.18), IL-6 (P=0.07), or IL-12 (P=0.07) had no significant effect on the proliferative response of CD8⁺ T cells induced by CD11a⁺ ALVC (Fig. 7 ). Furthermore, neither anti-IL-6 nor anti-IL-12 mAbs could block the increased proliferative response induced by supernatant (SN) derived from CD8/CD11a^- cultures (final dilution of supernatant, 1:8). However, addition of IL-1 to culture did significantly increase the proliferation of CD8⁺ T cells in response to CD11a^- ALVC (Fig. 7 ; P=0.03). Anti-IL-1 significantly reduced the increased proliferative response induced by supernatant derived from CD8/CD11a^- cultures, and also reduced the response of CD8⁺ T cells to CD11a^- ALVC (Fig. 8 ; P<0.05). Although addition of higher concentrations of IL-1ß (50 U/mL; Fig. 9 ) did slightly enhance the proliferation of CD8⁺ T cells in response to CD11a⁺ ALVC (P0.05), anti-IL-1ß antibody failed to block the increased proliferative response induced by SN derived from CD8/CD11a^- cultures, and did not reduce the response of CD8⁺ T cells to CD11a^- ALVC. Control mAb showed no significant blocking in either case (<10% reduction; data not presented). These results suggested that IL-1ß may be less important than IL-1 for the CD8⁺ T cell proliferation induced by ALVC.

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Figure 7. Addition of IL-1, IL-6, or IL-12 enhances the proliferative response of CD8+ T lymphocytes stimulated by CD11a+ ALVC. ALVC subsets were cultured with allogeneic CD8+ lymphocytes in the presence of the indicated cytokines (10 U/mL final concentration) or in the presence of supernatant derived from 4-day cultures of CD11a- ALVC with CD8+ lymphocytes (SN; 1:8 final dilution), either with or without the indicated mAb (25 µg/mL final concentration). Results are expressed as mean cpm ± SD of triplicate wells. *Significantly increased compared to CD8+/CD11a+ (P<0.05).

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Figure 8. Importance of IL-1 for the proliferative response of CD8+ T lymphocytes stimulated by ALVC. ALVC were cultured with allogeneic CD8+ lymphocytes in the presence of IL-1 (10 U/mL final concentration) or in the presence of supernatant derived from 4-day cultures of CD11a- ALVC with CD8+ lymphocytes (1:8 final dilution), either with or without the anti-IL-1 or control mAb (25 µg/mL final concentration). Results are expressed as mean cpm ± SD of triplicate wells. *Significantly decreased compared with relevant control (P<0.05).

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Figure 9. Role of IL-1 and IL-1ß in the proliferative response of CD8+ T lymphocytes stimulated by ALVC. ALVC were cultured with allogeneic CD8+ lymphocytes in the presence of IL-1 or IL-1ß (10, 50, or 100 U/mL final concentration) or in the presence of supernatant derived from 4-day cultures of CD11a- ALVC with CD8+ lymphocytes (1:8 final dilution), either with or without anti-IL-1, anti-IL-1ß or control mAb (25 µg/mL final concentration). Results are expressed as mean cpm ± SD of triplicate wells. Control mAbs were added as in Figure 8 , and no significant blocking was observed (<10% reduction; data not presented). *Significantly different from relevant control (P<0.05).

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bovine ALVC comprise two subpopulations characterized by differential expression of the MyD-1 antigen [¹⁸ ], CD11a, and the molecule recognized by mAb CC81 [² ]. These populations also display differences in their ability to stimulate T cells. Thus, although the major population of ALVC (CD11a^- CC81^- MyD-1⁺) are potent stimulators of both CD4⁺ and CD8⁺ T lymphocyte proliferative responses, the minor subpopulation of ALVC (CD11a⁺ CC81⁺ MyD-1^-), is significantly less efficient at stimulating CD8⁺ lymphocyte proliferation [² ]. A similar disparity in the stimulation of CD8⁺ lymphocyte proliferation has been reported for subsets of murine splenic DC [¹¹ ]. CD8⁺ lymphoid DC failed to stimulate the proliferation of CD8⁺ T lymphocytes, whereas myeloid DC were effective in stimulating CD8⁺ T cell responses. These data suggest that the CD11a⁺ CC81⁺ MyD-1^- subset may have similar functional properties to the murine lymphoid DC. However, the bovine CD11a⁺ CC81⁺ MyD-1^- ALVC lack expression of antigens such as CD8, which characterize murine lymphoid DC [⁹ ], and a conclusion on direct homology would be premature. The failure of murine lymphoid DC to induce CD8⁺ T cell proliferation was reported to be due to a lack of induction of IL-2 secretion by the responding lymphocytes [¹¹ ], and the response could be restored by the addition to culture of IL-2. In contrast, the response of cattle CD8⁺ T lymphocytes to the CD11a⁺ CC81⁺ MyD-1^- subset of ALVC could not be enhanced by the addition of IL-2, suggesting that the failure of CD11a⁺CC81⁺ MyD-1^- ALVC to induce CD8⁺ lymphocyte proliferation is not due to a lack of stimulation through the IL-2R. Recent evidence [³⁰ ] suggests that an IL-2-independent pathway of CD8⁺ T cell activation may be important. Furthermore, other cytokines that signal through components of the IL-2 receptor, such as IL-15, may be important in the absence of IL-2 [³¹ , ³² ]. However, addition of IL-15, which is known to be produced by human DC [³² ], had no enhancing activity on the proliferative response of CD8⁺ T cells to ALVC.

There was no evidence to suggest that the failure of CD11a⁺ CC81⁺ MyD-1^- ALVC to induce CD8 proliferation reflected the induction of anergy or death within the responder population, as was reported for CD4⁺ lymphocytes stimulated by mouse CD8⁺ DC [¹⁰ ]. Combinations of ALVC subsets were effective in the induction of T cell activation, indicating that the low-stimulatory ALVC subset does not deliver an inhibitory signal. CD8⁺ lymphocytes stimulated by CD11a⁺ CC81⁺ MyD-1^- ALVC were cytolytically active. In mice, both myeloid and lymphoid DC induced cytolytic CD8⁺ lymphocytes, although the lymphoid DC were lower in stimulatory efficiency [³³ ]. Induction of T cell tolerance by a DC subset has also been proposed from studies of ALVC in a rat model [²⁰ ]. Rat ALVC are OX41⁺ CD4⁺ or OX41^- CD4^- and the OX41 antigen is the rat homolog of the cattle MyD-1 antigen [³ , ¹⁹ ]. Thus, bovine MyD-1⁺ ALVC (CD11a^- CC81^-) and rat OX41⁺ ALVC may be equivalent populations of DC. Of particular importance from the studies of rat ALVC was the evidence for endocytosis of apoptotic intestinal cells by OX41^- DC and it was suggested that these cells may transport apoptotic cell debris to the draining lymph nodes where presentation of self antigen may induce T cell tolerance [²⁰ ]. However, apoptotic debris was only evident in DC associated with the intestine and it is not possible to conclude whether the CD11a⁺ CC81⁺ MyD-1^- cattle ALVC are the equivalent of the OX41^- rat ALVC.

The results suggest that the lack of response to CD11a⁺ CC81⁺ MyD-1^- ALVC reflects a lack of costimulation rather than a direct down-regulatory effect on the responding CD8⁺ T cell population. Differential expression of surface costimulatory molecules is unlikely to account for the divergent capacity of ALVC subsets to induce CD8⁺ T cell proliferation, as both subsets have been shown to express similar levels of MHC class II and CD80/86 [² ]. The likelihood that a secreted mediator is important for the stimulation of CD8⁺ T cells by ALVC was illustrated in experiments where the addition of culture supernatants derived from CD11a^-/CD8⁺ to CD11a⁺/CD8⁺ cultures significantly enhanced proliferation. The importance of cytokines as costimulatory molecules is well recognized [¹ ]. IL-12 is an important DC-derived cytokine that, in addition to costimulation of T cell activation, may direct the development of Th1-biased immunity [³⁴ ]. However, the addition of IL-12 to cultures of CD11a⁺ CC81⁺ MyD-1^- ALVC and CD8⁺ T cells failed to enhance stimulation, and anti-IL-12 antibodies did not block the effect observed upon addition of culture supernatant derived from the CD11a^- subset. Thus, although cattle DC may produce IL-12 [R. A. Collins, unpublished observations], this cytokine appears not to be important in the stimulation of allogeneic CD8⁺ T cell proliferation by ALVC. Other potentially important costimulatory cytokines include IL-1 and IL-6 [¹ ]. Early reports suggested that IL-1 and/or IL-6 could replace the requirement for antigen-presenting cells in the stimulation of both CD4 and CD8 lymphocytes [³⁵ ]. Both cytokines are produced by murine Langerhans cells and lymph node DC [³⁶ , ³⁷ ]. However, the addition of IL-6 to culture failed to significantly increase the CD8⁺ T cell proliferative response induced by the CD11a⁺ CC81⁺ MyD-1^- ALVC. In addition, expression of IL-6 mRNA was not detected in freshly isolated bovine ALVC [P. J. Chaplin, R. A. Collins, and C. J. Howard, unpublished observations]. Addition of IL-1ß was only effective at higher doses (50 U/mL), and antibodies to this cytokine did not block the enhanced proliferation observed upon addition of supernatant derived from CD8/CD11a^- cultures, nor did anti-IL-1ß block CD8⁺ T cell proliferation induced by the CD11a^- ALVC, suggesting that IL-1ß may not be an important element of the stimulation of CD8⁺ T cells by bovine ALVC. In contrast, there was a significant effect observed upon addition of IL-1, and the enhancement observed with CD11a^-/CD8⁺ cell culture supernatant was blocked with antibodies against IL-1. Moreover there was a significant reduction of the response to CD11a^- ALVC in the presence of anti-IL-1 mAb. Previous data have demonstrated that bovine ALVC express mRNA for IL-1, although it is not known whether this expression is subset restricted [P. J. Chaplin, R. A. Collins, and C. J. Howard, unpublished observations]. These results are similar to those reported by McCormack et al. [³⁸ ] who demonstrated that the differential capacity of murine macrophage clones to induce allogeneic CD8⁺ T lymphocyte responses was related to their capacity to secrete IL-1. The secretion of IL-1 by macrophage clones was associated with production of IFN- by CD8⁺ T cells [³⁸ ], which may be directly related to lymphocyte proliferation. Evidence also exists that IL-1 can up-regulate expression of CD40 by DC [³⁹ ], which would enhance the interaction with, and stimulation of, T lymphocytes. Although the expression of CD40 has previously been demonstrated to be similar on both subsets of bovine ALVC [² ], addition of IL-1 may enhance expression on the CD11a⁺ subset such that their costimulatory potential is significantly enhanced. Rissoan et al. [¹⁷ ] reported in studies of human DC that monocyte-derived DC, termed DC1, synthesized IL-1 but DC2, derived from plasmacytoid cells, did not. The important consequence of T cell stimulation by these two DC types was the induction of a Th1- or Th2-biased T cell response. A possible relationship of the ALVC subsets to the DC1 or DC2 of Rissoan et al. [¹⁷ ] would therefore have important consequences for T cell stimulation and differentiation. However, the CD11a⁺ CC81⁺ MyD-1^- ALVC express CD45RO [² ] and not the high-molecular-weight CD45R isoform, which is expressed by plasmacytoid DC in humans. The CD11a⁺ CC81⁺ MyD-1^- ALVC also express CD5, which has been reported on human myeloid DC [² , ⁴⁰ ]. Thus, we cannot conclude that the CD11a⁺ CC81⁺ MyD-1^- bovine ALVC are the plasmacytoid equivalent, although it remains possible.

Numerous other studies have reported the existence of subpopulations of DC with different phenotypic and functional properties. Thus, DC isolated from skin [⁴¹ ], bone marrow [⁴² ], blood [⁵ ], spleen [⁹ , ⁴³ ], Peyer’s patch [⁴⁴ ], and tonsil [⁴⁵ ] comprise several subpopulations with discrete features. Although maturation-induced differences may account in part for this heterogeneity it is clear that DC progenitors may directly differentiate into cells with distinct characteristics typical of specific types of DC such as Langerhans cells or dermal DC [⁵ ]. Lineage derivation may also be important [⁴⁵ ]. Determination of the functional properties of such DC subsets is central to the understanding of the initiation and regulation of immune responses by these cells.

We have demonstrated that the differential capacity of ALVC to induce CD8⁺ T lymphocyte proliferation is not due to the induction of death or anergy of the responding cells, nor due to a lack of IL-2 secretion, but rather is related to the failure of CD11a⁺ CC81⁺ MyD-1^- ALVC to provide adequate costimulation. The disparate stimulatory capacity of ALVC is likely to reflect, in part, the differential secretion of IL-1 by subsets of bovine dendritic cells and this may affect the type of response induced in vivo if presentation of antigen was predominantly by one of the ALVC subpopulations.

 
This work was supported by the Biotechnology and Biological Sciences Research Council and by the Ministry of Agriculture, Fisheries, and Food, United Kingdom. We thank members of IAH Compton staff for care of the cattle used within this study.

Received August 3, 2000; revised October 8, 2000; accepted October 18, 2000.

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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