The role of adjuvant on the regulatory effects of NK cells on B cell responses as revealed by a new model of NK cell deficiency

Dorothy Yuan1, Rula Bibi1 and Tam Dang1

1 Laboratory of Molecular Pathology, Department of Pathology, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA

Correspondence to: D. Yuan; E-mail: Dorothy.Yuan{at}UTSouthwestern.edu
Transmitting editor: P. Kincade


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have utilized a novel method to generate transgenic mice that are deficient in NK cells. The strategy entails introduction of the H and L chain genes encoding PK136, an antibody shown to be effective in the in vivo elimination of NK cells, into the mouse genome. Since the introduced H chain gene does not contain sequences encoding membrane exons, the transgenic Ig is not expressed on the cell surface, but is secreted by activated B cells. We show that these animals are chronically depleted of NK cells, but not B, T or NKT cells. Therefore, they are compromised in their ability to mediate NK-mediated cytotoxicity. In addition, the deficiency in NK cells reduces the level of switching to various downstream isotypes in response to T-independent type II antigens. However, this reduction is only apparent when antigens are injected in the presence of adjuvant. Since NKT cells are not depleted, the effect cannot be attributed to this subpopulation. These results help to resolve differences in previous findings regarding the role of NK cells in antibody responses.

Keywords: Ficoll, isotype switching, lung clearance, Pneumovax, transgenic


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NK cells constitute one of the key components of the innate immune system in that they can be rapidly activated without the need for stimulation of clonally distributed receptors. The activation of B lymphocytes, on the other hand, requires recognition of cognate antigen as well as the participation of various co-stimulatory signals. There is a significant body of evidence indicating that NK cells can exert an effect on B cell responses in vitro (13). Some subsets of B cells can in turn induce NK cell cytokine secretion (4,5). However, experiments attempting to obtain in vivo correlates of these interactions have not yielded consistent results. Depletion of NK cells by the injection of anti-NK1.1 antibodies in attempts to reveal modulation of antigen-specific B cell responses have shown different effects in a number of studies. In some cases, exhaustive depletion accompanied by careful verification of the depletion has failed to show changes in the response to different antigens (6). On the other hand, using similar depletion strategies, others have uncovered significant effects on the isotype distribution of specific antibody responses (7,8). The problem of variable and incomplete depletion, which may be the underlying cause of these inconsistent findings, might be overcome if genetically modified mice with a specific deletion of NK cells can be constructed. A number of genetically modified mouse strains exhibit defects in NK cell development and/or function, but these are usually accompanied by T cell defects as well (912). Although human CD3{epsilon} transgenic animals can be reconstituted with T cell precursors to create a mouse deficient only in NK cells, the cytokine constitution of these mice may be disturbed (12). The immune response of the Ly49A transgenic mouse (13), which exhibits a total defect in peripheral NK cells, may also be affected by the expression of high levels of transgenic Ly49 in many cell types. In view of the conflicting results derived from both post-natal and genetic depletion studies it is apparent that further investigation into this question is warranted.

In the absence of sufficient knowledge regarding regulatory genes that are uniquely restricted to the NK lineage we have adopted a novel method for developing a mouse strain that is deficient in only NK cells. The strategy is to construct a transgenic mouse that constitutively produces an anti-NK1.1 antibody identical to that produced by the PK136 hybridoma, which has a proven ability to delete NK cells specifically in vivo. The H and L chain gene constructs used for injection are virtually identical to the H and L chain genes that encode this antibody except that the membrane exons are deleted from the H chain gene so that the transgene cannot exert allelic exclusion (14) and should not greatly perturb the generation of a normal B cell repertoire. Therefore, B cell responses can be studied. In transgenic mice, sufficient anti-NK1.1 antibodies should be produced to kill all newly generated NK cells as soon as B cells are activated via endogenous or injected antigens. It should be noted that because the PK136 transgenes do not code for the antigen receptor of the B cell in which it resides, activation only occurs as a result of stimulation via the endogenous BCR. Plasma cells generated after immunization with T-independent (TI) antigens (15,16) and after virus infection have been shown to have long half-lives (17). Therefore, regardless of the initial stimulus, plasma cells producing the transgenic antibodies should persist in the animal and maintain the absence of NK cells. In mice carrying these transgenes we have now established that NK cells, but not NKT cells, are significantly reduced along with their ability to mediate natural cytotoxicity. More significantly, we show that several TI-2 responses under conditions of chronic NK depletion are partially compromised, but only when immunization is performed in the presence of adjuvant.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of anti-NK1.1 transgenic mice
Using degenerate primers and RT–PCR we identified and sequenced the VDJ segments used by the {gamma}2a H chain gene of anti-NK1.1 hybridoma PK136. The entire V region gene was substituted for that of a µ gene construct (pµ.Poly) that has been used successfully to generate H chain transgenic mice (18). The Cµ exons of the H chain were replaced with genomic sequences for the {gamma}2a constant region. After various unsuccessful attempts to clone the {kappa} variable region segment by the use of degenerate PCR primers, we isolated the PK136 IgG2a protein and determined the N-terminal sequence of the {kappa} chain. This information allowed us to design primers to isolate and sequence the V region gene utilized by the PK136 hybridoma. This gene segment was substituted for the V region of a {kappa} construct (S107 {kappa}) also used previously to generate a transgenic mouse (19). The final chimeric genes are illustrated in Fig. 1.



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Fig. 1. Schematic of H and L chain constructs used to generate transgenic mice. Vertical arrows indicate boundaries of fragments used for injection. Horizontal arrows indicate locations of PCR primers used to type animals carrying the transgene. See text for details of the constructs.

 
Excision of the fragment bounded by the arrows (SalI–BglII) ensures that the membrane exons are not included. This fragment was co-transfected with the linearized L chain gene construct into a plasmacytoma cell line, J558L, that produces only {alpha} and {lambda} chains. Stable integrants were selected based on G418 resistance. The majority of these clones were found to secrete IgG2a and the antibodies can be shown to specifically bind to IL-2-propagated NK cells (data not shown). Thus, the appropriate association of the H and L chains encoded by the transfected genes was not significantly disturbed by the presence of the other H and L chains present in the cell. The constructs were subsequently injected into C57BL/6 oocytes by the Transgenic Facilities of the Department of Molecular Genetics and Microbiology (University of Texas at Austin, TX). Only one founder was found to contain both the H and L chain genes by PCR analysis, whereas two other animals expressed only the L chain gene. Offspring typing of the founder mouse revealed that H and L chain genes were integrated into separated chromosomes. Therefore, every litter had to be typed for the presence of both H and L chain transgenes.

Animals
For adoptive transfer experiments, groups of 8- to 12-week-old C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) received 850 cGy 137Cs {gamma}-irradiation (Gamma Cell 40; Atomic Energy, Ottawa, Ontario, Canada). Each animal was injected i.v. with 1–2 x 107 bone marrow cells from 6-month-old PK136-transgene-positive or C57BL/6 control mice or mixtures thereof. Donor mice were sometimes immunized with 100 µg keyhole limpet hemocyanin (KLH) emulsified in Ribi adjuvant (Corixia, Seattle, WA) consisting of monophosphoryl Lipid A and synthetic trehalose dicorynomycolate. The extent of NK cell depletion in the chimeric animals was found to not be dependent on this immunization.

Cell preparations and analysis by FACS
For FACS analysis, peripheral blood lymphocytes were collected from animals by tail bleeds collected in Alsevers solution. Spleen and liver lymphocytes were prepared by grinding the organs between sintered glass slides followed by filtration through nylon mesh. Phycoerythrin (PE)–goat anti-mouse Ig, FITC–anti-CD19, biotinylated anti-CD3{epsilon}, FITC–anti-DX5 and PE–anti-NK1.1 as well as isotype control mAb were purchased from PharMingen (San Diego, CA). PE–anti-CD23 was a kind gift from Dr Thomas Waldschmidt (University of Iowa). Prior to staining spleen or liver cells they were first preincubated with 10 µg/ml rat anti Fc{gamma}RII antibodies, 2.4G2 (PharMingen). Stained cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Unlabeled anti-NK1.1 antibodies (PK136) were purified from hybridoma culture supernatants by Protein A affinity chromatography.

Lung clearance assay
The assay was performed as previously described (20). Briefly, YAC-1 tumor cells were labeled with 150 µC Na251CrO4 (Amersham Pharmacia Biotech, Piscataway, NJ) at 37°C for 90 min, resulting in a range of 40,000–80,000 c.p.m./106 cells. Then, 5 x 105 tumor cells were injected per mouse (i.v.), and 2 h later all lobes of the lung were isolated and the total radioactivity measured by a {gamma}-counter. To determine the 100% retention level (total lodging), lungs from one animal were collected 15 min after injection of the same cells. Results are reported as percent retention calculated as follows: percent retention = c.p.m. experimental lung/c.p.m. negative control x 100 where negative control = counts from lungs harvested after 15 min. To acutely deplete NK cells, animals were injected with 50 µg anti-NK1.1 mAb (PK136) 4 and 2 days prior to use in the lung clearance assay.

Immunization, serum Ig ELISA analysis
Groups of five to seven animals were each injected i.p. with 40 µg FITC-Ficoll or TNP-Ficoll (Solid Phase Sciences, San Rafael, CA) diluted in PBS or resuspended in Ribi as directed by the manufacturer (Corixia). Pneumovax 23 vaccine was purchased from Merck (West Point, PA). Each vial was resuspended in 1 ml of PBS or Ribi and used for five animals. Serum was obtained from tail bleeds at various times after immunization. Ig ELISA was performed as previously described (21). Briefly, flat-bottom flexible plastic ELISA plates were coated with saturating amounts of TNP-BSA, FITC–BSA or Pneumovax 23 overnight at 4°C. After washing and blocking with 5% dry milk (w/v) serial dilutions of serum samples were incubated overnight at 4°C. The isotypes and the subclasses of bound IgG were detected by horseradish peroxidase (HRP)-conjugated, isotype-specific goat anti-mouse Ig antibodies (Southern Biotechnology, Birmingham, AL) or with rabbit anti-mouse IgG2c (Nordic Immunochemicals, distributed by Accurate Chemicals, Westbury, NY) followed by HRP–donkey anti-rabbit IgG and developed with the substrate, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma, St Louis, MO). Results shown represent dilutions which are most sensitive to changes in antibody levels. It should be noted that in C57BL/6 mice the IgG2a antibodies contain H chains that are encoded by the {gamma}2c gene instead of the {gamma}2a gene (22). Since the majority of isotype-specific anti-IgG2a antibodies are made against myeloma proteins of BALB/c origin, they exhibit much greater activity against IgG2a antibodies carrying the Igh-1a allotype and lower activity against those with the Igh-1b allotypes.

ELISA plates were read by an automated ELISA reader (Molecular Diagnostics) at OD 405 nm. Total serum Ig was determined using the same reagents except that plates were coated with goat anti-mouse Ig(H + L) chains (Cappel-Cooper Biomedical, Malvern, PA). To estimate levels of total Ig of each subclass, ELISA units were compared to monoclonal standards using myeloma proteins.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-NK1.1 transgenic mice
We generated mice that carry transgenes containing the coding sequences for H and L chains derived from the PK136 mAb with specificity for NK1.1. The B cell specificity of the promoter and enhancer for the transgenes ensures that the antibodies will be secreted when the B cells are activated by either specific antigenic stimulation or overtly from environmental sources. The deletion of the membrane exons from the H chain gene encoding PK136 antibodies ensures that this Ig cannot be expressed on the B cell surface and does not mediate allelic exclusion of the endogenous H chain. Whereas it is possible that the rearranged L chain gene inhibits rearrangement of the endogenous L chain locus, a number of transgenic strains harboring rearranged L chain genes have indicated poor allelic exclusion (23,24). Therefore, the expression of the transgene should not extensively affect the repertoire or the function of B cells. Indeed, such absence of strong allelic exclusion has been shown in a transgenic animal constructed with a similar membrane-exon-deleted H chain gene together with a rearranged L chain gene (25) because tolerance to the particular V region was not induced. Therefore, the expression of the transgene should not affect the repertoire or the function of B cells.

To determine the effect of transgene expression on lymphocyte subpopulations we examined both splenocytes and liver cells from two transgene+ animals and compared the expression of their cell-surface markers with a control littermate as well as a control animal injected with anti-NK1.1 antibodies (Fig. 2). Neither the B nor the T cell compartments, as determined by anti-Ig and CD3 staining respectively, were significantly altered by the expression of the transgene (Fig. 2B and C). However, the number of NK1.1+ cells in the spleen was decreased by 80–90%, depending on the extent of background staining (Fig. 2A). The extent of decrease is greater than that in an animal assessed 24 days after injection with anti-NK1.1 antibodies. The equivalent levels of DX5 staining on NK1.1 cells indicate that the absence of NK1.1+ cells is probably not due to blocking of the NK1.1 determinants by the circulating antibodies. This is further confirmed by staining the spleens of each animal with anti-Ig antibodies (Fig. 2B). In addition to detection of B cells, the anti-Ig should also detect extrinsic antibodies on the same cells identified by anti-DX5 if there were Ig bound to the NK1.1 determinants. The low numbers of cells in the upper right quadrant indicate that only slightly higher than background numbers of cells are bound by anti-Ig. Therefore most of the cells expressing NK1.1 were eliminated in the transgenic mice. Co-expression of CD3 and DX5 (Fig. 2C) indicates that some of the residual DX5+ cells that do not stain with NK1.1 are NKT cells and that the abundance of the population is not greatly affected by depletion by anti-NK1.1 antibodies either due to acute injection or in the transgenic animals. The nature of the residual DX5+ cells that do not express NK, T or B cell markers is not known but these cells are absent in the peripheral blood. The abundance of NK1.1hi, DX5+ cells in the liver varies somewhat between animals, but was in general lower than that in the spleen (Fig. 2D). These cells are also reduced in the liver of transgene+ animals. The liver contains much higher numbers of CD3+ DX5+ cells (Fig. 2F), but the presence of the transgene did not alter their abundance. Staining with anti-Ig (Fig. 2E) also indicates that only a small fraction of the DX5+ cells in the liver were coated with Ig. The staining of 12-month and older animals (data not shown) also shows that the depletion of NK cells is maintained for the lifetime of the animal.



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Fig. 2. Comparison of the cell-surface phenotype of pK16 transgenic animals with an acutely anti-NK1.1-depleted animal. Spleen or liver cells from two 10-week-old transgene+ animals and a transgene littermate, as well as an animal injected 24 days previously with two consecutive doses of anti-NK1.1 antibodies (75 µg/injection) were double stained as indicated and analyzed on a FACScan flow cytometer. Profiles represent cells gated on the small lymphocyte population.

 
Effect of PK136 transgene expression on NK-mediated cytotoxicity
Due to difficulties in obtaining sufficient numbers of age-matched mice expressing both H and L chain transgenes we prepared chimeric animals using bone marrow cells from individual mice shown to carry both transgenes. The donor mice were hyperimmunized with KLH in adjuvant to ensure the generation of long-lived plasma cells that secrete both anti-KLH as well as PK136 antibodies. Recipient mice were lethally irradiated to eliminate extant NK cells in the host. Newly developing NK cells from both donor bone marrow as well as residual progenitors in the irradiated mice should be exposed to the antibodies and be eliminated as soon as they express the NK1.1 determinant. Six weeks after reconstitution, analysis of the spleen cells (Fig. 3) from the animals reconstituted with wild-type bone marrow cells revealed two populations of NK1.1+ cells. The single-positive cells stain with higher intensity than those that co-stained with anti-CD3. The latter population fits the characteristics previously described for NKT cells. In animal reconstituted with bone marrow cells from transgene+ animals only low-intensity staining NK1.1 cells that co-stained with CD3 were apparent, indicating that these cells were not deleted. Indeed, comparison of liver lymphocytes from representative mice from each group revealed similar numbers of NK1.1+CD3+ cells (Fig. 3A) and these were within the range of CD3+ DX5+ cells found in livers of intact animals (Fig. 2F). Before these animals were sacrificed their ability to clear CrO4-labeled YAC-1 tumor cells was evaluated. Figure 3(B) shows that the chimeric animals generated from the transgene mouse were able to clear the tumor cells as readily as non-manipulated animals (cf. Group B versus Group D). On the other hand, tumor rejection by most of the PK136 transgene-expressing chimeric animals was significantly compromised (Group A versus Group B, P < 0.015). The increase in tumor cell retention was similar in extent to a control mouse injected with anti-NK1.1 antibodies. Another set of chimeric animals reconstituted with bone marrow cells from a second transgenic mouse showed a similar defect (Fig 3B, Group C versus Group D, P < 0.0015). The reduction in natural cytotoxicity of the PK136 transgene-expressing mice can be directly correlated with a reduction in the abundance of mature NK cells.



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Fig. 3. Phenotype and cytotoxic function of PK136 and control chimeras. (A) Six weeks after reconstitution of irradiated recipients with bone marrow and spleen cells from the transgene+ or control animal the chimeric animals were used for the lung clearance assay. After sacrifice the spleens from each animal and the liver from one animal from each group was stained as indicated. Gated areas indicate NK1.1lowCD3low cells that represent the NKT cell populations. Gated areas in the chimeric liver FACS profiles indicate double-staining NKT cells. (B) Lung clearance assay was performed in either chimeric animals at 6 (Experiment 1) or 8 (Experiment 2) weeks after reconstitution with either transgene+ mice (Groups A and C) or a transgene mouse (Group B) or in intact, non-manipulated mice (Group D). One animal in Group B (indicated by large filled circle) was injected with anti-NK1.1 antibodies at 2 and 4 days prior to the assay. Student’s t-test assuming unequal variance was used to calculate the statistical significance between groups.

 
Effect of PK136 transgene expression on antibody responses
To ensure that the B cell repertoire is not skewed because of the possibility that the rearranged L chain can inhibit rearrangement of the endogenous L chain locus we generated mixed bone marrow chimeras using equal numbers of cells from the PK136 animals together with those from littermate controls. By 6 weeks after reconstitution analysis (Fig. 4) of the peripheral blood lymphocytes (PBL) shows that the B cell compartments in both mixed and control chimeras were completely regenerated [mean % of CD23+ cells in transgene+ and transgene animals = 45.7 and 52.8% respectively; P (T < 0.11)]. Furthermore all of the animals reconstituted with control bone marrow cells have regenerated significant numbers of DX5-expressing cells. However, NK cell numbers were uniformly reduced in the PBL of all of the mice reconstituted with transgene+ bone marrow cells. The T cell reconstitution at 10 weeks was also measured in the PBL of another set of chimeric animals generated in the same manner (Fig. 5A). It is interesting that the NKT cells are not as readily detectable in the peripheral blood as in the spleens of chimeric animals.



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Fig. 4. Cell-surface phenotype of PBL and serum Ig levels of chimeric animals generated from a transgene+ or transgene littermate. (A) Six weeks after reconstitution PBL of chimeras were analyzed by FACS staining as indicated. Six months later similar profiles were obtained. (B) Sera from the same transgene+ (filled symbols) and control (open symbols) animals were collected at 8 weeks, and analyzed for total Ig of each subclass as indicated. Total Ig was estimated by comparing ELISA units with measurement of myeloma proteins of each subclass.

 


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Fig. 5. Cell-surface phenotype of PBL and anti-pneumococcal vaccine responses of chimeric mice generated from PK136 transgene+ or transgene animals. (A) PBL from 10-week chimeras were stained as indicated. Numbers accompanying each FACS profile indicate the fraction of cells in the upper left (NK cells) versus the lower right quadrant (T cells). Background staining for the upper left quadrant varied between 0.02 and 0.04%. (B) The animals shown above as well as additional groups of chimeric animals were then immunized with Pneumovax in PBS or resuspended in Ribi as indicated. After 14–24 days serum collected was measured by isotype-specific ELISA analysis against the same antigen. Dilutions of serum used for assays shown are 1:16,000 for IgG1 and 1:4000 for IgG2a and IgG3. Similar differences between groups were found for 1:32,000 and 1:8000 dilutions, respectively. The mean of the responses in each group on day 14 are shown along with the SEM. P values of groups that score below 0.05 are indicated with asterisks.

 
We also compared the total Ig levels in each group of reconstituted animals. Eight weeks after cell transfer the levels of endogenous Ig for all of the isotypes measured were similar in PK136 transgene+ chimeras to that of non-irradiated animals. The two groups of chimeric animals also did not differ significantly from each other (Fig. 4B). However, measurement of IgG2a using an antiserum that detects both the products of Igh-1a as well as Igh-1c allelic genes (22) showed a significant increase in IgG2a levels in the recipients of transgene+ animals. The increased IgG2a serum levels indicate expression from the transgene, which carries the Igh-1a allotype, because when a reagent that detects only the IgG2ac allotype was used the IgG2a levels between the two groups of animals did not differ significantly (data not shown).

Groups of animals receiving either transgene+ or control cells were immunized with a TI antigen, TNP-Ficoll, in order to ascertain that the chimeric animals can mount antibody responses. Table 1 shows that at the peak day of the response (day 18) both groups produced similar IgG2a and IgG3 responses, but the extent of switching to IgG1 in both groups was too low for reproducible assessment. Therefore, there is no intrinsic defect in the ability of these animals to switch to downstream isotypes.


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Table 1. Effect of NK cell depletion on the response to TI-2 antigensa
 
A second experiment was performed using a separate set of chimeric animals generated from different donors. FACS evaluation of the PBL from chimeric animals revealed a similar extent of NK cell depletion as in Experiment 1 (data not shown). Immunization with FITC-Ficoll again showed no difference between the two groups (Table 1). IgG1 responses were also not detectable in this case.

Influence of adjuvant on the effect of NK cell depletion
These results showing that depletion of NK cells has no detectable effect on TI-II responses are similar to those obtained in transient depletion studies (16) and in the Ly49 transgenic model of NK cell depletion (13). However, since we have shown previously that stimulation of NK cells with poly(I:C) or with tumor cells preferentially increased IgG2a and IgG1 responses (21,26), it occurred to us that it may be possible to see an effect of the NK cell reduction if the antigen was injected in adjuvant. Table 1 (Experiment 3) shows that in the presence of adjuvant TNP-Ficoll induced a respectable IgG1 response. Significantly, however, in contrast to injection of the same antigen without adjuvant, responses of all isotypes including IgG1 were significantly compromised in animals that were depleted of NK cells.

We also tested the effect of NK cell depletion on immunization with another TI-II antigen, the polyvalent pneumococcal vaccine Pneumovax 23. Although Pneumovax is supposedly a TI-II antigen because of its purely carbohydrate nature, it is typically injected with adjuvant in animal studies. Chimeric animals exhibiting the phenotype shown in Fig. 5(A), generated from transgene+ and transgene animals were immunized with the vaccine. Figure 5(B) shows the serum antibody responses measured against the immunogen of three independent experiments. In the absence of adjuvant we were unable to detect above background levels of antigen-specific IgG1 responses, but neither the IgG2a or the IgG3 responses were affected by the reduction in NK cells. In the presence of adjuvant the responses in control animals were more heterogeneous than that to Ficoll, but the IgG1 responses of all animals with deficiencies in NK cells were significantly lower than that of control animals. Although IgG2a levels appear to be lower, the difference between experimental and controls were not statistically significant. Similarly, the IgG3 responses of transgene+ chimeras were reduced, but also did not reach statistical significance. Thus, isotype switching in response to a mixed antigen is also compromised in the absence of NK cells, but only in the presence of adjuvant.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have generated a transgenic mouse carrying B cells that can make anti-NK1.1 antibodies presumably as soon as the B cells are stimulated sufficiently to differentiate into antibody-secreting cells. Apparently, sufficient levels of antibodies are secreted by non-specifically activated B cells very early during development because a significant decrease in NK cell number can be detected in the peripheral blood of 6-week-old animals. NK1.1+ cells are also depleted in the spleen; however, residual NK1.1+ cells that co-express low densities of CD3 and DX5, indicating that they are NKT cells, remain in significant numbers. Furthermore, the numbers of double-staining cells in the liver are comparable to control animals. These results are consistent with previous indications that NKT cells are not readily killed by the anti-NK1.1 reagent (27). Nonetheless, the early and continuous presence of anti-NK1.1 antibodies allows us to examine the effect of chronic depletion of NK cells in vivo that may differ from that resulting from acute injections of anti-NK1.1 antibodies that have a finite half-life.

In order to obtain a large number of mice that have a homogenous phenotype we generated bone marrow chimeric mice from both PK136 transgene+ as well as transgene littermates. After a sufficient time period both T and B cells in the chimeric mice are regenerated, and the NK cells in control animals can mediate non-specific lysis of tumor cells as efficiently as intact mice. More importantly, the chimeric animals reconstituted with transgene-expressing bone marrow cells are as compromised in this lytic function as a mouse acutely injected with anti-NK1.1 antibodies. Therefore, the persistent NKT cells do not perform well in this aspect of NK function.

Previous attempts to alter in vivo B cell responses by acute depletion of NK cells using anti-NK1.1 antibodies have not revealed a definitive role for NK cells (69). The inconsistent findings could be attributed to differences in individual preparations of the antibodies or the exact protocol of antibody injection before immunization. The advantage of using bone marrow transplants of PK136 transgenic mouse is that the recipients seem to exhibit greater uniformity in the extent of depletion of NK cells. Furthermore, the phenotype remains constant from 6 weeks to >12 months (data not shown) after reconstitution. Therefore, any variability in antigen responses is more likely to be attributed to the responding cells. Using this approach we found that, if the antigen is injected in PBS, chronic depletion of NK cells has no effect on TI-2 responses, represented by the antigens, TNP- or FITC-Ficoll, or to the vaccine, Pneumovax 23, These results are similar to previous reports using both acute depletion (21) and an alternative chronic depletion model (13).

Interestingly, however, when the antigens are injected together with adjuvant the reduction in NK cells results in decreases in antibody responses of all subclasses with the IgG1 response being most significantly affected. The increases in IgG1 responses as a result of adjuvant injection differ from our previous findings using poly(I:C) to transiently stimulate NK activity which resulted in an enhancement of predominantly the IgG2a response to either the TI-1 antigen TNP-lipopolysaccharide (21) or the TI-2 antigen TNP-Ficoll (16). In another model of chronic depletion, the human CD3{zeta} model, the production of only IgG2a antibodies in response to DNP-Ficoll (28) and another TI-II antigen, also introduced in adjuvant, as well as to the polyoma virus was decreased (29). However, it should be noted that the cytokine composition of the reconstituted human CD3{zeta} transgenic mouse, used in both of these experiments, may not be totally normal (9). In fact, despite the decrease in IgG2a responses it was found that after immunization the levels of IFN-{gamma} in the transgenic mice did not differ from control mice (30). Thus, the cytokine generally associated with NK cells and the IgG2a switch does not seem to be involved.

The requirement for adjuvant for the effect of NK cell depletion to be detectable suggests that the mechanism of NK cell involvement is associated with activation events initiated by adjuvant. The action of adjuvant is multifactorial (31) involving both direct activation of various antigen-presenting cells as well as secondary activation via cytokine secretion. Our findings here indicate that NK cells are implicated in these circuits. Part of the action of adjuvants appears to be similar to that of poly(I:C), at least insofar as accessory cell activation (3234). However, the observation that IgG1 responses could also be affected even to typical TI antigens such as TNP-Ficoll and pneumococcal vaccine suggests that adjuvant exerts additional effects that require NK cells. These effects can occur either directly or indirectly via other cell types. Indirect activation could occur via cytokine production by antigen-presenting cells activated by adjuvant. Direct stimulation of NK cells may be derived from dendritic cells (35) as well as activated B cells (4,5,26). Response to some TI-2 antigens involves the capture of antigen by blood-borne dendritic cells and subsequent transport to the marginal zone (36); however, NK cells have not been implicated in this process. On the other hand, IFN-{alpha}/ß produced by activated macrophages has been shown to elevate co-stimulatory molecules on B cells in the context of BCR ligation (37). Thus, it is conceivable that marginal zone B cells activated by TI-II antigens can be further stimulated by cytokines initiated by adjuvant. These B cells can then up-regulate NK cell cytokine production which has been shown to include not only IFN-{gamma} but, additionally, other cytokines such as IL-13 (38), which may exert opposite effects to IFN-{gamma}. Whereas it is well documented that switching to downstream isotypes occurs in germinal centers, careful studies by Toellner et al. (39) suggest that the bulk of switching occurs elsewhere. More B cells expressing switched transcripts are actually found outside of the follicles. Interestingly, this would be the site where B cells have the opportunity to come into contact with NK cells that are mainly localized in the red pulp (40).

We have initiated experiments to examine the T-dependent antigen response in these animals. However, the results so far have been quite variable in that in some experiments the reduction mainly affected IgG2a responses, while in others only IgG1 responses were altered. One possible reason for these inconsistencies, which has been also observed in transient depletion experiments (6,21), is that the NK1.1 marker may be expressed on activated T cells. Although most of the findings document co-expression on CD8 T cells (41,42), LCMV infection appears to induce NK1.1 on CD4 T cells as well (43). Experiments are in progress to determine if this occurs for T cells activated by protein antigens when the inflammatory process is less pronounced. It is unlikely that T cells contribute significantly to the pure TI-2 antigens utilized here. Although a large number of T cell-derived cytokines can increase antibody responses to TI-2 antigens in vitro (44), it is difficult to define a pathway for their activation in vivo.

Finally, it is worthy of note that for many disease models, the experimental elicitation of symptoms requires the injection of antigen in the presence of strong adjuvants (7,8,45). Therefore, the role of NK cells in these models may not be identical to their involvement in the course of the natural disease. We are examining alternative means of investigating this question using our model of NK1.1 depletion.


    Acknowledgements
 
We acknowledge the efforts of Shan D. Maika (University of Texas at Austin) in generating the transgenic mice and Marilyn Gibson for editorial assistance. We are also indebted to Dr Michael Bennett (UT Southwestern Medical Center) for helpful discussions. Supported by National Institutes of Health grant AI38938.


    Abbreviations
 
HRP—horseradish peroxidase

KLH—keyhole limpet hemocyanin

PBL—peripheral blood lymphocyte

PE—phycoerythrin

TI—T independent


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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