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The Journal of Immunology, 2001, 167: 2011-2018.
Copyright © 2001 by The American Association of Immunologists

IFN-{gamma}-Dependent and -Independent Initiation of Switch Recombination by NK Cells1

Ning Gao, Tam Dang and Dorothy Yuan2

Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have examined the effect of IL-2-propagated NK or NK-T cells on each of the steps required for B cell switch recombination leading to IgG2a production. The results indicate that NK cells, on their own and in the absence of IFN-{gamma}, can induce germline transcription in resting, IgG- B lymphocytes from the {gamma}2a locus as well as mRNA for activation-induced cytidine deaminase (AID) via a process that requires cell-cell interactions. The results also show that, in contrast to induction by T cells, activation by NK cells does not involve CD40-CD40 ligand interactions and does not extend to the induction of I{gamma}1 transcription. Furthermore, in contrast to stimulation by LPS and IFN-{gamma} or by T cells, the activation events initiated by NK cells do not result in significant synthesis of functional {gamma}2a mRNA in resting B lymphocytes even in the presence of IFN-{gamma}. Thus, induction of germline and AID transcripts are necessary but not sufficient events for functional switching to IgG2a. These experiments, showing that NK cells themselves cannot induce IgG2a production but can polyclonally program B lymphocytes so that they preferentially switch to this isotype may explain how activated NK cells can skew the Ag-specific immune response toward IgG2a. The findings also provide further demonstration of the definitive yet limited extent of how a non-Ag-specific component of the innate system can modulate the direction of the adaptive immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Switch recombination leading to production of downstream isotypes during B cell differentiation involves complex cellular and molecular mechanisms and may differ for each isotype. It is believed that at least three independent control points are needed for the complete process. First, B cells have to be induced to go into cell cycle (1, 2). Second, the specific locus needs to be transcriptionally activated so that germline transcripts upstream of the candidate constant region can be synthesized (reviewed in Ref. 3). Differential enhancement of germline transcription also provides the means by which different cytokines can either stimulate or inhibit the ensuing switch recombination (reviewed in Ref. 4). In addition to the initiation of germline transcription, recent reports indicate that induction of a RNA-specific activation-induced cytidine deaminase (AID)3 gene (5) is absolutely required for all class switch recombination (6). Finally, in some cases additional signals may be needed to induce the actual switch recombination (7, 8, 9, 10). In vitro, the proliferative signal for switching can be LPS, anti-CD40, CD40 ligand (CD40L), or the cross-linking reagent dextran-anti-IgD (4). Early reports indicate that, for the switch to IgG2a, with LPS as a proliferative signal, the addition of IFN-{gamma} is sufficient to induce even resting B cells to switch to IgG2a (11). However, there is variable, but significant, levels of IgG2a responses to infectious agents as well as defined Ags in both IFN-{gamma}-/- (12, 13, 14, 15) as well as IFN-{gamma}R°/° mice (16, 17, 18). Furthermore, although IL-12 has been shown to amplify effects mediated by IFN-{gamma} (12, 19), injection of anti-IL-12 completely abrogated the IgG2a Ab response to a T cell-independent (TI) Ag (20). In addition, injection of IL-12 into IFN-{gamma}-/- mice can increase the level of IgG2a Abs in response to a TI-2 Ag (21). Therefore, IFN-{gamma}-independent pathways may also function in the induction of the switch to IgG2a.

A survey of a number of viral infections has indicated a skewing of the Ab response toward IgG2a (22). It is interesting that in many of these infections NK cells have been shown to play a role in the early response (23, 24, 25, 26). Thus, one reason for the preferential switch to IgG2a has been attributed to the ability of NK cells to secrete IFN-{gamma}, which in turns predisposes the T cell response to a Th1 type. However, it is not clear whether NK cells play a role independent of its ability to produce cytokines. We have shown that activation of NK cells before immunization with either TI or T cell-dependent (TD) Ags can increase the levels of Ag-specific IgG2a produced and that the enhancement is dependent on NK cells as well as IFN-{gamma} (20, 27). However, the basal level of the IgG2a response was not affected by prior NK cell depletion. In contrast, in the CD3{epsilon}-transgenic animal with a deficiency in NK cells, a total defect in the IgG2a response to a TD Ag was reported (28), although in the same strain the switch to IgG2a induced by TI Ags does not seem to be affected (21). In view of such contrasting results, we have examined more closely the role of NK cells in this switching event.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell preparations

For B cell preparations, T lymphocytes were depleted from splenocytes of BALB/c-Ifg<tm1 (GKO) mice (Ref. 29 ; The Jackson Laboratory, Bar Harbor, MA) and fractionated by Percoll gradient centrifugation as previously described (30). After staining with F(ab')2 of fluorescein-conjugated goat anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL) and incubation with anti-fluorescein-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA), the IgG+ cells were depleted according to manufacturer’s specifications. After two rounds of binding, IgG+ cells were no longer detectable by FACS analysis. SCID NK cells were propagated by culturing bone marrow cells as previously described (31) for 7 days from C.B-17 or CBF1 SCID mice deficient in both B and T cells. Bone marrow cells were used because the small size of the SCID spleen precluded recovery of large numbers of cells. However, propagated cells from bone marrow or spleen do not differ functionally. NK cells from GKO spleens were prepared by first depleting T cells by complement lysis followed by depletion of other non-NK cells by negative selection using the StemSep system (StemCell Technologies, Vancouver, BC) according to manufacturers’ instructions. The remaining cells were cultured in 500U/ml IL-2 in the same manner as SCID bone marrow cells. Activated T cells were prepared by depleting splenic B cells by negative selection using bio-anti-B220 (BD PharMingen, San Diego, CA) and the StemSep system. The remaining cells were stimulated with plate-bound (20 µg/ml) anti-TCR{beta} Abs (32) and 200 U/ml IL-2. Disruption of the IFN-{gamma} gene in GKO mice was confirmed by PCR analysis of tail DNA as well as by RT-PCR assessment for the absence of IFN-{gamma} mRNA (32) in cultured NK cells.

Cell culture

B lymphocytes were either cultured alone (1 x 106 per ml), or together with NK cells (1 x 106 per ml) in the presence of 200 U/ml IL-2 with or without other additives in 24- or 48-well Falcon tissue culture plates (BD Biosciences, Cockeysville, MD). Anti-CD40 (33) was added at optimal concentrations previously determined to induce proliferation but not secretion of resting B cells. Recombinant IL-12, provided by Dr. R. O’Hara (Genetics Institute, Andover, MA), was added at 10 ng/ml, a concentration previously shown to enhance LPS-induced IgG2a responses (data not shown). Recombinant IFN-{gamma} (Schering-Plough, Kenilworth, NJ; 50 U/ml) or rIL-4 (20 U/ml; Sterling Research Group, Great Valley, PA) were added at optimal concentrations for B cell-IgG2a or IgG1 production, respectively, when added together with LPS (50 µg/ml; Difco, Detroit, MI). Anti-CD40L (CD154; Ref. 34) was provided by Dr. R. Noelle (Dartmouth Medical School, Dartmouth, NH). F(ab')2 were prepared from anti-CD40L and from anti-H2-Dd (hybridoma 34-5-8S purchased from American Type Culture Collection (Manassas, VA)) as previously described (35). Rat anti-mouse IFN-{gamma} mAb (R46A2; BD PharMingen) was added at the previously titrated optimal concentrations for inhibition of switching to IgG2a in LPS-stimulated cultures.

Semiquantitative RT-PCR analysis

RNA was prepared using the TRIzol reagent (Life Technologies, Grand Island, NY), and RT-PCR was performed as previously described (36). For assessment of germline transcription, the forward primer, obtained from published data (I{gamma}2a-4; Ref. 37), located close to the 3' end of the I{gamma}2a exon, was used together with a reverse primer located in the first exon of the {gamma}2a gene (5'-GGCCAGGTGCTCGAGGTT). This primer differs from the homologous {gamma}2b region by 4 bp; consequently, no {gamma}2b sequences were amplified from B cells stimulated with LPS or LPS and IFN-{gamma} as determined by direct sequencing of the RT-PCR product. The sequence was also found to be identical to the published sequence for one of the I{gamma}2a germline splice variants (37). The same reverse primer was used together with a forward primer (5'-TATGGACTACTGGGGTCAAG) located in the JH4 segment for assessment of cDNA from switched {gamma}2a (S{gamma}2a) mRNA. Primers for AID cDNA, designed from published sequences (forward, 5'-CTTCCTTTGGCCTAAGAC; reverse, AGGCGCGCGGTGAAAATC), spanned an intron (5) precluding amplification of genomic DNA. Amplified products for both AID and I{gamma}1 (38) were authenticated by size and restriction enzyme analysis. Primers for µM were forward, 5'-GGTATGCAAAATCCACTACGGAGGC, located in CH1; and reverse, 5'-GATAAAAGCTGGAGGGCAAC, located in µMII. Primers for GAPDH have been described (32). To quantify RT-PCR products, one of each primer pair was 3' end-labeled with [{gamma}-32P]ATP and used to spike reaction mixtures. Amplified products were quantified using the ImageQuant software package (Molecular Dynamics, Sunnyvale, CA). For all primer pairs, titration curves were performed to ascertain that the cycle number used fell within the linear range as cDNA concentrations were increased. Fig. 1Go depicts representative titration curves for four sets of primers. Control reactions performed with non-reverse-transcribed RNA revealed no priming with any of the primer sets.



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  		FIGURE 1. Amplification of RT-PCR products as a function of RNA concentration. Various concentrations of cDNA were used in 30-cycle PCR using 32P-labeled primers as described in Materials and Methods. The relative intensity of the specific bands obtained was quantified using ImageQuant software. It should be noted that absolute levels of each message cannot be determined by this method due to differences in specific activity of the primers.

 
FACS analysis

Cultured cells were stained and analyzed using the FACScan flow cytometer (BD Biosciences, San Jose, CA) as previously described (39). Biotin-conjugated anti-CD3, fluorescein-conjugated anti-CD19, and fluorescein-conjugated DX5 were all purchased from BD PharMingen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of germline {gamma}2a transcripts by NK cell

We have previously shown that IL-2-propagated NK cells can induce low levels of polyclonal IgM production from resting B lymphocytes and that the secretion can be increased by the addition of IL-5 (40). Therefore, they must be able to activate at least a small fraction of the cells in the absence of any other B cell activator. In these cultures, we failed to detect significant levels of IgG2a production even in the presence of LPS (41). Therefore, the low amount of IFN-{gamma} secreted by the propagated NK cells is apparently insufficient for induction of switch recombination of resting B cells. Nevertheless, there is a possibility that NK cells can induce earlier events that lead to switch recombination, thus predisposing cells to undergo further differentiation upon interaction with other cell types and or cytokines. To eliminate the possibility that any observed effects in B lymphocytes mediated by NK cells is attributed to amplification of a minor population of differentiated cells containing preexisting RNA, we extensively purified the starting population. Thus, high-density, resting B lymphocytes were first prepared by Percoll gradient centrifugation, then the IgG-expressing cells were depleted by staining them with fluorescein-conjugated anti-IgG followed by negative selection on anti-fluorescein-coupled magnetic beads. We evaluated the cells for the presence of germline (I{gamma}2a) and switched (S{gamma}2a) {gamma}2a transcripts by semiquantitative RT-PCR analysis. Fig. 2GoA shows that the starting B cell preparation expressed both I{gamma}2a transcripts and S{gamma}2a mRNA despite the fact that they were obtained from GKO mice that cannot produce IFN-{gamma}; however, neither of the transcripts could be detected after the depletion step. Upon stimulation by LPS, low amounts of each transcript were induced by LPS that were further enhanced by the addition of IFN-{gamma}.



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  		FIGURE 2. Induction of {gamma}2a germline (I{gamma}2a) or cytidine deaminase (AID) transcription in B lymphocytes by NK cells. A, Percoll gradient fractionated, high-density B lymphocytes from GKO spleens labeled with FITC-conjugated goat anti-mouse Ig F(ab')2, either before or after depletion by anti-fluorescein-coated magnetic beads, were analyzed by semiquantitative RT-PCR analysis using primers specific for each transcript as indicated on the left. S{gamma}2a, Products from switched {gamma}2a mRNA. GAP, Products from a housekeeping gene, GAPDH. The size of each amplified product is indicated on the right. RNA was also extracted from the same purified cells after a 48-h culture period in the presence of LPS or LPS and IFN-{gamma}. B, Semiquantitative RT-PCR analysis of the resting, IgG- lymphocytes cultured for various periods with day 7 IL-2-propagated NK cells from CBF1 SCID bone marrow cells or with IFN-{gamma} only.

 
Resting B cells prepared by Percoll density fractionation followed by depletion of IgG+ cells were then incubated with NK cells propagated from SCID mice that are devoid of both B and T cells. Fig. 2GoB shows the semiquantitative RT-PCR analysis of RNA isolated from the cocultures. We found that a surprisingly high abundance of I{gamma}2a transcripts was induced as early as 24 h after activation. Because the B lymphocytes were obtained from GKO mice, the results show that initiation of I{gamma}2a transcription does not require prior exposure of B cells to IFN-{gamma}. Greater than 80% of the PCR products amplified by our primers were authenticated to be I{gamma}2a by restriction digestion. Low levels of the I{gamma}2a transcripts were detected in B cells incubated with IFN-{gamma} alone, but none could be amplified from NK cells cultured alone. The µM products amplified from NK cells are derived from the low levels of sterile µ transcripts made in SCID mice (42).

The initiation of germline transcripts cannot be attributed solely to amplification of some preactivated B lymphocytes by the IFN-{gamma} produced by NK cells because the level of induction by direct addition of IFN-{gamma} to the cultures was much lower than that obtained by addition of NK cells (Fig. 2GoB). Alternatively, the induction could be due to survival or proliferative signals provided by the NK cells such that some of the B lymphocytes are now receptive to stimulation. Therefore, to determine the role of IFN-{gamma} for the induction, we included anti-IFN-{gamma} Abs in the NK-B cell cocultures (Fig. 3GoA). Although the level of induction was reduced by the neutralization of IFN-{gamma}, not all of the transcripts were eliminated. Increasing the Ab concentration did not further decrease the extent of induction (data not shown). Therefore, NK cells themselves appear to confer an additional signal. To determine whether NK cells can induce germline transcripts in the complete absence of IFN-{gamma}, we propagated NK cells from GKO splenocytes and prepared cocultures with the resting, IgG-depleted B lymphocytes in the same manner. These cells were found to be also effective in inducing significant levels of germline transcripts, albeit the time period required for induction was somewhat delayed (Fig. 3GoB). For equivalent numbers of B lymphocytes the level of I{gamma}2a transcripts was quantified to be similar to that obtained by LPS addition to the same cells (data not shown).



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  		FIGURE 3. Induction of {gamma}2a germline or cytidine deaminase transcripts does not require IFN-{gamma}. A, B lymphocytes purified as in Fig. 2Go were cultured with propagated NK cells from syngeneic C.B-17 SCID bone marrow cells in the presence or absence of 25 µg/ml rat anti-mouse IFN-{gamma}. B, Purified B lymphocytes were cultured with day 10 NK cells propagated from GKO spleen cells and harvested at the indicated times for semiquantitative RT-PCR analysis.

 
Upon comparison of the surface phenotype of the NK cells propagated from GKO mice (Fig. 4GoB) with that propagated from SCID mice (Fig. 4GoA), we found that >90% of the cells in each preparation expressed the pan-NK marker, DX5, and were negative for a B cell marker, CD19. However, in contrast to those from SCID mice, NK cells from TCR-intact mice (Fig. 4GoB) continued to express low levels of CD3 on most of the cells; therefore, as has been noted by others (43), these culture conditions favor the propagation of NK-T cells.



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  		FIGURE 4. FACS analysis of propagated NK cells or anti-TCR-stimulated T lymphocytes. Day 7 IL-2-propagated NK cells from C.B-17 SCID bone marrow, day 10 IL-2-propagated NK cells from GKO spleen, or day 2 anti-TCR-activated T lymphocytes from GKO spleen were stained with the pan-NK Ab, FITC-DX5, FITC-CD19, or biotinylated anti-CD3 followed by PE-streptavidin. Shaded profiles represent staining by isotype controls. The size vs scatter profile for each cell population is also shown. For comparison, the gated region indicates size of nonactivated small lymphocytes. The intensity of CD3 staining of cultured T cells is approximately a log lower than that on freshly isolated T cells (data not shown) because of down-regulation of the receptor.

 
Induction by activated T cells

To test whether activated T cells from GKO mice can also activate B lymphocytes in a similar manner, we purified T cells from both GKO and IFN-{gamma}-intact mice and stimulated them on anti-TCR{beta}-coated plates for 2 days. As expected, these activated cells were completely devoid of DX5 staining (Fig. 4GoC) because propagation of NK cells requires high concentrations of IL-2. As shown in Fig. 5GoA, IFN-{gamma}-/- T cells also induced I{gamma}2a transcripts. Furthermore, although the germline transcripts induced by both NK and T cells can be augmented by the addition of IFN-{gamma}, it is clear that both activated T as well as NK-T cells can induce these transcripts in the absence of IFN-{gamma}.



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  		FIGURE 5. Induction of germline {gamma}2a (I{gamma}2a) and switched {gamma}2a (S{gamma}2a) transcripts by NK or T cells. A, Resting IgG- B lymphocytes were cultured with day 7 IL-2-propagated NK cells from IFN-{gamma}-intact SCID NK cells or with activated T cells obtained from either GKO or BALB/c mice in the presence or absence of IFN-{gamma}. After 48 h, RNA was extracted and subjected to semiquantitative RT-PCR analysis using the specific primers as indicated. B, For each sample in A, the relative intensity of each of the amplified I{gamma}2a or S{gamma}2a products was determined by ImageQuant analysis and normalized to the relative intensity of the amplified µM product in the same sample.

 
Specificity of I{gamma}2a germline transcription induction

The initiation of {gamma}2a germline transcription by NK and T cells suggests the possibility that such cell-cell interaction with B lymphocytes results in the nonspecific opening up of more than one downstream isotype locus. To address this question, we assessed the cocultures for the presence of {gamma}1 germline (I{gamma}1) transcripts. To confirm the authenticity of the species amplified by the primers, B lymphocytes were first stimulated with LPS or anti-CD40. As shown previously (44, 45), a low abundance of I{gamma}1 mRNA of the appropriate size can be induced by anti-CD40 stimulation that is increased upon the addition of IL-4 (Fig. 6GoA). Cocultures of NK-B cells were then analyzed for the presence of I{gamma}1 transcripts. Interestingly, whereas NK-T cells propagated from GKO mice can induce low levels of this transcript that is increased upon addition of IL-4, none could be detected in cocultures containing NK cells from SCID mice. The absence is not due to the antagonistic effect of IFN-{gamma} (46, 47) secreted by NK cells because neither addition of anti-IFN-{gamma} nor addition of IL-4 resulted in induction. Not surprisingly, GKO-T cells induced high levels of I{gamma}1 transcription (Fig. 6GoB). Although T cells from intact mice produced equivalent levels of IFN-{gamma} mRNA as NK cells (data not shown), they could also initiate I{gamma}1 transcription, albeit at lower levels than GKO-T cells. To obtain a semiquantitative assessment of the induction by different cell types, the intensity of each amplified germline band was quantified by ImageQuant analysis and normalized against the amplified product of µM-specific primers within each sample (Fig. 6GoC). The level of µM amplification was used for normalization because mRNA from a housekeeping gene such as GAPDH is present in both cell types and differential recovery of either would affect the normalization.



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  		FIGURE 6. Comparison of the induction of germline {gamma}1 or {gamma}2a transcripts by NK and T cells. A, Percoll-fractionated, resting, IgG- B lymphocytes were cultured with LPS or anti-CD40 or with IL-2-propagated NK cells from GKO or SCID cells for 48 h in the presence of cytokines or Abs as indicated. RT-PCR was performed using the I{gamma}1 or µM primers. B, The same B cells prepared for the experiment in Fig. 5Go were cultured with or without NK cells from either SCID or GKO mice in the presence of cytokines or Abs as indicated. Forty-eight hours later, RT-PCR analysis was performed in the same manner. C, For each sample in B, the relative intensity of each of the amplified I{gamma}2a or I{gamma}1 products was determined as in Fig. 5GoB.

 
Induction of germline transcripts by NK cells is not mediated via CD40-CD40L interaction

It is possible that the induction of I{gamma}2a by NK cells is mediated by interactions between CD40 on B cells and CD40L on NK cells because CD40L has been shown to be present on human NK cells (48), although we have not been able to detect CD40L on murine NK cells. Nevertheless, we tested whether interactions mediated by a low abundance of this ligand could be eliminated by including Abs to CD40L in the NK-B cocultures. Fig. 6GoB shows that neither the inclusion of F(ab')2 of anti-CD40L Abs nor control Abs affected the induction of I{gamma}2a transcription by NK cells but partially inhibited that mediated by GKO T cells (Fig. 6Go, B and C). Anti-CD40L Abs also did not inhibit the induction of I{gamma}2a transcription by IFN-{gamma}-intact T cells, probably because of the ability of these cells to produce IFN-{gamma}. Conceivably, this is also the reason NK cell-mediated induction is not inhibited by anti-CD40L. However, anti-CD40L also failed to inhibit the induction by NK cells propagated from GKO mice. The dramatic decrease in I{gamma}1 transcription induced by GKO-T cells confirmed that optimal amount of anti-CD40L Abs were added and suggests that, whereas activation of the I{gamma}1 locus requires the participation of the CD40-CD40L interaction, induction of I{gamma}2a transcription may involve additional interaction molecules.

Induction of AID mRNA

Because transient expression of a specific cytidine deaminase gene, AID (5, 6), may be a necessary step before switch recombination, we examined the expression of this gene to assess whether induction of germline transcription is correlated with the activation of the gene. Fig. 2GoA shows that LPS, on its own, induced the expression of AID mRNA that is presumably associated with switching to IgG3 and IgG2b (1). Addition of IFN-{gamma} further enhanced the abundance. Incubation of the IgG-, resting B lymphocytes with NK cells clearly resulted in the induction of this mRNA in a time-dependent manner (Fig. 2GoB). Furthermore, the same effect was mediated by NK cells propagated from GKO mice (Fig. 3GoB). Thus, the initiation of germline transcription of the {gamma}2a locus is correlated with the next step required for switch recombination.

Contact dependence of induction

To determine whether direct cell-cell interaction is necessary for the induction of {gamma}2a germline transcription, NK from GKO mice were cocultured either together with B lymphocytes or in compartments separated by a semipermeable membrane. Forty-eight hours later, B cells were harvested and analyzed for the relative expression of µM and I{gamma}2a transcripts. Fig. 7GoA shows that the induction of I{gamma}2a transcription mediated by GKO NK cells was eliminated by the physical separation of the two cell types; therefore, direct contact is needed when NK cells do not secrete IFN-{gamma}. In contrast, separation of NK cells from IFN-{gamma}-intact SCID mice still resulted in the induction of some germline transcripts that is likely due to IFN-{gamma} secretion (Fig. 7GoB).



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  		FIGURE 7. Contact dependence of induction of germline and AID transcripts by NK cells. A, Purified B lymphocytes from GKO splenocytes were separated by a semipermeable membrane from day 10 IL-2-propagated GKO NK-T cells placed in transwells, or cultured together, as indicated. B, The same types of cultures were set up for IL-2-propagated IFN-{gamma}-intact SCID NK cells as indicated. C, Purified B lymphocytes were cocultured either with non-NK cells FACS sorted from SCID spleen in the proportions indicated or with day 6 IL-2-propagated NK cells. Cells were harvested for RT-PCR analysis after 48 h of culture.

 
The abundance of µM transcripts in cultures containing only B cells was consistently lower than in those cocultured with NK cells, suggesting that the recovery of B cells by 48 h appears to be improved by the presence of the stimulator cells. Therefore, a mechanism for the induction of germline transcripts may simply be a function of the maintenance of B cell viability. To test this possibility, we depleted NK1.1-expressing cells from SCID splenocytes by FACS sorting and cultured the negative cells with purified B cells. Fig. 7GoC shows that the abundance of µM mRNA increased as a function of the number of stimulator cells, suggesting that these non-B, non-T, non-NK cells can improve B cell viability. However, it is clear that under these conditions no germline transcripts could be detected unless propagated NK cells were added.

Induction of switch recombination to IgG2a

Removal of IgG+ cells reduced the presence of preswitched cells that contained {gamma}2a mRNA to nondetectable levels, both by FACS analysis (data not shown) and by RT-PCR analysis. Therefore, we could use RT-PCR analysis to determine the level of induction of switch recombination by assessing the appearance of switched {gamma}2a mRNA using primers for JH4 and C{gamma}2a. Fig. 5Go shows that despite the induction of I{gamma}2a transcription by both NK cells as well as T cells from GKO mice, the level of switched transcripts that could be detected was only minimal and significantly lower than in cells induced by IFN-{gamma}-intact T cells. The abundance of switched transcripts was increased upon addition of IFN-{gamma}. Interestingly, whereas the cytokine increased the level of induction of germline transcription by NK-T or T cells from GKO mice to the same extent, the induction of switch recombination by T cells was much more effective. This is depicted graphically by calculating the relative intensity of respective {gamma}2a products as a function of µM expression (Fig. 5GoB). Therefore, whereas either NK or T cells can open up the {gamma}2a locus, a cytokine is needed for effective switch recombination and, in the absence of IFN-{gamma}, T cells and or its products may be able to substitute for this cytokine more effectively.

Effect of IL-12

In the case of GKO animals, the nature of the cytokine that can induce the final step of switch recombination is not known. Because in vivo neutralization of IL-12 before immunization with TI Ags completely abrogated the Ag-specific IgG2a response (20), this cytokine is a good candidate for the final induction step. To test this possibility, we added IL-12 to cocultures of NK and B lymphocytes from GKO mice. Fig. 8GoA shows that IL-12 did not significantly alter the extent of I{gamma}2a and AID transcription and did not induce switching to {gamma}2a in the presence of NK cells. Interestingly, addition of IL-12 to B lymphocytes cocultured with NK cells from intact mice also only minimally increased the abundance of S{gamma}2a, despite the fact that addition of IL-12 greatly enhanced the expression of IFN-{gamma} mRNA (Fig. 8GoB). Therefore, IL-12 does not appear to be a cytokine that can substitute for IFN-{gamma} in this event.



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  		FIGURE 8. IL-12 does not induce or augment switch recombination of purified B lymphocytes. A, Purified B lymphocytes were cultured either with IL-12 or day 10 IL-2-propagated NK-T cells from GKO splenocytes as indicated. At 48 h, cells were harvested for assessment of µM, I{gamma}2a, and AID transcripts by semiquantitative RT-PCR. The same number of cells cultured for the same time period with IFN-{gamma}-intact NK cells propagated from SCID bone marrow were analyzed by semiquantitative RT-PCR analysis using primers for either S{gamma}2a (A) or IFN-{gamma} and GAPDH (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vivo-activated NK cells can enhance Ag-specific IgG2a production by B lymphocytes (20, 27). This function of NK cells has been attributed to their production of IFN-{gamma}; however, whether cytokine production alone is sufficient has not been established. Furthermore, elimination of NK cells has not consistently resulted in alterations in the Ag-specific IgG2a response to some Ags (23, 27, 28, 49, 50, 51). The experiments described herein showing that the effect of NK cells themselves can be dissociated from cytokine secretion may add further insight to these issues. For these studies, we have extensively used RT-PCR analysis to effectively analyze RNA from low numbers of cells necessitated by the rigorous isolation procedures. The conclusions from this approach should be valid due to confirmation of both the authenticity of the PCR products as well as maintenance of appropriate PCR cycle numbers (Fig. 1Go). The isolation procedures for B lymphocytes ascertained that the starting populations do not contain memory B cells that have already undergone switch recombination and could be amplified if they survived the culture period. The assessment of mRNA should be as sensitive as determination of switched DNA by methods such as digestion circularization PCR (7) because the accumulated mRNA of a small number of cells may be easier to detect than DNA. Admittedly, the method cannot detect cells that may have possibly undergone switch recombination but yet require additional signals for initiation of RNA synthesis. It is also apparent from Fig. 2Go that the isolation procedure removed cells that still contain germline transcripts as well. However, it is not possible to eliminate cells that may have received the signal for switch recombination but do not as yet produce sufficient levels of the transcript to allow detection by this method. These cells may account for the induction by IFN-{gamma} of germline but not switched transcripts. A similar effect of addition of cytokine alone has been previously observed for the induction of the I{gamma}1 locus by IL-4 (44, 45).

Using these methods, we have shown that IL-2-propagated NK cells from SCID mice can interact with B lymphocytes to induce both germline transcription of the {gamma}2a locus as well as enhance the level of AID mRNA production, initial steps that are required for switch recombination to {gamma}2a (Fig. 2GoB). The induction is not attributable to maintenance of B cell viability (Fig. 7GoB) and requires activated NK cells. To dissociate the effects of IFN-{gamma} produced by NK cells from those mediated by the cells themselves, we have used propagated NK cells from GKO mice that do not produce IFN-{gamma}. Despite the complete removal of T cells in the starting population, the majority of the resultant propagated cells display, along with the NK marker DX5, CD3, albeit at a lower intensity than T cells. Whether the presence of CD3 on virtually all of the cells represents the preferential outgrowth of a minor population or differentiation in culture is yet to be determined. Nonetheless, our results indicate that these NK-T cells can also induce I{gamma}2a transcription in B lymphocytes. The induction requires direct cell contact (Fig. 7GoA), despite the documentation that these NK-T cells produce a number of cytokines (52). The longer time required for the appearance of the transcripts indicates also that the mechanism differs from that of induction by IFN-{gamma}. Therefore, NK cells can function in a manner similar to other B cell polyclonal activators such as LPS or anti-CD40 to induce opening up of the I{gamma}2a locus. A major difference is that although they can initiate low levels of IgM secretion (40), NK cells do not induce significant proliferation of B lymphocytes (T. Dang and D. Yuan, unpublished observations).

Without overt antigenic stimulation of the TCR on NK-T cells, they differ from T cells activated via the TCR in the manner of their interaction with B lymphocytes. Although they can both initiate germline transcription of the IgG2a locus, induction by activated T cells is at least in part mediated by the CD40-CD40L interaction (Fig. 6Go). However, it is unlikely that the induction by NK or NK-T cells also involves this ligand pair for several reasons. First, Abs to CD40L do not inhibit the activation. Second, NK cells cannot induce I{gamma}1 germline transcription even in the presence of anti-IFN-{gamma} and IL-4, whereas NK-T cells can only induce low levels of I{gamma}1 germline transcription and even in the presence of additional IL-4 the level is some 8-fold lower than that induced by GKO T cells (Fig. 6GoA). Finally, unlike stimulation by anti-CD40 or CD40L of B cells, NK cells do not induce detectable B cell proliferation. Interestingly the inhibition of I{gamma}1 by anti-CD40L Abs is much more effective than the inhibition of I{gamma}2a transcripts; therefore, the induction of I{gamma}2a by T cells may also involve ligand-receptor pairs other than the CD40-CD40L interaction. Whereas both anti-CD40 and LPS have been shown to induce NF-{kappa}B/Rel translocation (53, 54), the exact make-up of the transcription factors required for initiation of I{gamma}1 or I{gamma}2a transcription is still not completely defined. Therefore, it will be of interest to determine the extent and composition of NF-{kappa}B/Rel proteins induced by NK stimulation of B cells.

Although the participants of this interaction are not known at present, a number of ligand receptors pairs known to be expressed by these two cell types are presently being explored. We have preliminary evidence to indicate that the interaction may be inhibited by the addition of CTLA4-Ig, and experiments are in progress to determine the exact ligands involved. It is intriguing that transfection of the B cell ligands, B7-2 or CD40, into tumor cells can activate the NK cell lytic machinery via receptors that are not CD28 or CD40L (55). Our evidence that CD40 is not the ligand necessary for activation of B cell germline transcription suggests that activation of NK cells by B lymphocytes is likely to implicate different ligand-receptor pairs than those involved in activation by NK cells.

The use of RT-PCR to quantify switched, processed transcripts derived from the product of this rearrangement should allow us to detect not only newly switched cells, but also selective increases of possible residual previously switched cells in the preparation. It is interesting that, despite the opening up of the {gamma}2a locus and the concomitant induction of AID mRNA by NK cells, the extent of synthesis of the switched {gamma}2a transcripts is detectable but remains low. Furthermore, the level is lower than that induced by T cells from GKO mice even in the presence of added IFN-{gamma}. Thus, as we have shown previously for Ig secretion (41), despite the synthesis of IFN-{gamma} by propagated NK cells the amount produced is insufficient to induce productive switch recombination of resting B cells (Fig. 5Go, A and B). Furthermore, addition of IL-12 does not enhance germline or switched {gamma}2a transcripts. Thus, despite the presence of IL-12 receptors on B lymphocytes (56) IL-12 does not appear to activate B cells directly in this respect. These results are somewhat surprising in light of our previous finding that anti-IL-12 administration in vivo completely abrogated the Ag-specific IgG2a response. To account for these findings, it is important to note that we have used activated NK cells in all of these studies. Resting NK cells, freshly isolated from SCID spleens, were not effective in the induction of germline transcripts (N. Gao and D. Yuan, manuscript in preparation). Therefore, in vivo NK cells need to be activated before they can initiate germline {gamma}2a transcription polyclonally if and when they contact B lymphocytes. IL-12, with or without other cytokines, produced by infected macrophages or APCs is the most likely means for this activation. Thus, the effectiveness of NK cell activation depends on the nature and extent of cytokine production by accessory cells, which is governed by the type of immune stimulation. Upon activation, IFN-{gamma} produced by NK cells can further stimulate accessory cells to complete a cytokine circuit to increase IFN-{gamma} production to such levels that they can function in inducing switch recombination of only B lymphocytes that have encountered Ag. The dependence on the cytokine circuit would account for the inefficient induction of switch recombination by NK cells alone. The dependence on Ag stimulation would prevent the undesirable effects of polyclonal activation of B cells. Furthermore, this hypothesis would account for an essential role of NK cells in some but not all immune responses as well as the previously documented dependence of IL-12 function on IFN-{gamma} in the induction of IgG2a (21).

Finally, it is interesting that NK cells from GKO mice can induce I{gamma}2a transcripts as effectively as those from intact mice. Ag-specific IgG2a is produced during the response to a number of pathogens in GKO or in IFN-{gamma}R°/° mice (13, 14, 15, 16, 17, 18, 19), although the levels are decreased to variable levels when compared with intact mice. Thus, clearly another cytokine(s) other than IFN-{gamma} can induce progression to switch recombination after the locus has been opened up. The nature of this cytokine is not known but is probably not produced by NK cells because activated T lymphocytes from GKO mice can induce progression of B cells to S{gamma}2a mRNA synthesis much more effectively than NK cells.

In conclusion, we have shown that activated NK cells can initiate events required for B cell switch recombination to IgG2a even in the absence of IFN-{gamma}. However, this interaction is insufficient for driving the reaction to completion that would result in the production of IgG2a Abs. Nonetheless, these studies show how NK cells can program B cells to skew the isotype distribution upon appropriate stimulation by either TD signals or by appropriate antigenic cross-linking derived from TI Ags.


   Acknowledgments
 
We thank Dr. Michael Berton (University of Texas Health Science Center, San Antonio, TX) and Dr. Laurie Davis (University of Texas Southwestern Medical Center, Dallas, TX) for helpful discussions. We thank Dr. Richard O’Hara (Genetics Institute, Cambridge, MA) for providing rIL-12.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI38938. Back

2 Address correspondence and reprint requests to Dr. Dorothy Yuan, Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390. E-mail address: Yuan.Dorothy{at}pathology.swmed.edu Back

3 Abbreviations used in this paper: AID, activation-induced cytidine deaminase; CD40L, CD40 ligand; TI, T cell independent; TD, T cell dependent; GKO, BALB/c-Ifg<tm1. Back

Received for publication April 19, 2001. Accepted for publication June 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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