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The Journal of Immunology, 2002, 169: 2020-2025.
Copyright © 2002 by The American Association of Immunologists

Reversal of Coccidioidal Anergy In Vitro by Dendritic Cells from Patients with Disseminated Coccidioidomycosis

John O. Richards*, Neil M. Ampel*,{dagger},{ddagger} and Douglas F. Lake1,*,§

* Department of Microbiology and Immunology, Arizona Health Sciences Center, Tucson, AZ 85724; Departments of {dagger} Medicine and {ddagger} Microbiology and Immunology, Southern Arizona Veterans Affairs Health Care System, Tucson, AZ 85723; and § Department of Microbiology and Immunology, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Coccidioides immitis is a pathogenic, dimorphic fungus found in the southwestern United States and is the causative agent of coccidioidomycosis. Extrathoracic dissemination of coccidioidomycosis is associated with a lack of cellular immunity. Dendritic cells (DCs) have been shown to initiate and modulate cellular immune responses. To determine whether DCs could modulate or initiate the immune response in this disease, monocyte-derived DCs were generated from coccidioidal Ag nonresponsive patients with disseminated coccidioidomycosis and healthy nonimmune individuals. DCs generated from both groups demonstrated phenotypes characteristic of DCs and stimulated strong allogeneic MLR. DCs from patients and healthy nonimmune individuals pulsed with the coccidioidal Ag preparation T27K induced lymphocyte proliferation. Mature DCs were much more efficient than immature DCs in these stimulations. Furthermore, restimulation of T27K-primed PBMC with Ag-pulsed DCs generated a C. immitis-specific cellular immune response in PBMC from patients with disseminated coccidioidomycosis as well as healthy nonimmune individuals. These results show that 1) DCs have the capacity to stimulate specific cellular immune responses from patients with disseminated coccidioidomycosis who are nonresponsive to coccidioidal Ag and healthy nonimmune individuals in vitro; 2) DCs can be used to screen coccidioidal Ags as candidates for human vaccine development; and 3) DC therapy may be useful in the treatment of disseminated coccidioidomycosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Coccidioides immitis is a dimorphic, pathogenic fungus found in the southwestern United States and focal regions of Central and South America (1, 2, 3). Fungal infection occurs when susceptible individuals inhale airborne arthroconidia (4). Individuals with resolved coccidioidomycosis after primary infection demonstrate evidence of cellular immunity in response to coccidioidal Ags characterized by the production of Th1 cytokines IFN-{gamma} and IL-2 (5). On the other hand, patients with active coccidioidomycosis that has disseminated beyond the thoracic cavity usually demonstrate diminished cellular immunity to coccidioidal Ags (6, 7). Treatment of individuals with disseminated coccidioidomycosis has proven to be difficult and many individuals relapse after fungal therapy is discontinued (8, 9, 10).

Dendritic cells (DCs)2 have been described as initiators and modulators of the immune response (11). Mature DCs are able to prime naive and polarize them toward a Th1 response, while immature DCs have been shown to induce tolerance (12). Immunization of humans with mature DCs loaded with keyhole limpet hemocyanin followed by immune evaluation has demonstrated the ability of DCs to activate naive lymphocytes in vivo (13). In cancer patients, DCs loaded with tumor Ags have been shown to stimulate Ag-specific lymphocytes, and in some cases mediated tumor regression (14, 15). In vitro studies have demonstrated that Ag-pulsed mature DCs can also stimulate naive lymphocytes (16). These human studies demonstrate that DCs are capable of initiating and modulating lymphocyte responses in both infectious diseases and cancer.

The ability of DCs to activate the immune response stems from their efficiency to both capture Ag and to activate lymphocytes. DC function reflects the degree of cell maturation, as immature DCs efficiently take up Ag while mature DCs activate lymphocytes (11, 17). Immature DCs have been found in virtually all tissue types (18). In tissues, immature DCs perform the function of Ag uptake by macropinocytosis, phagocytosis, and receptor-mediated endocytosis (11). Interactions with microbial products or inflammatory cytokines induce DCs to mature (19). Mature DCs decrease Ag uptake, undergo a change in chemokine receptor expression that promotes the migration to secondary lymphoid organs, up-regulate MHC molecules, costimulatory molecules, and adhesion molecules, and secrete chemokines to attract naive or memory T lymphocytes (11, 20). In secondary lymphoid organs, mature DCs interact with and activate Ag-specific T lymphocytes (18). Activated T lymphocytes then migrate from secondary lymphoid organs to the site of infection where they perform effector functions leading to the elimination of the pathogen (20).

In a previous study, we demonstrated that DCs pulsed with a coccidioidal Ag preparation, toluene spherule lysate, activate lymphocytes from nonimmune individuals to proliferate in response to coccidioidal Ags (21). In the present study, we have expanded this work by investigating the stimulatory effects of DCs pulsed with T27K, a second coccidioidal Ag preparation, using PBMC from both healthy nonimmune individuals and from patients with disseminated coccidioidomycosis who demonstrated anergy, in vitro, to the T27K Ag preparation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects

Human blood was obtained according to the guidelines of the Human Subjects Committee of the University of Arizona from five patients with disseminated coccidioidomycosis, six healthy nonimmune individuals, and three healthy immune donors. Individuals with disseminated coccidioidomycosis and healthy nonimmune individuals donated blood for the generation of DCs and lymphocytes for these studies. PBMC from nonimmune donors and patients with disseminated coccidioidomycosis did not respond in lymphocyte proliferation assays to the coccidioidal Ag preparation T27K.

Generation of DCs

DCs were generated from PBMC by a modification of the methods described by Romani et al. (22). Briefly, 100 ml of blood was obtained from patients with disseminated coccidioidomycosis and healthy nonimmune volunteers. Whole blood was layered on Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) to obtain PBMC. Approximately 1 x 105 PBMC were added to T-75 flasks in 12 ml of AIM-V medium (Life Technologies, Grand Island, NY) and allowed to adhere for 2 h in a 37°C incubator containing 5% CO2. To remove the nonadherent PBMC fraction, flasks were washed several times with PBS. Nonadherent PBMC were collected and frozen for future use. X-Vivo 15 (BioWhittaker, Walkersville, MD) medium supplemented with 2-ME (Life Technologies), 1000 IU/ml GM-CSF (Immunex, Seattle, WA), and 500 IU/ml IL-4 (Schering-Plough, Kenilworth, NJ) was added to adherent PBMC and cultured for 4 days. On day 4, DCs were harvested and split into wells and fresh X-Vivo 15 containing GM-CSF and IL-4 was added to the cultures. Half the plated DCs were matured with 500 IU/ml TNF-{alpha} (R&D Systems, Minneapolis, MN) and 10 µM PGE2 (Sigma-Aldrich, St. Louis, MO) or left untreated for 48 h.

T27K Ag

The C. immitis Ag preparation used in these experiments is a soluble, aqueous supernatant that is obtained after mechanically disrupting thimerosal-preserved spherules are centrifuged at 27,000 x g. Preliminary experiments to determine an appropriate concentration of T27K demonstrated that thimerosal did not affect proliferation or viability of the stimulatory or responding cells. Previous work with Formalin-fixed spherules showed that it protected mice from experimental coccidioidal infection when combined with alum (23). To date, the components of T27K are not fully defined but several coccidioidal Ags have been identified in the preparation, including chitinase, chitobiase, aspartyl protease, Ag2/proline-rich Ag, alkaline phosphatase, serine protease, and tube precipitin (D. Pappagianis, unpublished data).

Flow cytometric analysis

Immature and mature DCs were phenotyped for HLA-DR, CD1a, CD14, CD40, CD54, CD80, CD83, and CD86. The phenotype of activated lymphocytes was evaluated using anti-CD3, -CD4, -CD8, and -CD69 Abs. Both activated lymphocytes and DCs were stained using three-color flow cytometry and combinations of Abs containing the conjugated fluorochromes FITC, PE, CyChrome (BD PharMingen, San Diego, CA), and Tri-Color (Caltag Laboratories, Burlingame, CA). Cells were stained for 45 min on ice and washed three times with PBS before analysis on a FACScan flow cytometer (BD Biosciences, San Jose, CA).

Allogeneic MLR

To assay for allogeneic lymphocyte proliferation, immature and mature DCs (2 x 104 DC/well) were plated in triplicate wells of a 96-well flat-bottom plate (Falcon 3072; BD Labware, Franklin Lakes, NJ) and irradiated with 3000 rad from a 60Co source. Lymphocytes from various donors were prepared as follows. Nonadherent allogeneic PBMC (2 x 105 cells) were added to the DCs in a total volume of 200 µl of X-Vivo 15. On the fifth day of culture, cells were pulsed with 1 µCi/well [3H]thymidine (NEN Life Science Products, Boston, MA) for 16 h and then frozen. Later, plates containing the cells were thawed and harvested onto a Unifilter GF/C filter plate (Packard Instrument, Meriden, CT) using a Filtermate Harvester (Packard Instrument). [3H]Thymidine incorporation was measured after the addition of 25 µl Microscint 0 scintillation fluid (Packard Instrument) on a TopCount scintillation counter (Packard Instrument). The results of these experiments are presented as the mean plus/minus SEM of triplicate wells.

Ag presentation assays

Immature and mature DCs were prepared as described above except the DCs were pulsed with or without 20 µg/ml T27K on day 4. On day 7, T27K-pulsed immature and mature DCs were plated at 2 x 104 in triplicate wells of a 96-well flat-bottom plate and irradiated with 3000 rad from a 60Co source. Autologous nonadherent PBMCs were added at 2 x 105 cells/well. Proliferation of Ag-specific PBMC was evaluated after 5 days by measuring [3H]thymidine uptake during the last 16 h of the assay and then frozen. [3H]Thymidine uptake was performed as described above. Net cpm was calculated by subtracting the autologous MLR (DC plus PBMC) from Ag-pulsed DCs plus autologous PBMC.

Restimulation of DC-primed PBMC

Mature DCs were generated as described above except the DCs were pulsed with 20 µg/ml T27K or left untreated on day 4 as controls. On day 7 of culture, DCs were washed twice with PBS and irradiated as described above. Autologous nonadherent PBMCs were added to DCs at a 10:1 ratio. The coculture was incubated for 11 days. On the eighth day of coculture, additional autologous immature DCs were plated in a 24-well plate and were pulsed with either T27K or left untreated as a restimulation control. After 72 h, the Ag-pulsed immature DCs were irradiated and washed twice with PBS and plated in a 48-well plate. Autologous lymphocytes primed by either T27K or unpulsed mature DCs were then harvested and plated onto the Ag-pulsed immature DCs. The second stimulation of lymphocytes proceeded for 24 h. Supernatants were collected, and cytokine analysis was performed using the human Th1/Th2 cytokine bead array (BD Biosciences) according to the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PBMC from subjects with disseminated coccidioidomycosis and nonimmune subjects generate functional immature and mature DCs

DCs were generated from patients with disseminated coccidioidomycosis and healthy nonimmune donors. After 7 days in culture, large cells with DC morphology were visualized under microscopic examination. Phenotypic evaluation by flow cytometry showed that the large cells expressed HLA-DR, CD54, various levels of CD1a, low levels of CD40, CD80, CD86, and CD83, and no CD14 (Fig. 1Go). Upon addition of maturation factors, TNF-{alpha} and PGE2, there was an increase in surface expression of HLA-DR, CD40, CD54, CD80, CD83, and CD86 and a decrease in CD1a. The phenotype of these cells was generally identical to the phenotype of DCs generated from healthy nonimmune individuals. In addition, these immature DCs from both donor groups stimulated strong allogeneic lymphocyte proliferation (Fig. 2Go). DCs matured with TNF-{alpha} and PGE2 stimulated higher levels of allogeneic PBMC proliferation compared with DCs that were maintained in GM-CSF and IL-4 (Fig. 2Go). Based on morphology, phenotype, and function, these data indicate that DCs can be generated in vitro from patients with disseminated coccidioidomycosis.



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  		FIGURE 1. Generation of monocyte-derived DCs from patients with disseminated coccidioidomycosis. DC phenotype is represented by the following: filled histograms are mature DCs, bold lines are immature DCs, and thin-lined histograms are isotype controls. Results are representative of five independent experiments.

 


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  		FIGURE 2. DCs induce allogeneic mixed lymphocyte proliferation. Allogeneic PBMC were cocultured with immature or mature DCs and the proliferation of allogenic PBMC was measured by incorporation of [3H]thymidine. Normal indicates allogeneic PBMC and DCs from a healthy nonimmune individual and Patient indicates DCs from a patient with disseminated coccidioidomycosis. Results are representative of five independent experiments from five different patients.

 
DCs induce Ag-specific lymphocyte activation with the coccidioidal Ag preparation T27K

The effective generation of DCs from anergic patients with disseminated coccidioidomycosis allowed us to evaluate autologous PBMC responses to T27K-pulsed DCs. DCs were pulsed with T27K and maintained as immature or matured with TNF-{alpha} and PGE2 followed by coculture with autologous PBMC (Fig. 3GoA). T27K-pulsed DCs that were matured with TNF-{alpha} and PGE2 stimulated significantly stronger autologous PBMC proliferation from anergic patients (p = 0.006) and healthy nonimmune individuals (p = 0.037) than did immature DCs (Fig. 3Go, B and C). Additionally proliferative responses from patient PBMC were almost 2-fold stronger than PBMC from nonimmune donors responding to T27K-pulsed, mature DCs. PBMC proliferation shown in Fig. 3Go, B and C, indicate that DCs can reverse lymphocyte nonresponsiveness observed in vitro in patients with disseminated coccidioidomycosis.



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  		FIGURE 3. T27K-pulsed DCs stimulate autologous PBMC proliferation. A, Experimental design showing the time line for pulsing Ag onto DCs followed by maturation with TNF-{alpha} and PGE2 or maintained as immature. On day 7, autologous PBMC were cocultured with DCs. Proliferation of autologous PBMC was measured by [3H]thymidine incorporation. B, T27K-pulsed mature DCs from patients with disseminated coccidioidomycosis induce greater lymphocyte proliferation than immature DCs. Data are representative of four of five patients tested. C, T27K-pulsed mature DCs from healthy nonimmune individuals stimulate greater lymphocyte proliferation than immature DCs. Lower graph is representative of experiments with six nonimmune individuals. Net cpm was calculated as described in Materials and Methods. cpm for lymphocytes plus unpulsed DCs (autologous MLR) was 953 cpm for B and 209 cpm for C.

 
Fig. 4Go summarizes and clearly demonstrates the different responses among immune donors, nonimmune donors, and patients with disseminated disease in lymphocyte assays and DC-mediated stimulations. When mature DCs from anergic patients with disseminated coccidioidomycosis were incubated with autologous PBMC, reversal of Ag-specific anergy occurred as measured in proliferation assays (Fig. 4Go). The PBMC proliferative responses stimulated by mature DCs from patients (p < 0.001) and nonimmune individuals (p = 0.028) were significantly greater than the proliferative response in the respective lymphocyte transformation (LT) (proliferation) assays (Fig. 4Go). As expected, however, PBMC proliferation in LT assays from immune individuals responded much more strongly to T27K than PBMC stimulated by T27K-pulsed mature DCs from patients with disseminated disease (p = 0.028) and nonimmune individuals (p = 0.031).



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  		FIGURE 4. Reversal of Ag-specific anergy by DCs pulsed with T27K in vitro. LT assays were performed with PBMC from immune, nonimmune individuals, and patients with disseminated coccidioidomycosis. PBMC from nonimmune individuals and patients were stimulated with mature DCs (mDC) pulsed with T27K. Results are the mean ± SEM from each population.

 
To demonstrate the specificity of the response, PBMC incubated with T27K-pulsed mature DCs were stimulated a second time either in the presence of immature DCs pulsed with T27K or incubated with immature DCs not pulsed with T27K. After a 24-h incubation, supernatant from the second stimulation was analyzed for the presence of Th1 and Th2 cytokines. Restimulation of T27K-primed PBMC from a representative disseminated patient resulted in secretion of 5000 pg/ml IFN-{gamma} compared with 1448.2 pg/ml IFN-{gamma} from a second stimulation of unpulsed immature DCs (Fig. 5GoA and Table IGo). The IFN-{gamma} levels from the restimulation were also much higher than those observed in the primary stimulation, which contained 694.6 pg/ml IFN-{gamma}. Upon restimulation, primed PBMC from healthy nonimmune donors secreted 4830.3 pg/ml IFN-{gamma} compared with 41.0 pg/ml IFN-{gamma} produced from a second stimulation of PBMC in the presence of unpulsed immature DCs and 122.6 pg/ml IFN-{gamma} from the primary stimulation (Fig. 5GoB and Table IGo). The increased levels of cytokine secretion found in supernatants from restimulated PBMC populations but not in unpulsed populations indicate that the response was Ag specific.



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  		FIGURE 5. Restimulated PBMC show specificity and a Th1 response. PBMC were primed on T27K-pulsed mature DCs. A second stimulation was performed on T27K-pulsed immature DCs (T27K/T27K) or unpulsed DCs (T27K/UP). A second stimulation of PBMC primed on unpulsed mature DCs restimulated on unpulsed immature DCs was used as a control (UP/UP). Primed PBMC (T27K) alone without further stimulation were also used as a control. Evaluation of cytokines was performed using a cytokine bead array in which each cytokine is detected simultaneously in a supernatant. On the y-axis (FL-3) from brightest (upper) to dimmest (lower), cytokines are IL-2, 4, 5, 10, IFN-{alpha}, and IFN-{gamma}. The quantity of cytokine produced is shown on the x-axis (FL-2). Brighter intensities correlate with an increase in cytokine levels found in the supernatant. A, Data shown are representative of three independent experiments performed using PBMC from different individual patients with disseminated coccidioidomycosis. B, One of two experiments performed with healthy nonimmune individuals.

 

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  		Table I. Quantitation of cytokine data

 
The type of immune response was evaluated by comparing the levels of Th1 cytokines with Th2 cytokines from restimulated PBMC. IFN-{gamma} predominated in culture supernatants from PBMC that were restimulated on T27K-pulsed immature DCs from patients with disseminated coccidioidomycosis and healthy nonimmune individuals (Fig. 5Go, A and B, and Table IGo). The levels of IFN-{gamma} were 10-fold higher than any other cytokine evaluated in all patients and healthy nonimmune subjects. Another Th1 cytokine, IL-2, was secreted at higher levels than the type 2 cytokines IL-4, IL-5, and IL-10, while TNF-{alpha} levels remained similar to Th2 cytokine levels. The strong predominance of IFN-{gamma} suggests that the DCs mediate a cellular immune response in this in vitro system.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have shown for the first time that phenotypically and functionally normal monocyte-derived DCs can be generated from patients with disseminated coccidioidomycosis. We also demonstrate reversal of Ag-specific PBMC anergy mediated by DCs pulsed with a C. immitis spherule Ag preparation, T27K. Furthermore, DCs pulsed with T27K elicit a Th1 cytokine response from PBMC obtained from anergic patients with disseminated coccidioidomycosis as well as from healthy nonimmune individuals.

As the most potent APC, DCs play an important role in initiating or modulating immune responses (11). To further our understanding of the role of DCs in human coccidioidal immunity, DCs were generated from anergic patients with disseminated coccidioidomycosis. DCs from these patients expressed HLA-DR, CD40, CD54, CD80, CD83, CD86, and CD1a, but not CD14. Maturation of DCs with TNF-{alpha} and PGE2 stimulated a strong increase in the surface markers listed above except for CD1a, which decreased in surface expression and CD14 which remained negative (Fig. 1Go). This result shows that DCs can be generated from anergic patients with disseminated coccidioidomycosis and that the number of DCs differentiated from adherent PBMC are equal to the numbers obtained from healthy individuals. Functional analysis showed that DCs generated from anergic patients stimulated allogeneic MLR in a manner equal to DCs generated from healthy individuals (Fig. 2Go).

To determine whether DCs stimulated autologous PBMC to respond to coccidioidal Ag, DCs were pulsed with T27K and then cocultured with autologous PBMC. T27K was chosen because of its ability to stimulate type 1 immune responses from primary lymphocyte stimulations obtained from healthy immune donors but not from patients with disseminated coccidioidomycosis or healthy nonimmune donors (6). T27K has also been shown to be protective in an experimental vaccine model in mice (23). To take advantage of the dual nature of DCs (processing and priming) and the fact that T27K did not mature DCs, they were pulsed with T27K before maturation with TNF-{alpha} and PGE2 and compared with T27K-pulsed DCs that were not purposely matured. Both immature and mature DCs pulsed with T27K induced PBMC proliferation from anergic patients with disseminated coccidioidomycosis and nonimmune donors in vitro (Fig. 3Go). However, DCs that were pulsed with T27K followed by maturation with TNF-{alpha} and PGE2 stimulated significantly higher PBMC proliferative responses than both immature DCs and non-DC-based LT assays (Table IGo). These results agree with previous findings that pulsing DCs with protein or mRNA before DC maturation result in increased lymphocyte responsiveness compared with immature DCs (16, 24). The finding that DCs stimulate PBMC proliferation from anergic patients with disseminated coccidioidomycosis is significant considering that previous studies have demonstrated a limited or complete lack of responsiveness to coccidioidal Ags (25, 26) while retaining immune competence to other recall Ags. Furthermore, the ability of T27K-pulsed DCs to stimulate PBMC proliferation from healthy nonimmune donors supports our previous finding showing that DCs pulsed with toluene spherule lysate, a similar coccidioidal Ag preparation, induces healthy nonimmune individuals to respond to coccidioidal Ags (21).

Although T27K-loaded, mature DCs stimulate PBMC proliferation in patients with disseminated coccidioidomycosis (and in nonimmune donors), the PBMC proliferation response was weaker than the response observed from LT of immune donors (Fig. 4Go). One likely explanation for the strong proliferation from healthy immune donors over patients with disseminated coccidioidomycosis is the increased Ag-reactive T cell precursor frequency in immune donors (25). Immune donors have a mean Ag-reactive cell frequency of 3.7 per 1 x 105 compared with 1.7 per 1 x 105 in patients with disseminated disease (25). Another explanation is that memory T cells may be anergized in patients with disseminated disease. This possibility is currently under investigation in our laboratory. Alternatively, DC function in vivo may be suppressed in an Ag-specific manner by certain unknown suppressive C. immitis components. One obvious explanation for the difference in proliferative responses between immune and nonimmune individuals is that immune individuals have memory T cells while nonimmune individuals do not. Memory T cells have less strict requirements for activation than naive T cells, resulting in enhanced proliferation and cytokine production (27).

Cytokine analysis of supernatants from primed PBMC that were restimulated with T27K-pulsed DCs demonstrated polarization toward Th1 immunity. This may suggest that T cells present in these individuals have been previously activated against C. immitis, but have developed Ag unresponsiveness. In a guinea pig model of coccidioidal anergy, sensitized animals were rendered unresponsive after continuous high-dose administration of coccidioidin (28). Anergy in this model continued as long as high-dose Ag was administered but was transient upon removal of high-doseinjections of coccidioidin. Similarly, Shurin et al. (29) noted that modulations of immune responses have been associated in cancer patients. Specifically, as tumors progress, the immune response shifts from type I to a type II and upon successful therapy shifts back toward a type I immune response (29).

The induction of PBMC proliferation and polarization toward a type I cytokine profile suggests that DCs can stimulate an appropriate immune response against C. immitis in patients with disseminated coccidioidomycosis in vitro. On the other hand, in a toleragenic setting, treatment of disseminated patients with immature DC may exacerbate disease. However, the finding that DCs activate anergic PBMC from patients with disseminated disease toward a type I immune response might suggest a role for DCs in the treatment of disseminated coccidioidomycosis. Clinical trials in cancer patients using DCs support this position, as DC-based immunotherapy has demonstrated objective clinical responses as high as 41–45% (14, 15). The results of this study also suggest that DCs may be a useful tool for testing different coccidioidal Ags as potential vaccine candidates for use in humans. Future studies are ongoing in our laboratory examining the role of the DC-T cell interaction in the presence of different C. immitis components. These studies will position us for using DCs in the treatment of disseminated coccidioidomycosis.


   Acknowledgments
 
We thank Dr. Demo Pappagianis and Kay Kerekes for supplying T27K Ag preparations and for helpful review of this manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Douglas F. Lake, University of Arizona Cancer Center, 1515 North Campbell Avenue, Room 4921, Tucson, AZ 85724. E-mail address: dlake{at}azcc.arizona.edu Back

2 Abbreviations used in this paper: DC, dendritic cell; LT, lymphocyte transformation. Back

Received for publication February 22, 2002. Accepted for publication June 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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