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Ly49I NK Cell Receptor Transgene Inhibition of Rejection of H2^b Mouse Bone Marrow Transplants^{1 ,2}

Jingxuan Liu^3,*,, Margaret A. Morris^3,*,, Paul Nguyen^*, Thaddeus C. George^*,, Elena Koulich^*, Wayne C. Lai^*, John D. Schatzle^*, Vinay Kumar^* and Michael Bennett^4,*

^* Department of Pathology, Laboratory of Molecular Pathology, and Graduate Program in Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235

	Abstract

Top Abstract Introduction Materials and Methods Results and Discussion References

 
The Ly49 family of genes encode NK cell receptors that bind class I MHC Ags and transmit negative signals if the cytoplasmic domains have immunoregulatory tyrosine-based inhibitory motifs (ITIMs). 5E6 mAbs recognize Ly49C and Ly49I receptors and depletion of 5E6⁺ NK cells prevents rejection of allogeneic or parental-strain H2^d bone marrow cell (BMC) grafts. To determine the function of the Ly49I gene in the rejection of BMC grafts, we transfected fertilized eggs of FVB mice with a vector containing DNA for B6 strain Ly49I (Ly49I^B6). Ly49I^B6 is ITIM⁺ and is recognized by 5E6 as well as Ly49I-specific 8H7 mAbs. Normal FVB H2^q mice reject H2^b but not H2^d BMC allografts, and the rejection of H2^b BMC was inhibited partially by anti-NK1.1 and completely by anti-asialo GM1, but not by anti-CD8, Abs. In FVB mice, NK1.1 is expressed on only 60% NK cells. FVB.Ly49I^B6 hosts failed to reject H2^d or H2^b BMC, but did reject class I-deficient TAP-1^-/- BMC, indicating that NK cells were functional. Nondepleting doses of anti-Ly49I Abs reversed the acceptance of H2^b BMC by FVB.Ly49I^B6 mice. FVB.Ly49I^B6+/- mice were crossed and back-crossed with 129 mice—H2^b, 5E6^-, poor responders to H2^d BMC grafts. While transgene-negative H2^b/q F₁ or first-generation back-crossed mice rejected H2^b marrow grafts (hybrid resistance), transgene-positive mice did not. Thus B6 strain Ly49I receptors transmit inhibitory signals from H2^b MHC class I molecules. Moreover, Ly49I^B6 has no positive influence on the rejection of H2^d allografts.

	Introduction

Top Abstract Introduction Materials and Methods Results and Discussion References

 
H2-specific bone marrow cell (BMC)⁵ allograft rejection can be mediated by NK cells in mice (1). According to the missing self hypothesis, NK cells are capable of lysing target cells that lack self class I MHC molecules (2). Proposed almost a decade ago, the missing self hypothesis was also able to explain the phenomenon of hybrid resistance, whereby parental bone marrow grafts are rejected by F₁ hybrids. The discovery of immunoregulatory tyrosine-based inhibitory motif (ITIM)-containing Ly49 receptors validated the missing self hypothesis, as well as explaining how hybrid resistance occurred. This family of receptors consists of type II transmembrane proteins (intracellular N termini) with C-type lectin domains. Two subfamilies exist within this family: those with or without ITIMs. The ITIM⁺ Ly49 receptors transmit negative signals to prevent NK-mediated lysis of target cells and presumably rejection of BMC allografts. In vivo, Ly49A binds to D^d and D^k (3, 4, 5), Ly49G2 binds to D^d or L^d (3, 6), and Ly49C binds to H2^d, H2^k, H2^b (possibly K^b), and H2^s (3, 7, 8). In vitro binding studies have shown that Ly49I interacts with H2^b, H2^d, H2^k, H2^q, H2^r, H2^s, and H2^v (3). Functional in vitro studies have suggested that Ly49I transmits an inhibitory signal from H2^b, but not from H2^d (9). However, until the mAb 8H7, which binds Ly49I but not Ly49C, was developed, one could not be sure of the function of this NK cell subset. Blocking interactions between these Ly49 receptors and their class I ligands using anti-Ly49 or anti-class I mAbs restores killing of the target cell by the NK cell (10).

To study the function of individual Ly49 family members, several investigators have made Ly49 transgenic mice. This system has the advantage of studying one Ly49 gene that is separated from other closely linked Ly49 genes. Two Ly49A transgenics have been made using two different promoter systems (11, 12). The first Ly49A transgenic was used as a tool to study Ly49A function and specificity in vivo (11). The transgene in these mice abrogated the ability of B6 mice to reject H2^d bone marrow grafts. Another Ly49A transgenic mouse was used to look at regulation of Ly49A expression in the presence of its ligand; this study provided evidence for "receptor calibration" (12). We decided to use this approach to study Ly49I, one of the inhibitory Ly49 receptors.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Mice

FVB mice were purchased from The Jackson Laboratory (Bar Harbor, ME) or Harlan Sprague-Dawley (Indianapolis, IN). (C.B-17 x B6)F₁ SCID mice were obtained from the Carolinas Medical Center (Charlotte, NC). All other mice were bred and maintained in our Microbiology Department Colony. BMC chimeras were made by exposing FVB mice to 7.5 Gy ¹³⁷Cs in a Gamma Cell 40 irradiator (Atomic Energy, Ottawa, ON, Canada) and infusing inocula of 5 x 10⁶ BMC from either FVB or FVB.Ly49I^B6 mice. The mice were given antibiotics in their drinking water for 10 days to prevent infection. Chimeras were challenged with donor marrow grafts 4–8 wk after reconstitution. To produce F₁ mice, FVB. Ly49I^B6 (H2^q, Ly49I^+/-) and 129/Sv EMS (129) mice (H2^b, Ly49I^-/-) were mated. Back-cross (BC₁) mice were then produced by mating F₁ Ly49I^+/- mice with 129 mice. Experiments were performed with mice housed in conventional and in specific-pathogen-free facilities. The Institutional Animal Care and Research Advisory Committee of the University of Texas approved all procedures.

The development of transgenic mice

A two-step cloning strategy was used to insert B6 Ly49I cDNA into an expression vector that has XhoI as the only cloning site (13). Ly49I cDNA from B6 mice was obtained by RT-PCR. Messenger RNA preparation and reverse transcription were conducted as described (5). The following two primers were designed based on published B6 Ly49I sequences (5, 6): 5'-AGC CTC GAG CCG GTA GAG ACA CAG AGA ACA-3' and 5'-AGC CTC GAG TAG ATA GGA GAG TAC AGT CCC-3'. XhoI sites (underlined) were added for the convenience of subcloning. The B6 Ly49I cDNA was amplified by PCR as follows: denaturation at 95°C for 2 min, followed by 35 cycles of denaturing at 94°C for 1 min, annealing at 60°C for 1 min, and extending at 72°C for 90 s. The final product was extended at 72°C for 5 min, then incubated at 4°C before running the product out on a 1% agarose gel. A 0.94-kb band of DNA was removed under UV light and electroeluted to obtain pure PCR products (14). The electroeluted PCR product was ligated into a TA cloning vector and transformed into competent cells (Invitrogen, San Diego, CA). Plasmid DNA was purified with a Plasmid Maxi Kit (Qiagen, Chatsworth, CA). The B6 Ly49I cDNA was sequenced with a DNA sequencing kit (U.S. Biochemical, Cleveland, OH) using T7 and Sp6 primers in the TA cloning vector and primers synthesized based on known sequences inside the cDNA. The one amino acid difference between this protein sequence and the reported sequence (7) was in the transmembrane region at position 59, an I (isoleucine) instead of a V (valine).

The B6 Ly49I cDNA was excised with XhoI from the TA cloning vector, purified, and subcloned into the XhoI cloning site in the Thy-1 gene expression cassette (13) (kindly provided by Dr. van der Putten, Ciba Geigy, Basel, Switzerland) as described (14) to obtain the transgenic construct pTS-Ly49I (see below).

View larger version (14K): [in this window] [in a new window]   FIGURE S1.

Enrichment of NK cells from fresh splenocytes

Enrichment of NK cells from fresh splenocytes employed StemSep gravity feed columns (StemCell Technologies, Vancouver, BC, Canada) according to their instructions and as previously described (15). Essentially, all non-NK cells are biotinylated with biotin-labeled mAbs to B, T, and myeloid cells, then aggregated with an anti-biotin tetramer. Incubation with StemSep magnetic colloid allows the biotinylated cells to adhere to the magnetic column. NK cells not bound by the colloid and trapped by the magnet are enriched from 1 to 5% to about 50% of splenocytes.

Growth of lymphokine activated killer cells

Splenocytes enriched for NK cells were cultured in DMEM medium (Life Technologies, Grand Island, NY) supplemented with 500-1000 U/ml human IL-2 (Cetus Corporation, Emeryville, CA) and cultured for 4–5 days as described (15).

Abs and flow cytometry

Staining of T and NK cells and typing of mice were done with PBL as described (16). For two-color staining of T and NK cells (Fig. 1), PBL were first incubated with Fc receptor blocking Ab, 2.4G2 (PharMingen, San Diego, CA) and then stained with anti-NK1.1 and anti-Ly49I/C (5E6), anti-CD3 and 5E6, anti-CD4 and 5E6, anti-CD8 and 5E6, or anti-B220 and 5E6 in a sequential manner (Fig. 1). FITC-conjugated DX5 was also used (see Fig. 4). The mAbs were obtained from PharMingen.

View larger version (48K): [in this window] [in a new window]   FIGURE 1. NK and T, but not B, cells express the Ly49IB6 transgene. After lysis of RBC, PBL from FVB. Ly49IB6 transgenic mice (a, c, e, g, and i) or FVB mice (b, d, f, h, and j) were stained with mAb 5E6 and a panel of mAbs against T cell and B cell-surface Ags (a and b, anti-NK1.1; c and d, anti-CD3; e and f, anti-CD4; g and h, anti-CD8; i and j, anti-B220). The percentages depicted in the figure are values following deduction of percentages of cells stained with isotype control Abs. When the percentage of cells stained with an Ag-specific Ab was equal to or lower than those stained with an isotype control Ab, the value 0.01% was used.

View larger version (48K): [in this window] [in a new window]   FIGURE 4. NK/T cells and NK cell phenotypes in FVB and B6 mice. a and b, FVB.Ly49I splenocytes were stained with CD3 PE, DX5 FITC, and 5E6 biotin/Streptavidin Red 670. a, CD3 vs DX5 staining. Region 2 reflects NK/T cells. Cells were then collected in this gate. b, Histogram representing the 5E6 staining of NK/T cells from a, with thick gray line representing isotype-matched control for 5E6 staining and the thin black line representing 5E6 staining. FVB (c) and B6 (d) NK cells were prepared as in Fig. 2, then stained with PE-conjugated anti-NK1.1 mAb (PK136) followed by FITC-conjugated DX5.

The production of the Ly49I/J-specific 8H7 mAb is described elsewhere (17, 18). BW5147 cells transfected with the B6 strain Ly49I were strongly positive when stained with the 8H7 mAb. BW5147 cells transfected with Ly49C^BALB/c, which are strongly positive when stained with 5E6 or Ly49C-specific 4L03311 mAbs, had minimal shift when stained with 8H7. For in vivo studies, we grew 8H7 hybridoma cells in (C.B-17 x B6)F₁ SCID mice to obtain ascitic fluids that have high titers of mAbs. The fluid was filtered over glass wool filters to remove any particulate matter. The ascites fluid was used to "block" Ly49I receptors on host NK cells at a dose that did not deplete NK cells.

The hybridomas GK1.5 (anti-CD4 mAb) and 2.43 (anti-CD8 mAb) were obtained from American Type Culture Collection (Manassas, VA). Ammonium sulfate precipitated PK136, GK1.5, and 2.43 mAbs were prepared as described (19). The depletion efficiency of PK136 and anti-asialo GM1 (Wako Chemicals, Dallas, TX) was tested by reversal of NK cell-mediated BMC graft rejections. The depleting efficiency of GK1.5 and 2.43 was confirmed by elimination of CD4⁺ and CD8⁺ cells, respectively, of PBL. 2.43 has also reversed CD8⁺ T cell-mediated H2^k bone marrow cell transplants (20).

BMC transplantation, isotope assay, and statistics

These procedures were performed as described (21). Briefly, irradiated mice (8 Gy) were infused with 2.5 or 3.5 x 10⁶ donor BMC. PK136 (anti-NK1.1) mAbs were injected i.p. 2 and 1 days, and 2.43 (anti-CD8) mAbs were injected i.p. 6 and 4 days before transplantation to deplete NK cells and CD8⁺ T cells, respectively.

Proliferation of transplanted BMC in recipients was judged in terms of splenic uptake (%) of [¹²⁵I]-iododeoxyuridine (¹²⁵IUdR), a specific DNA precursor and thymidine analogue 5 days after cell transfer (21).

The statistics used are described in detail (21). The percentage of injected ¹²⁵IUdR incorporated into each spleen was calculated and converted to log₁₀ values. Geometric means (95% confidence limits) values of groups (four to six mice) are presented. The significance of differences between any two groups was calculated by parametric and nonparametric methods using the Vax computer UTSTAT NGROUP program provided by the Academic Computing Service at the University of Texas.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Ly49I transgene is expressed by CD4⁺ and CD8⁺ T cells and NK cells

Thy-1 molecules are expressed on about 50% of fresh NK cells and nearly 100% of IL-2-activated NK cells (22, 23, 24, 25, 26). We con-cluded that the Thy-1 promoter was a good candidate to be used in the development of Ly49I^B6 transgenic mice because expression would be limited to NK and T cells. We placed the Ly49I^B6 transgene into FVB H2^q mice because NK cells of FVB mice were negative by staining with mAb 5E6 in preliminary experiments, an advantage in screening for transgenic mice. Moreover, FVB mice reject H2^b, but not H2^d, BMC grafts.

Four Ly49I^B6 transgenic mouse lines were derived with the transgene expressed at different levels and on different percentages of PBL as determined by staining with mAb 5E6. Results of studies using line 2120 are reported here; 2120 mice had the highest percentage of Ly49I^B6+ PBL, but other lines had higher intensity of staining on fewer Ly49I^B6+ PBL. Staining of PBLs from either transgenic FVB.Ly49I^B6 mice or wild-type FVB mice with 5E6 and a panel of Abs against T and B cell-surface Ags were compared (Fig. 1). About 50% of the PBL in 2120 mice express Ly49I^B6+, and the expression on T cells is brighter than on NK cells (Fig. 1a). Two-color staining of PBL with 5E6 and anti-CD3 mAbs demonstrated that most of cells expressing high levels of the B6 Ly49I transgene were T cells (Fig. 1c). CD4⁺ and CD8⁺ T cells are Ly49I positive (Figs. 1, e and g). B cells do not express Ly49I^B6 (Fig. 1i).

To detect Ly49I expression on NK cells, we enriched NK cells from either FVB.Ly49I^B6 or FVB splenocytes to nearly 50% purity. These cells were then placed in culture for 4 days with IL-2 to generate activated NK cells. After culture, the cells were stained with PE-conjugated NK1.1. They were also stained with FITC-conjugated 5E6, or FITC-conjugated 8H7. Compared with the staining by an isotype control Ab, most transgenic NK cells are 5E6 positive (Fig. 2a) and 8H7 positive (Fig. 2c), indicating that most activated NK cells express the Ly49I transgene. In contrast, there was no difference between the histograms of FVB cells stained with 5E6, 8H7, or their respective isotype control Abs (Fig. 2e, data not shown). Similar results were obtained using (FVB x 129)F₁ and (FVB. Ly49I^B6 x 129)F₁ NK cells (Fig. 2, b, d, and f), although the staining intensity of (FVB. Ly49I^B6 x 129)F₁ NK cells is somewhat lower than that seen in FVB.Ly49I^B6 NK cells. The level of Ly49I staining is lower than that seen in B6 or B6D2F₁ mice, where this epitope is expressed endogenously (Ref. 27 and data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 2. NK cells of FVB.Ly49IB6 or (FVB.Ly49IB6 x 129)F1 mice express the Ly49IB6 transgene. Enriched, IL-2-cultured NK cells from transgene-positive FVB.Ly49IB6 (a) and (FVB.Ly49IB6 x 129)F1 (b) mice were stained with and gated on cells that were positive for PE-conjugated anti-NK1.1 mAb. They were also stained with FITC-conjugated 5E6 mAb (solid line, a and b) or FITC-conjugated 8H7 mAb (solid line, c and d) or a mouse isotype control Ab (dotted line). In the same experiment, NK cells of FVB (e) and (FVB x 129)F1 (f) mice were also stained with these same Abs. 8H7 and 5E6 staining of control FVB and F1 NK cells are equivalent.

Expression of the B6 Ly49I transgene in FVB hosts affects rejection of H2^b but not H2^d BMC grafts

To determine the ability of NK cells of FVB mice to reject allogeneic BMC, we transplanted B6 H2^b, BALB/c H2^d, or 129 H2^b BMC into lethally irradiated FVB hosts. FVB mice reject inocula of 3.5 x 10⁶ B6 BMC (Fig. 3a). This rejection can be partially reversed by anti-NK1.1 but not by anti-CD8 mAbs, suggesting that NK cells are responsible for marrow graft rejection. Whereas FVB mice rejected B6 or 129 (both H2^b) marrow grafts, the FVB.Ly49I^B6 mice failed to reject H2^b BMC (Fig. 3c). It may be noted that B6 BMC grafts grew much better in FVB.Ly49I^B6 hosts than in FVB hosts treated with the anti-NK1.1 (PK136) mAbs. Recent reports indicate that anti-NK1.1 (PK136) mAbs bind to NKR-P1B in Swiss.NIH mice and NKR-P1C in B6 mice (28); NKR-P1C is expressed on all NK cells in B6 mice, whereas NKR-P1B is expressed on about 50–60% NK cells (28). Because FVB are inbred Swiss mice, their NK cells might well express NKR-P1B rather than NKR-P1C. We stained FVB NK cells for both the pan NK marker, DX5, and PK136 (Fig. 4). Only 60% of DX5⁺ cells coexpressed PK136 (NK1.1), while almost all NK1.1⁺ NK cells expressed DX5. By comparison, all DX5⁺ cells in B6 mice express NK1.1 (Fig. 4). Greater than 98% of the DX5⁺ cells were CD3^-, indicating that only rare DX5⁺ cells are NK/T cells (Fig. 4, a and b; Ref. 29). We have also stained for NK/T cells by three-color analysis of anti-CD3, anti-DX5, and anti-5E6 mAbs. From this stain, most NK/T cells also express 5E6, and, therefore, we cannot exclude NK/T cells as effectors in the bone marrow grafts. We tentatively conclude that FVB NK cells express NKR-P1B and not NKR-P1C. Because NKR-P1B is not expressed on all NK cells of FVB mice, PK136 should not deplete all NK cells in this strain; this explains the partial rather than full loss of ability to reject H2^b BMC in irradiated FVB hosts injected with PK136. In contrast, because Ly49I^B6 is expressed on all NK cells in the FVB transgenic mice, they lose the ability to reject H2^b BMC "completely."

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Bone marrow cell transplants in Ly49IB6 transgenic mice. A total of 3.5 x 106 B6, 129, or BALB/c BMC were transplanted into lethally irradiated hosts as indicated. The isotope assay was performed 5 days after cell transfer. Open circles represent individual mice in each group, and solid vertical lines represent the geometric mean of each group. a, Donor B6 BMC were transplanted into either syngeneic or FVB hosts. Anti-NK1.1 PK136, anti-CD8 2.43, or both mAbs were injected into separate groups of mice. b, Donor BALB/c or 129 BMC were transplanted into syngeneic, FVB, or FVB.Ly49IB6 hosts. c, FVB.Ly49IB6 and FVB radiation BMC chimeras were generated as described in Materials and Methods. Donor B6 BMC were transplanted into the chimeras to look at the properties of transgenic hemopoietic cells.

Depletion of the 5E6⁺ NK cells in irradiated hosts reversed the rejection of BALB/c (H2^d), but not B6 (H2^b), marrow grafts (30, 31), which led to the hypothesis that molecules recognized by 5E6 mAbs on B6 NK cells could be receptors for H2^d. Because 5E6 mAb reacts with both Ly49C and Ly49I, one or both 5E6⁺ NK cell subsets could be responsible for the rejection. To test if Ly49I of B6 origin is a stimulatory NK receptor for H2^d, 3.5 x 10⁶ BMC from BALB/c (H2^d) mice were transplanted into FVB.Ly49I^B6 transgenic or FVB hosts. The expression of the transgene failed to confer upon FVB mice the ability to reject BALB/c H2^d BMC (Fig. 3c), refuting the hypothesis that Ly49I^B6 functions as an activating NK cell receptor for H2^d.

Inhibition of hybrid resistance by the Ly49I transgene

FVB. Ly49I^B6+/- (H2^q) mice were crossed with 129 (H2^b) mice to produce F₁ (H2^b/q) mice. Because NK cells of both FVB and 129 mice are negative for 5E6 (Fig. 2 and Ref. 31), only Ly49I^B6 transgenic F₁ mice should have 5E6⁺ NK cells. Ly49I^B6 transgenic and control F₁ hosts were lethally irradiated and challenged with B6 BMC. Transgene-positive F₁ hosts accepted B6 BMC, while transgene-negative F₁ hosts strongly rejected B6 BMC (Fig. 5a), indicating that B6 Ly49I molecules inhibited hybrid resistance. To ensure complete depletion of NK cells in (FVB x 129)F₁ mice, rabbit anti-asialo GM1 serum was used. All NK cells and a small portion of CD8⁺ T cells express asialo GM1. Depletion of CD8⁺ T cells by themselves had only a slight effect on the rejection of B6 BMC by (FVB x 129)F₁ mice (data not shown). Depletion of asialo GM1⁺ cells reversed the rejection of B6 BMC by (FVB x 129)F₁ hosts, suggesting that NK (and/or NK/T) cells are the major effector cells responsible for rejecting B6 BMC in (FVB x 129)F₁ hosts (data not shown). The inability to reject B6 BMC by transgene-positive (FVB x 129)F₁ mice was due to the expression of the transgene on NK cells (Fig. 2, b and d).

View larger version (14K): [in this window] [in a new window]   FIGURE 5. BMC transplants in (FVB.Ly49IB6 x 129)F1 and (F1 x 129)BC1 mice. a, Donor B6 BMC were transplanted into B10 (H2-identical), (FVB x 129)F1, or (FVB.Ly49IB6 x 129)F1 hosts to assess hybrid resistance. b, A total of 2.5 x 106 donor B6 BMC were transplanted into syngeneic, [(FVB x 129)F1 x 129]BC1, or [(FVB. Ly49IB6 x 129)F1 x 129]BC1 hosts. c, Donor TAP1-/- BMC were transplanted into both nontransgenic and transgenic F1 hybrid hosts. *, Geometric mean significantly less than other groups, p < 0.05.

Blocking Ly49I leads to rejection of B6 BMC grafts in FVB. Ly49I^B6 mice

To verify that the transgene is responsible for transmitting a negative signal to the NK cell, we used 8H7 mAbs to block interactions between Ly49I^B6 and prospective class I molecules. To determine whether 8H7 anti-Ly49I^B6 mAbs could bind to NK cells without depleting them, several doses were given to mice in preliminary experiments. We determined that 50 µl of 8H7 ascites fluid sufficiently coats NK cells without depleting them (Fig. 6a). FVB. Ly49I^B6 mice were injected with 50 µl of 8H7. Two days later, splenocytes were removed, enriched, and stained with anti-NK1.1PE, mouse anti-rat IgG FITC, and 5E6 biotin plus Streptavidin Red 670. In untreated mice, as well as control FVB mice treated with 8H7, the mouse anti-rat IgG FITC does not stain the splenocytes (data not shown). However, in FVB.Ly49I^B6 mice injected with 8H7, the mouse anti-rat IgG FITC detected 8H7 remaining on the surface of the splenocytes (Fig. 6a). After gating on live NK cells, we confirmed that the cells stained with mouse anti-rat IgG FITC were Ly49I^B6+ by staining the same cells with the 5E6 mAb. Because 5E6 mAbs detect a different epitope of Ly49I^B6, all cells positive for mouse anti-rat IgG FITC should also be positive for 5E6 (Fig. 6b). The orthogonal distribution of stained cells results from the double stain of 5E6 and mouse anti-rat IgG. Thus, cells that bound 8H7 were still present, and 8H7 could have blocked Ly49I receptors at the 50-µl dose. We used the 50-µl dose in bone marrow transplantation experiments to block Ly49I^B6 interactions with H2^b class I molecules. Injection of 8H7 mAbs i.p. on the day of BMC transfer allowed transgene-positive (FVB x 129)F₁ hosts to reject B6 BMC (Fig. 6c). This result suggests that the anti-Ly49I Abs blocked interactions between Ly49I^B6 on NK cells and H2^b class I molecules on B6 BMC rather than depleting Ly49I^B6+ NK cells (Fig. 6). Higher doses (e.g., 250 µl) of 8H7 ascites fluid did deplete all Ly49I^B6+ NK cells because there were minimal numbers of rat IgG⁺ or 5E6⁺ cells present (data not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Monoclonal Ab to Ly49IB6 (8H7) blocks interactions between Ly49I and H2b class I molecules. a, FVB.Ly49IB6 mice treated with 8H7 mAb on day 0 were sacrificed on day 2. Their spleens were removed, splenocytes enriched for NK cells as described in Materials and Methods, and stained with FITC-conjugated mouse anti-rat IgG and PE-conjugated NK1.1. A gate for NK1.1-positive cells was determined. The mouse anti-rat IgG stains only cells treated with 8H7 (a), and not those that are untreated (data not shown). b, NK cells from a stained with biotin-conjugated 5E6 followed by Streptavidin Red 670. Dot plot shows 5E6 (y-axis) vs mouse anti-rat IgG (x-axis) expression. In the transgenic mice, all NK cells that are positive for the mouse anti-rat IgG are also 5E6+, indicating that 8H7 mAbs block, rather than deplete, transgenic NK cells at this dose. c, FVB or FVB.Ly49IB6 mice were transplanted with B6 BMC as in Fig. 3. One group of FVB.Ly49IB6 mice was given the 50-µl dose. Bone marrow transplantation was assayed as described in Materials and Methods. *, Geometric mean greater than other groups, p < 0.05, Geometric mean value less than (p < 0.05) FVB.Ly49IB6 mice not injected with 8H7 mAbs.

	Acknowledgments

 
We thank Dr. van der Putten (Ciba Geigy, Basel, Switzerland) for provision of the transgenic vectors, Drs. Dorothy Yuan, Mathew A. Porunelloor, and Jiabin An for excellent advice and helpful discussion, and Silvio and Maria Peña for the breeding and care of mice.

	Footnotes

 
¹ This work was supported by Grants CA36922, AI38938, CA70134, AI20451, and CA09082 from the National Institutes of Health.

² A preliminary report appeared in FASEB J. 12:A601 (Abstr. 3492). Morris, M., J. Liu, V. Kumar, and M. Bennett. 1998. Natural killer (NK) and T cell function in Ly49I transgenic FVB mice.

³ J.L. and M.A.M. contributed equally to this study.

⁴ Address correspondence and reprint requests to Dr. Michael Bennett, Department of Pathology NB6.440, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9072. E-mail address:

⁵ Abbreviations used in this paper: BMC, bone marrow cells; ITIM, immunoregulatory tyrosine-based inhibitory motif(s); ¹²⁵IUdR, [¹²⁵I]-5'iodo-2'deoxyuridine.

Received for publication January 29, 1999. Accepted for publication December 3, 1999.

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