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1
* Laboratory of Host Defense, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan;
Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan;
Center of Excellence, Department of Pathology II, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan;
# Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan;
¶ Leprosy Research Center, National Institute Infectious Disease, Tokyo 189-0002, Japan; and
Toyama Institute of Health, Toyama, 939-0363, Japan
1Correspondence: Division of Host Defense, Medical Institute of Bioregulation, Kyushu University, 3–1-1, Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. E-mail: yoshikai{at}bioreg.kyushu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by T cells stimulated with anti-T cell receptor cross-linking, and increased susceptibility to Mycobacteium bovis infection, were prevented in IL-15 Tg mice inoculated with LP-BM5 MuLV. Cytotoxic T lymphocyte response to a highly antigenic 10-mer peptide encoded by LP-BM5-defective virus gag p12 gene was detected in the spleen and peritoneal exudate cells from IL-15 Tg mice infected with LP-BM5 MuLV. Intramuscular injection of cDNA encoding secretable IL-15 also prevented the development of MAIDS. These results indicate that IL-15 prevents the progression of MAIDS and may provide insight into an immunotherapeutic approach using the IL-15 gene for controlling retrovirus-induced immunodeficiency.—Umemura, M., Nishimura, H., Yajima, T., Wajjwalk, W., Matsuguchi, T., Takahashi, M., Nishiyama, Y., Makino, M., Nagai, Y., Yoshikai, Y. Overexpression of interleukin-15 prevents the development of murine retrovirus-induced acquired immunodeficiency syndrome.
Key Words: IL-15 • transgenic mice • murine AIDS • CD8+ CTL • NK cells • gene therapy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chains of IL-2R (1
2
3)
and its own specific high-affinity binding 
chain (designated IL-15R) (4
, 5)
. Mice genetically lacking IL-15R or IL-15 are reported to have no natural killer (NK) cells and severely reduced numbers of 
intestinal intraepithelial lymphocytes (i-IEL), NKT cells, and memory-type CD8+ T cells (6
, 7)
. It thus appears that IL-15 has a potential role in the development and maintenance of significant fractions of lymphocytes including NK, NKT, i-IEL, and memory-type CD8+ T cells. We have recently generated transgenic (Tg) mice expressing IL-15 cDNA encoding a secretable isoform IL-15 precursor protein under the control of an MHC class I promoter and found that the IL-15 Tg mice contained a large number of central memory-type CD8+ T cells expressing CD44high CD62L+ Ly6C+ in peripheral lymphoid tissues (8)
. The IL-15 Tg mice showed strong resistance to infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG) accompanied by enhanced Tc1 responses to the bacteria (9)
. Thus, it appears that IL-15 serves to protect against microbial infections through activation of memory-type CD8+ T cells.
Infection with LP-BM5 murine leukemia virus (MuLV), including both replication-competent and replication-defective viruses, results in the development of a fatal immunodeficiency syndrome in mice known as murine AIDS (MAIDS). MAIDS is characterized by activation and proliferation of T and B cells, impaired T and B cell function, an aberrant regulation of cytokine production, hypergammaglobulinemia, decreased NK cell function, the development of B cell lymphoma, and susceptibility to opportunistic infections (10
, 11)
. Several lines of evidence indicate that specific antiviral CD8+ cytotoxic T lymphocytes (CTL) may play a major role in resistance to development of MAIDS (12
13
14)
. Makino et al. reported that in vivo depletion of CD8+ T cells from MAIDS-resistant A/J mice made them susceptible to LP-BM5 MuLV-initiated MAIDS (15)
. Adoptive transfer of CTL clones generated against terminal MAIDS lymphoma protected mice against LP-BM5 MuLV-induced MAIDS (16)
. A MAIDS-resistant strain of mice was able to generate vigorous CTL responses for the defective gag product, whereas MAIDS-susceptible C57BL/6 (B6) mice appear to be CTL unresponsive to the antigens (Ags) (14
, 17)
. Taken together, these findings suggest that the inability of B6 mice to generate strong antiviral CTL responses in the early stage of LP-BM5 MuLV infection is a major factor contributing to the prototypic susceptibility of this strain to MAIDS.
To clarify the role of IL-15 in protecting against development of MAIDS, we examined the progression of MAIDS in IL-15 Tg mice after inoculation with LP-BM5 MuLV and found that overexpression of IL-15 prevented the progression of MAID in MAIDS-susceptible strain B6 mice accompanied by an increased LP-BM5 MuLV-specific CTL response. Furthermore, intramuscular injection of IL-15 cDNA under the control of a CMV promoter prevented the development of MAIDS. Implications of these findings for a therapeutic approach using the IL-15 gene to control retrovirus-induced immunodeficiency are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A full-length cDNA encoding murine IL-15 gene with the originally described exon 5 was inserted into a transgenic cassette that contained the H-2K promoter, Ig enhancer, and ß-globin splice site and poly(A) signal. The resulting IL-15 transgenic constructs were injected into C57BL/6 blastocysts. Three and two founders from 113 and 81 independent mice of each group were found to be incorporated with the IL-15 transgene with the originally described exon 5. Homozygous transgenic mice were generated by breeding heterozygous littermates in order to obtain maximal levels of IL-15 mRNA expression. All lines of each transgenic group were born healthy and survived normally up to 12 months. As the analysis of offspring of each of the three normal IL-15 transgenic lines showed similar results in each group, we demonstrate the representative results obtained with offspring from normal IL-15 transgenic line 3, which contain 
10 copies of the transgene. In each experiment, age- and sex-matched C57BL/6 (B6) mice purchased from Charles River Japan (Hino, Japan) were used as controls.
Microorganisms
Four-week-old mice were inoculated intraperitoneal (i.p.) with 0.1 mL of LP-BM5 viral stock containing 104 XC plaque-forming units and 102 mink cell focus-inducing units per milliliter (18)
. Mycobacterium bovis (BCG), strain Tokyo, was grown in Middlebrook 7H9 medium (Difco, Detroit, MI) supplemented with ADC enrichment (Difco) and Tween 80 (Difco) at 37°C. At the midlog phase, bacteria in the culture were stored in Middlebrook 7H9 medium supplemented with ADC enrichment, Tween 80, and 20% (v/v) glycerol at -80°C until use. Mice were inoculated i.p. with 0.1 mL of LP-BM5 viral stock and infected i.p. 68 days later with 2 x 107 CFU of BCG. Mice were analyzed individually and compared with age-matched, LP-BM5 MuLV-inoculated, or BCG-infected control mice.
Ab and flow cytometry analysis
Cy-chrome-conjugated anti-CD4, fluorescence isothiocyanate (FITC)-conjugated anti-CD3
mAb, FITC-conjugated anti-Thy1.2 mAb, phycoerythrin (PE) -conjugated anti-CD8 mAb, PE-conjugated anti-B220 mAb PE-conjugated anti-CD95, PE-conjugated anti-T cell receptor (TCR) mAb, PE-conjugated anti-NK1.1 mAb, biotin-conjugated anti-TCRß mAb, biotin-conjugated anti-immunoglobulin (Ig) 
light chain, and Cy-chrome-conjugated streptavidin were purchased from PharMingen (San Diego, CA). Anti-TCRß mAb (H57–597) was a gift from Dr. R. Kubo (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO). Anti-CD3 mAb (145–2C11) was a gift from Dr. J. A. Bluestone (University of Chicago, Chicago, IL). Anti-FcRII/III-specific mAb (2.4G2) was obtained from American Type Culture Collection (Rockville, MD). The stained cells were analyzed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA).
Detection of LB-PM5 MuLV genome integration by DNA-PCR
Template DNA extracted from spleen cells was amplified by PCR as described elsewhere (19
, 20)
. The specific primers were 5'-CCTCTTCCTTTATCGACACT-3' and 5'-ATTAGGGGGGGAATAGCTCG-3'. These primers correspond to the sequences in the defective LP-BM5 MuLV gag-encoded genes p15 and p12, respectively. The conditions for PCR were at 94°C for 1 min, at 54°C for 1 min, and at 72°C for 30 s. Before the first cycle, a denaturation step of 5 min at 94°C was included. After 30 cycles, PCR products were subjected to electrophoresis on a 1.5% agarose gel.
Bacterial counts
Bacterial counts in the peritoneal cavity, liver, and spleen on days 7, 14, and 28 after BCG infection were determined as described (9)
. Peritoneal exudates were obtained from the peritoneal cavity by lavage with 4 mL of HBSS (Nissui, Tokyo, Japan). Serial dilutions of the exudate samples were plated on Middlebrook 7H10 medium (Difco) supplemented with OADC enrichment (Difco) and 20% (v/v) glycerol. To enumerate viable counts, the liver was perfused with 20 mL of sterile HBSS to wash out bacteria in the blood vessels immediately after the mice had been bled. Bacterial counts in the liver were measured as described above. The numbers of colonies were determined after incubation for 3 wk.
In vitro stimulation assay and cytokine ELISA
Peritoneal exudate cells (PEC) were allowed to adhere for 1 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. Nonadherent PEC were used for FACS analysis. Peritoneal T cells prepared by passing cells over a nylon/wool column of nonadherent PEC were cultured at a concentration of 2 x 105 cells/well with immobilized anti-TCRß mAb (100 µg/mL) or anti-CD3 mAb (50 µg/mL) for 48 h at 37°C. The cytokine level in the culture supernatant was determined using a Duo Set ELISA development system (Genzyme-TECHNE, Cambridge, MA) for IL-4 or IFN-. ELISA for IL-15 in each serum sample was performed in triplicate using purified anti-mouse IL-15 mAb (G277–3588, PharMingen), biotin-conjugated anti-mouse IL-15 mAb (G277–3960, PharMingen), and peroxidase-conjugated streptavidin (Genzyme Diagnostics, Cambridge, MA).
Measurement of NK activity
YAC-1 lymphoma target cells were incubated for 1 h with 200 µCi of Na2 51CrO4 (Amersham, Arlington Heights, IL). The effector cells were obtained from the spleen at various times after LP-BM5 MuLV injection. The percentage of specific release of 51Cr after 4 h incubation was calculated by the following formula: % = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100%.
LP-BM5 MuLV gag p12-specific CTL responses
Peptide, a 10-mer peptide (P12–10; TENLPNLPPL) containing the class I (H-2Db) binding motif sequence, was purchased from Sigma Genesys Japan (Tokyo, Japan) (15)
. For liposomal conjugation, P12–10 was dissolved in serum-free sterile PBS and mixed with a cationic lipid transfection reagent (Roche, Gipf-Oberfrick, Switzerland), N-[1-(-2,3-dioleoyloxy)propyl]-N, N,N-trimethylammonium methyl-sulfate to a final concentration of 200 µg/mL in a total volume 1 mL for 20 min at room temperature (P12–10-DOTAP) (21)
. Mice were immunized i.p. with P12–10-DOTAP containing 100 µg peptide; CD8+ T cells from the spleens were positively selected 1 wk later using a magnetic cell separation device (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+ T cells were also purified from the spleens of mice inoculated with LP-BM5 MuLV. Purity, as assessed by flow cytometry, was always > 95%. CD8+ T cells (1x106 cells/well) were cultured with 1 x 106 mitomycin-treated spleen cells/well in the presence of P12–10 (100 mM) in RPMI containing rat concanavalin A (ConA) supernatant at 37°C for 5 days. Cultured cells were harvested and their cytotoxic activity was measured by a conventional 4 h 51Cr release assay using EL4 pulsed with 50 µg/mL of P12–10 as targets. In some experiments, mice inoculated with LP-BM5 MuLV were injected i.p. with P12–10-DOTAP together with 10% proteose peptone (1 mL, Difco, Detroit). Four days later, CD8+ T cells were purified from nonadherent PEC; their cytotoxic activity was measured directly by the 4 h 51Cr release assay.
Construction and injection of the IL-15 expression vector
cDNA encoding a secretory form of IL-15 was cloned into pEGFP-N1 (Clontech Lab., Palo Alto, CA), yielding pEGFP-N1::IL-15, and into pCR3.1 (Invitrogen, Carlsbad, CA), yielding pCR3.1::IL-15. pCR3.1, pCR3.1::IL-15, and pEGFP-N1::IL-15 were prepared using a Wizard PureFection plasmid DNA Purification System (Promega, Madison, WI) and administered by particle bombardment with DNA-coated gold beads (4–5 µg/dose) using a helium-powered Helios Gene Gun delivery system (Bio-Rad Lab., Richmond, CA). For expression of IL-15-green fluorescence fusion protein (GFP) on adherent lymph node (LN) cells, axillary and inguinal LN cells were allowed to adhere for 2 h at 37°C in a humidified atmosphere of 95% air and 5% CO2. Adherent cells were collected by scraping with a rubber policeman, washed, and counted; > 95% of the cells obtained by this procedure were macrophages/dendritic cells. Fluorescent images were viewed recorded using a Bio-Rad MRC-series confocal imaging system (Bio-Rad Laboratories, Hercules, CA).
Statistical analysis
The statistical significance of the data was determined by the generalized Wilcoxon’s test for survival rate or Student’s t test. A P value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, n=30, P<0.001). We then investigated integration of the defective LP-BM5 MuLV genome by PCR in spleen cells of mice inoculated 68 days earlier, when all non-Tg mice showed apparent MAIDS. Integration levels of the defective LP-BM5 MuLV gene were clearly reduced in all samples of IL-15 Tg mice to levels below those in non-Tg mice (Fig. 1B
). We compared signs of MAIDS-related symptoms, including splenomegaly and appearance of abnormally activated lymphocytes in the LN, in IL-15 Tg and non-Tg mice on day 68 after LP-BM5 MuLV inoculation. Non-Tg mice infected with LP-BM5 had marked splenomegaly, whereas IL-15 Tg mice had significantly reduced spleen weight (P<0.01, Table 1
). The relative numbers of abnormally activated lymphocytes such as Thy1- TCRß T cells and Fas+ cells were increased in the LN of non-Tg mice inoculated with LP-BM5 MuLV, whereas IL-15 Tg mice had reduced numbers of these unusual lymphocytes in the LN (P<0.01, Table 1
). We conclude that overexpression of IL-15 inhibits progression of MAIDS.
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Retained NK and T cell functions in IL-15 Tg mice inoculated with LP-BM5 MuLV
We next examined NK activity in the spleens of mice inoculated with LP-BM5 MuLV. NK activity was severely impaired in the spleens of non-Tg mice inoculated with LP-BM5 68 days earlier (Fig. 2
A). NK activity was markedly augmented in the spleens of uninfected IL-15 Tg mice, and an appreciable level of NK activity remained in the spleens of IL-15 Tg mice that had been inoculated with LP-BM5 68 days earlier (Fig. 2A
).
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The ability of peritoneal T cells to produce IFN- in response to immobilized anti-CD3 mAb or anti-TCRß mAb was impaired in non-Tg mice on day 68 after viral inoculation (Fig. 2B
). On the other hand, peritoneal T cells from IL-15 Tg mice inoculated with LP-BM5 MuLV produced IFN- in response to these stimuli at levels comparable to those in normal naive B6 mice (P<0.05). Thus, overexpression of IL-15 prevented virus-induced functional abnormalities in NK and T cells.
Protection against BCG infection was not impaired in IL-15Tg mice inoculated with LP-BM5 MuLV
We recently reported that MAIDS mice are highly susceptible to BCG infection accompanied by impaired IL-15 production (22)
. To further compare the immunological functions in non-Tg mice and IL-15 Tg mice inoculated with LP-BM5MuLV, we examined susceptibility to M. bovis BCG infection in mice with MAIDS. Two x107 BCG were i.p. infected into non-Tg mice and IL-15 Tg mice that had been i.p. inoculated with LPBM-5 68 days earlier. Since the BCG we used in the present study was an avirulent Tokyo strain, the bacteria were cleared within 8 days after infection with this dose of BCG in both non-Tg mice and IL-15 Tg mice without MAIDS (data not shown). However, the numbers of bacteria in the livers of non-Tg mice with MAIDS gradually increased by day 28 after BCG infection whereas the bacteria were efficiently cleared in IL-15 Tg mice inoculated with LP-BM5 (Fig. 3
A).
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We next monitored IL-15 levels in the sera of non-Tg mice and IL-15 Tg mice inoculated with LP-BM5 MuLV after BCG infection. Consistent with our previous finding (22)
, IL-15 production was severely impaired in non-Tg mice with MAIDS during the course of BCG infection (Fig. 3B
). On the other hand, IL-15 Tg mice that had been inoculated with LP-BM5 MuLV had increased levels of IL-15 in serum after BCG infection (Fig. 3B
). IL-15 has been shown to be important for proliferation and maintenance of NK cells, NKT cells, and memory CD8+ T cells (6
7
8)
. To monitor the kinetics of peritoneal lymphocyte populations after BCG infection, FACS analysis of the expressions of CD3, CD4, CD8, and NK1.1 was carried out on nonadherent PEC on days 0, 7, 14, and 28. The relative number of CD3- NK1.1+ cells in IL-15 Tg mice inoculated with LP-BM5 was increased on day 14 after BCG infection, whereas the number was severely reduced in the PEC of non-Tg mice with MAIDS (Fig. 4
A). The numbers of CD8+ T cells were larger in IL-15 Tg mice inoculated with LP-BM5 before and after BCG infection than those in non-Tg mice with MAIDS (Fig. 4B
, data not shown).
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LP-BM5 MuLV-specific CTL response in IL-15 Tg mice inoculated with LP-BM5 MuLV
We examined the CTL response to a 10-mer peptide (P12–10) encoded by defective LP-BM5 MuLV gag p12 gene in mice inoculated with LP-BM5 MuLV. A significant level of CTL response to P12–10 was detected in B6 mice after inoculation with liposomal conjugates with P12–10 (P12–10-DOTAP) (P<0.05, Fig. 5
A), indicating that P12–10 containing a class I (H-2Db) binding motif sequence is highly antigenic in B6 mice. We scarcely detected a peptide-specific CTL response in peptide-stimulated splenic CD8+ T cells from non-Tg mice at an early stage (28 days) after LP-BM5 MuLV inoculation, but detected a significant level of CTL response in those cells from IL-15 Tg mice that had been inoculated with LP-BM5 28 days earlier (P<0.05, Fig. 5B
). No cytotoxic activity was detected against P12–10 unpulsed target cells in the CD8+ T cells that were restimulated with P12–10 plus APC in the presence of ConA supernatants. We further confirmed CTL responses by peritoneal CD8+T cells induced by an i.p. injection of P12–10-DOTAP together with 10% proteose peptone. The peritoneal CD8+ T cells were directly used for 51Cr release assay without in vitro stimulation with p12–10 and APC. A significant level of peptide-specific CTL response was detected in non-Tg mice that had been inoculated with LP-BM5 28 days earlier and the level was much higher in IL-15 Tg mice (P<0.05, Fig. 5C
). Thus, virus-specific CTL responses were generated at an increased level in IL-15 Tg mice after inoculation with LP-BM5 MuLV.
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IL-15 gene therapy for MAIDS
We constructed an IL-15 expression vector using cDNA encoding a secretory form of IL-15 under the control of a CMV promoter, and DNA-coated microcarriers were delivered into the abdominal epidermis of mice using a Gene Gun. We confirmed that IL-15-GFP was expressed by adherent LN cells 3 days after a single injection with pEGFP-N1::IL-15GFP (data not shown). The survival of mice was significantly prolonged by a single injection of pCR3.1::IL-15 (P<0.05, Fig. 6
A) and further prolonged by 10 injections given every week after LP-BM5MuLV inoculation (P<0.005, Fig. 6B
). Thus, the IL-15 gene may be useful for an immunotherapeutic approach to treat retrovirus-induced immunodeficiency diseases.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7
8)
. We have already reported that mice with advanced MAIDS showed impaired ability for IL-15 production (22)
. We demonstrated in the present study that MAIDS progression was severely retarded in IL-15 Tg mice. NK and T cell functions remained at normal levels in IL-15 Tg mice after LP-BM5MuLV infection. A significant level of CTL response to a highly antigenic peptide encoded by the BM5def gag p12 gene was detected in CD8+T cells from IL-15 Tg mice infected with LP-BM5 MuLV. Intramuscular injection with IL-15 cDNA also prevented MAIDS progression. Thus, overexpression of the IL-15 gene may be useful for the development of a new immunotherapeutic approach against retrovirus-induced immunodeficiency diseases.
There are several possible mechanisms for delayed progression of MAIDS in IL-15 Tg mice. IL-15 plays roles in proliferation and maintenance of B cells, including B1 cells, which are target cells for LP-BM5 MuLV infection (19
, 23)
. Therefore, it is possible that IL-15 may inhibit LB-PM5 MuLV genome integration via altering B cell functions. However, we did not find any phenotypically and functionally abnormal B cells in naive IL-15 Tg mice (8
, 21
; data not shown). IL-15 functions in maintaining NK cells and memory CD8+ T cells, which play important roles in protecting against viral infection (5
6
7
8)
. IL-15 Tg mice had increased numbers of NK cells and central memory CD8+ T cells before inoculation, and these cells remained at increased levels after LP-BM5 MuLV inoculation. IL-15 Tg mice had increased numbers of NK cells and central memory CD8+ T cells before inoculation; these cells remained at increased levels after LP-BM5 MuLV inoculation. IL-15 Tg mice showed markedly increased NK activity before LP-BM5 infection and the high level of NK activity remained after infection. Furthermore, we detected a significant level of BM5def gag p12-specific CTL response by CD8+T cells in IL-15 Tg mice infected with LP-BM5 MuLV. We and others have reported accelerated progression of MAIDS in Fas-mutant C57BL/6 lpr/lpr mice, suggesting that Fas/FasL-mediated cytotoxicity is involved in the host defense mechanisms against the retrovirus infection (24
25
26
27)
. Together, these findings suggest that cytotoxic mechanisms mediated by NK cells and CD8+ CTL contribute to clearance of L-BM5 MuLV-infected host cells.
The Th1-type cytokine has also been reported to play a vital role in the induction of protective immunity against retrovirus-induced immunodeficiency (28
, 29)
. In vivo treatment with recombinant IL-12 protected mice from several immune defects induced by LP-BM5 infection in an IFN--dependent manner (29)
. Mizuochi et al. suggested that Th1 type cytokine-producing CD8+T cells rather than CTL contribute to prevention of MAIDS progression (30)
. We earlier reported that IL-15 Tg mice showed dominant Tc1 responses after BCG infection (9)
or OVA immunization (21)
. In the present study, we found that the ability of T cells to produce IFN- on TCR triggering remained at a normal level in IL-15 Tg mice inoculated with LP-BM5 MuLV. Therefore, it is possible that overexpression of IL-15 induces a permanent switch from Tc0 to Tc1 cells, providing protection against the diseases.
It is notable that CTL response to a highly antigenic 10-mer peptide encoded by LP-BM5-defective virus gag p12 gene was detected in peritoneal CD8+T cells induced by peptide-pulsed APC in non-Tg mice infected with LP-BM5 MuLV. A MAIDS-resistant strain of mice was able to generate CTL responses for the defective gag product whereas MAIDS-susceptible B6 mice appeared to be CTL nonresponsive to the Ags (14
, 17)
. Consistent with these findings, in B6 mice with MAIDS we could not detect virus-specific CTL in the splenic CD8+ T cells stimulated with the peptide in vitro. On the other hand, a significant level of virus-specific CD8+CTL was detected in the inflammatory site induced by the Ag with inflammatory agents. Central memory CD8+T cells with low cytotoxic activity located in secondary lymphoid tissues such as the spleen require restimulation with Ag to exhibit strong cytotoxicity, whereas effector/memory CD8+ T cells with high cytotoxic activity are located in nonlymphoid tissues such as the peritoneal cavity (31
, 32)
. It is well known that T cells from mice with advanced MAIDS are easily subjected to apoptosis after in vitro culture (26
, 33)
. Therefore, we speculate that most of the central memory T cells in the spleen may die by apoptosis during the in vitro stimulation, whereas effector/memory CD8+ T cells in the peritoneal cavity of B6 mice with MAIDS showed strong CTL activity without in vitro restimulation with Ag. We have previously reported that in vivo response of T cells to a superantigen remained intact in mice with advanced MAIDS whereas the response of T cells was severely impaired in in vitro response to the superantigen (34)
. Taken together, the results suggest that LP-BM5 MuLV-specific CTL are apparently generated in MAIDS mice in vivo. IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8+T cells, especially central memory T cells, after microbial exposure via promotion of cell survival and homeostatic proliferation (35
, 36)
. We found that IL-15 Tg mice infected with LP-BM5 MuLV exhibited a significant level of CTL response in the spleen and a substantial level of CTL response in the PEC. Overexpression of IL-15 in vivo may maintain the level of central memory CTL specific for LP-BM5 MuLV in the spleen by protection against apoptosis and promotion of cell division.
Prevention of MAID progression by IL-15 overexpression is seen not only in IL-15 Tg mice, but also in mice given intramuscular injection of IL-15 cDNA. We confirmed that a single injection of IL-15 cDNA induced local production. We previously reported that IL-15 cDNA transfected into a tumor made it highly immunogenic and protected tumor growth in an Ag-specific manner (37)
. It has been reported that IL-15 expression plasmid as a DNA vaccine adjuvant enhances CTL activity specific for HIV-1 (38)
. Taken together, the results suggest that IL-15 gene could be useful for immunotherapy against infection by a retrovirus, intracellular parasite, and tumor, for which protection primarily depends on CD8+ T cell responses.
IL-15 also plays important roles in proliferation and maintenance of NKT cells (39)
. Fehninger et al. recently reported that fatal lymphocytic leukemia derived from an NKT cell lineage followed early expansions in NK cells and memory phenotype CD8+ T cells in most IL-15 Tg mice with IL-15 cDNA with an IL-2-signal peptide coding sequence (40)
. Although we did not detect fatal lymphocytic leukemia in IL-15 Tg mice inoculated with LP-BM5 MuLV, we should use IL-15 cautiously for immunotherapy to control lymphocytic leukemia.
In conclusion, IL-15 can exert beneficial effects favoring development of effective cell-mediated functions against LP-BM5 MuLV. The IL-15 gene may be useful in treatment of retrovirus-induced immunodeficiencies.
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ACKNOWLEDGMENTS |
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Received for publication March 25, 2002. Accepted for publication July 19, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13,2822-2830[Medline]
chain of the IL-2 receptor. EMBO J. 14,3654-3663[Medline]
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