CELLULAR
IMMUNOLOGY
135,445-453
(199 1)
Studies on Murine Natural Killer (NK) Cells V. Genetic Analysis
ROBERTC.BURTON,*
of NK Cell Markers
Y.CHENGSMART,* ANDHENRY J. WINN$
GLORIACKOO,~
*Discipline qf‘Surgicai Science, Faculty qf Medicine, University of Newcastle, New South Wales 2308. Australia: TDepartment of Immunology, Merck, Sharp, & Dohme Research Laboratory, Rahway, New Jersey 07065: and *Transplantation Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 Received
October
16, 1990; accepted
January
31. 1991
This paper attempts to clarify the number and nomenclature of murine natural killer (NK) cell specific alloantigens by defining the genetic relationships between them, that is, are they coded by loci which are independent, allelic, or linked. Strain typing and Fz analysesusing five altoantisera (C3H X BALB/c)F, anti-CE, CE anti-CBA, NZB anti-BALB/c, C3H anti-ST, and BALB/c antiDBA/2 revealed that (a) the alloantigens NK-1.1 and NK-3.1 are determined by distinct loci which are linked on the same chromosomes, (b) the alloantigen NK-2.1 is determined by an independently segregating locus to those coding for NK- 1.1 and NK-3.1, (c) the alloantisera, CE anti-CBA and NZB anti-BALB/c, which have been designated anti-NK-2.1 alloantisera recognize different alloantigens coded by independent genetic loci. Thus, these five alloantisera detect four NK cell specific alloantigens which, based on the chronology of their discovery, have been designated NK- 1.1-(C3H X BALB/c)F, antiCE, NK-2.1-CE anti-CBA, NK-3.1 -C3H anti-ST, and BALB/c anti-DBA/Z and NK-4. I-NZB anti-BALB/c. o 1991 Academic press, I~C.
INTRODUCTION A number of cell membrane alloantigens, NK-1, NK-2, and NK-3 expressed by murine natural killer (NK) cells which kill the murine T lymphoma YAC in vitro, have been described (l-7). Identification of these three NK cell specific alloantigens has been based on four criteria: (i) they are expressed by less than 5% of murine splenic leukocytes, (ii) this subset of splenic leukocytes contains all of the in vitro spontaneous lytic activity against YAC, (iii) appropriate backcross and/or F2 genetic analyses have shown that a particular alloantigen is determined by a single genetic locus, and/or (iv) monoclonal antibodies (MoAb) have been produced which fulfill the first two criteria. In this paper we seek to examine the relationship between the loci coding for these alloantigens, that is, are they independent, allelic, or linked. The data presented herein will show that the alloantigens are determined by independent loci, and that two of these appear to be linked on the same chromosome. We will also report that CE antiCBA and NZB anti-BALB/c antisera, which have been described as detecting the NK2.1 alloantigen (5, S), detect alloantigens determined by independent nonlinked loci indicating that, in fact, there are at least four murine NK cell specific alloantigens. 445 0008-8749/9
I $3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduaion in any form reserved
446
BURTON
MATERIALS
ET
AND
AL.
METHODS
Mice. The inbred mice used in these studies were obtained from the Jackson Laboratory (Bar Harbor, ME) and from the Central Animal House of the University of Newcastle (New South Wales). BlO.A, C57Br, C58, and 129 were obtained from the Walter and Eliza Hall Institute (Melbourne, Victoria). The Fr and F2 hybrids were raised in our laboratory and in the Central Animal House of the University of Newcastle. Mice of both sexes were used in NK assays at 6- 15 weeks of age. Assayfor NK activity. The activity of splenic NK cells was measured by an in vitro “Cr release assay against YAC target cells, a Moloney virus induced T lymphoma, and a prototype NK cell target, as described elsewhere (3,4). The tissue culture medium (TCM) was RPM1 1640 supplemented with 2 m&f glutamine, 1% nonessential amino acids, 20 mM Hepes buffer, 50 &ml of gentamicin, and 10% fetal calf serum. All assays were performed in V-bottom microtiter trays in quadruplicate as a minimum of three twofold dilutions of effector spleen cells against lo4 “Cr-labeled YAC target cells in 200 ~1 TCM. The microtiter trays were centrifuged at 15Og for 1 min and incubated at 37°C in 5% CO* in air for 16 hr. At the end of the assay, 100 ~1 of supematant was removed from each well and the level of radioactivity was counted in a Cobra gamma-counter (Packard Instrument Co., IL). The results were calculated as the mean percentage specific lysis + SEM for each group of four replicates as Percentage specific lysis =
cpm test sample - cpm background total cpm - cpm background
x 100.
Background represents the release of “Cr from tumor cells incubated alone. Total cpm is the total 51Cr activity in 5 X lo3 labeled tumor cells. In the assay, spontaneous (background) release of “Cr from YAC was 25 to 30% of total cpm. For each sample tested, a straight line was obtained for the percentage specific lysis versus effectortarget ratio. The percentage specific lysis at one or more effector-target ratios was used in calculating a significant reduction (P < 0.0 1, Student’s t test) by the antiserum and C compared to C alone control according to our previously defined criterion (4, 7). Results herein were represented as percentage specific lysis or as positive (+) or negative (-) depending on whether the P value was < or >O.O 1, respectively. Preparation of antisera. The antisera used herein were all produced by hyperimmunization of mice as previously described (4, 5, 7). Sera from individual mice and/ or pools of sera from groups of mice were tested for complement (C) dependent depletion of splenic NK activity against “Cr-labeled YAC targets in vitro before the final pools of sera were made for use in these experiments. Testing of antisera. The effects of antisera and C on NK cells were usually tested in a two-stage procedure. Antiserum was added to 1O7 spleen cells in 1O-ml centrifuge tubes and the mixtures were incubated at 37°C in 5% CO2 in air for 30 min. The tubes were then centrifuged at 600g and the cells were washed once with 10 ml of TCM and resuspended in 1 ml of dilute rabbit C (Pel Freez Biologicals, Inc., Rogers, AK). They were incubated again for 30 min at 37°C in 5% CO2 in air, washed twice with TCM, and resuspended to the original (pretreatment) volume for use in NK analyses. In the analysis of the back-cross mice, a microtray was used in place of the conical tubes and the procedure was carried out as follows. Quadruplicate samples of 1O6and 5 X 1O5spleen cells in 100 ~1 of culture medium were placed in the tray wells
GENETIC
STUDIES ON MURINE
NATURAL
KILLER
447
CELLS
with 100 ~1 of antiserum; the trays were incubated at 37°C in 5% CO* in air for 30 min and then centrifuged at 150 g for 3 min. The cells were washed once, resuspended in 200 ~1 diluted C, and again incubated for 30 min, after that they were again washed and resuspended. FCS was omitted from the TCM during the time of treatment with antiserum and C, but was introduced immediately after the C had been removed and the cells were being prepared for analysis of NK activity. To avoid enriching the surviving cells and potentially introducing artifacts, the antiserum and C or C alone treated spleen cells were resuspended to the starting volume (200 ~1) in all assays. RESULTS
AND
DISCUSSION
In our past genetic analysis of CE anti-CBA alloantisera we used as indication of a positive result, a reduction of 50% or more of in vitro lysis of YAC by splenic leukocytes treated with alloantiserum and C as compared to the C control (4). In our analysis of C3H anti-ST alloantisera we applied the same criterion (7). These reductions in lysis were always highly statistically significant (P < 0.0 1 or 0.00 1, Student’s t test). In the analysis of (C3H X BALB/c)F, anti-CE and NZB anti-BALB/c alloantisera, Pollack and Emmons (5) used the criterion of a 30% reduction with the first antiserum and a 25% reduction with the second, this being significant for the genetic analysis of those antisera. We have produced these alloantisera but have found that their anti-NK titers were usually lower than those of CE anti-CBA and C3H anti-ST alloantisera. We have therefore applied the one criterion (P < 0.01, Student’s t test) to the genetic analyses and strain typing of all four alloantisera. This is illustrated in Table 1 which shows the results of typing (CE X CBA)F2 mice for simultaneous expression of NK- 1.1, NK2.1, and NK-3.1 with three anti-NK alloantisera (C3H X BALB/c)F, anti-CE, CE anti-CBA, and C3H anti-ST. Table 2 sets out the strain distributions reported for the mm-me NK cell alloantigens, NK- 1.1, NK-2.1, and NK-3.1 in 26 strains of mice (2,4-9). In this report spleen cells from 11 strains were treated with (C3H X BALB/c)F, anti-CE (anti-NK- 1. l), CE antiCBA and NZB anti-BALB/c (anti-NK-2. l), and C3H anti-ST (anti-NK-3.1) antisera TABLE 1 Expression of NK Cell Alloantigens Recognized by Three Anti-NK Alloantisera in Seven (CE X CBA) F2 Mice Mouse No.
C control
NK-1.1 (C3H X BALB/c)F, anti-CE
I 2 3 4 5 6 7
46f la 64-c 1 54 -c 2 43 + 2 58 f 3 39 f 3 28 f 2
48 + I (-)* 21 * 1 (+) 2 IL 2 (+) 38 + 2 (-) 53 + 2 (-) 8 + 2 (+) 6 zk 1 (+)
NK-2.1 CE anti-CBA 17 + 44 * 52 -t 37 f 41 It 25 + 2+
2 (+)” 3 (+) 2 (-) 3 (-) 3 (+) 2 (+) 2 (+)
NK-3. I C3H anti-ST 46 50 37 44 54 26 22
+ 3 (-) f 2 (+) f 2 (+) z!z0 (-) Ii 4 (-) +- 1 (+) -t 4 (-)
a Mean specific lysis t SEM of four replicates at the highest effector-target ratio. ’ Mouse scored negative because P > 0.01 for antiserum + C versus C control (Student’s t test). ’ Mouse scored positive because P < 0.01 for antiserum + C versus C control (Student’s f test).
448
BURTON
ET AL.
TABLE 2 Strain Distribution of Murine NK Cell Alloantigens Recognized by Alloantisera: (C3H X BALB/c)F, Anti-CE (anti-NK-1. 1), CE Anti-CBA a NZB Anti-BALB/c (Anti-NK-2. l), and C3H Anti-ST (Anti-NK3.1) NK-2.1 Group I”
Strain C5lBL/6
NK-1.1
CE anti-CBA
NZB anti-BALB/c
NK-3.1
+
+
+
+ + + NT
IIb
NZB Ma/My SJL CE CBA C3H BALB/c A
+ + + + -
+ + + +
IIF
C58 BIO.A 129 C57Br
+ + + +
IV*
DBA/ 1 DBA/Z RF AKR
+ + NT -
+ + + + + -
+ + + +
+ + NT NT
+ + + +
V*
LP ST SM C57L C57BL/lO BlO.Br BlO.DZ AL BLB
+ + + NT NT NT NT NT
+ + NT NT + + + + -
+ NT NT NT NT NT NT
NT + NT NT + + + NT NT
+ + + + -
* Strains expressing all alloantigens. b NK- 1.i and NK-3.1 are clearly different from NK-2.1. ’ NK-2.1 defined by CE anti-CBA is different from that defined by NZB anti-BALB/c. *These strains type differently for NK- 1.1 and NK-3.1. e Other strains.
made in our laboratory, and the results of 10 of these strains-C57BL/6, NZB, CE, C58, BlO.A, DBA/2, 129, CBA, C3H, and BALB/c-confirmed the published results. For strain DBA/ 1 the result with (C3H X BALB/c)Fr anti-CE (anti-NK- 1.1) was in accord with the results published by Koo and Peppard for their anti-NK- 1.1 monoclonaf antibody (6), and this was negative. This, however, conflicts with the data of Pollack and Emmons who reported that DBA/l NK cells were NK-1.1 positive by sorting studies (5) although by C depletion with an anti-NK-1.1 antiserum was negative. We would argue that the NZB anti-BALB/c antiserum they produced could possibly contain non-C fixing alloantibodies to Qa-5, Ly-1 1, Ly-7.3, and Ly-24.1 (8, 10) whose
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CELLS
449
expression by NK cells is unknown. These antibodies could have produced the positive results obtained by the cell sorter. It is important to stress that the genetic characterizations of the NK specific alloantigens NK- 1.1, NK-2.1, and NK-3.1 have been performed using alloantisera and Cmediated removal of NK activity in back-cross and F2 mice (4, 5, 7). In order to be certain that the same antibodies were being detected by flow cytometry, similar genetic analyses, or, at the very least a strain distribution analysis, would have to be performed by flow cytometry. Thus, we believe that DBA/l should be designated as NK- 1.1 negative. We had previously reported that CE anti-CBA sera and C did not significantly decrease the NK activity of DBA/l spleen cells (4). The pool of CE anti-CBA serum used in the genetic analyses described herein was used to type strain DBA/I positive in 3/3 experiments. Therefore, we have revised the typing of strain DBA/l from negative to positive with CE anti-CBA sera. Of the 26 strains listed in Table 2, 20 have been typed with anti-NK- 1. I and antiNK-3.1,24 with CE anti-CBA antisera, and 16 with both CE anti-CBA and NZB antiBALB/c antisera. This Table has been arranged into five groups based on whether the alloantigens were similarly expressed in the strains and whether alloantisera, CE antiCBA and NZB anti-BALB/c typed similarly for NK-2.1. As can be seen, only strain C57BL/6 was positive for all four antisera (Group I). Eight strains typed the same (Group II), and five (Group IV and strain 129) typed differently for NK- 1.1 and NK3.1. NK- 1.1 and NK-3.1 were easily distinguished from NK-2.1 by the very different strain typing (Group II). CE anti-CBA and NZB anti-BALB/c have both been proposed as anti-NK-2.1 alloantisera (5, 8). As can be seen in Group III, four strains typed differently with these two antisera for C-dependent removal of NK activity as was previously reported (8). To confirm that these antisera detect different NK alloantigens we have performed the relevant F2 genetic analysis as proposed by Pollack and Emmons (5). Table 3 summarizes the results of the genetic analyses in the (CE X CBA)F2 mice tested with the three antisera. A total of 35 mice were tested with the same pools of anti-NK- 1.1, anti-NK-2.1, and anti-NK-3.1 alloantisera. An additional 2 1 mice were tested with a second pool of CE anti-CBA (anti-NK-2.1) antiserum. As can be seen, the results indicate that NK-1.1 and NK-2.1 are determined by independently segregating loci as are NK-2.1 and NK-3.1. This, however, is not the case for NK-1.1 and NK-3.1 since of the 35 mice tested 33 typed either NK-1.1 and NK-3.1 positive (29 mice) or NK-1.1 negative and NK-3.1 negative (4 mice). The remaining 2 mice were clearly NK- 1.1 positive and NK-3.1 negative. These results are not compatible with independently segregating loci and, taken together with the strain distribution result for NK-1.1 and NK-3.1 (Table 2) indicate that the most likely relationship between these two loci is that they are distinct but linked on the same chromosome. Using BXD recombinant inbred mice and anti-NK- 1.1 MoAb, it has been shown that the gene which codes for NK- 1.1 is located on chromosome 6 (9). These results indicate that there are at least three murine NK specific alloantigens, NK-1, NK-2, and NK-3, determined by independent loci, but that two of these, NK1.1 and NK-3.1, are linked on the same chromosome. The strain distribution in Table 2 suggests, however, that there may be a fourth alloantigen because of the discrepant typing of four strains with two putative anti-NK-2.1 alloantisera CE anti-CBA and
450
BURTON
ET AL.
TABLE 3 Inheritance of NK Phenotypes in (CE X CBA)Fz Mice NK- 1.1 and NK-2.1
F2 Observed F2 Expected”
NK-l+ NK-2+
NK- I + NK-2-
NK- l- NK-2’
40 (24) 31.5 (20)
10 (17) 10.5 (6.5) P=NSh
3 (2) 10.5 (6.5)
NK-1
NK-2-
3 (2) 3.5 (2)
NK-2. I and NK-3.1
Fz Observed F, Expected
NK-2+ NK-3+
NK-2+ NK-3-
NK-2- NK-3+
NK-2- NK-3.
23 20
5 6.5 P=NS
6 6.5
I 2
NK-1.1 and NK-3.1
F, Observed F, Expected
NK-l+ NK-3+
NK-l+ NK-3-
NK-I- NK-3’
NK-lm NK-3-
29 20
2 6.5 P
IMMUNOLOGY
135,445-453
(199 1)
Studies on Murine Natural Killer (NK) Cells V. Genetic Analysis
ROBERTC.BURTON,*
of NK Cell Markers
Y.CHENGSMART,* ANDHENRY J. WINN$
GLORIACKOO,~
*Discipline qf‘Surgicai Science, Faculty qf Medicine, University of Newcastle, New South Wales 2308. Australia: TDepartment of Immunology, Merck, Sharp, & Dohme Research Laboratory, Rahway, New Jersey 07065: and *Transplantation Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 Received
October
16, 1990; accepted
January
31. 1991
This paper attempts to clarify the number and nomenclature of murine natural killer (NK) cell specific alloantigens by defining the genetic relationships between them, that is, are they coded by loci which are independent, allelic, or linked. Strain typing and Fz analysesusing five altoantisera (C3H X BALB/c)F, anti-CE, CE anti-CBA, NZB anti-BALB/c, C3H anti-ST, and BALB/c antiDBA/2 revealed that (a) the alloantigens NK-1.1 and NK-3.1 are determined by distinct loci which are linked on the same chromosomes, (b) the alloantigen NK-2.1 is determined by an independently segregating locus to those coding for NK- 1.1 and NK-3.1, (c) the alloantisera, CE anti-CBA and NZB anti-BALB/c, which have been designated anti-NK-2.1 alloantisera recognize different alloantigens coded by independent genetic loci. Thus, these five alloantisera detect four NK cell specific alloantigens which, based on the chronology of their discovery, have been designated NK- 1.1-(C3H X BALB/c)F, antiCE, NK-2.1-CE anti-CBA, NK-3.1 -C3H anti-ST, and BALB/c anti-DBA/Z and NK-4. I-NZB anti-BALB/c. o 1991 Academic press, I~C.
INTRODUCTION A number of cell membrane alloantigens, NK-1, NK-2, and NK-3 expressed by murine natural killer (NK) cells which kill the murine T lymphoma YAC in vitro, have been described (l-7). Identification of these three NK cell specific alloantigens has been based on four criteria: (i) they are expressed by less than 5% of murine splenic leukocytes, (ii) this subset of splenic leukocytes contains all of the in vitro spontaneous lytic activity against YAC, (iii) appropriate backcross and/or F2 genetic analyses have shown that a particular alloantigen is determined by a single genetic locus, and/or (iv) monoclonal antibodies (MoAb) have been produced which fulfill the first two criteria. In this paper we seek to examine the relationship between the loci coding for these alloantigens, that is, are they independent, allelic, or linked. The data presented herein will show that the alloantigens are determined by independent loci, and that two of these appear to be linked on the same chromosome. We will also report that CE antiCBA and NZB anti-BALB/c antisera, which have been described as detecting the NK2.1 alloantigen (5, S), detect alloantigens determined by independent nonlinked loci indicating that, in fact, there are at least four murine NK cell specific alloantigens. 445 0008-8749/9
I $3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduaion in any form reserved
446
BURTON
MATERIALS
ET
AND
AL.
METHODS
Mice. The inbred mice used in these studies were obtained from the Jackson Laboratory (Bar Harbor, ME) and from the Central Animal House of the University of Newcastle (New South Wales). BlO.A, C57Br, C58, and 129 were obtained from the Walter and Eliza Hall Institute (Melbourne, Victoria). The Fr and F2 hybrids were raised in our laboratory and in the Central Animal House of the University of Newcastle. Mice of both sexes were used in NK assays at 6- 15 weeks of age. Assayfor NK activity. The activity of splenic NK cells was measured by an in vitro “Cr release assay against YAC target cells, a Moloney virus induced T lymphoma, and a prototype NK cell target, as described elsewhere (3,4). The tissue culture medium (TCM) was RPM1 1640 supplemented with 2 m&f glutamine, 1% nonessential amino acids, 20 mM Hepes buffer, 50 &ml of gentamicin, and 10% fetal calf serum. All assays were performed in V-bottom microtiter trays in quadruplicate as a minimum of three twofold dilutions of effector spleen cells against lo4 “Cr-labeled YAC target cells in 200 ~1 TCM. The microtiter trays were centrifuged at 15Og for 1 min and incubated at 37°C in 5% CO* in air for 16 hr. At the end of the assay, 100 ~1 of supematant was removed from each well and the level of radioactivity was counted in a Cobra gamma-counter (Packard Instrument Co., IL). The results were calculated as the mean percentage specific lysis + SEM for each group of four replicates as Percentage specific lysis =
cpm test sample - cpm background total cpm - cpm background
x 100.
Background represents the release of “Cr from tumor cells incubated alone. Total cpm is the total 51Cr activity in 5 X lo3 labeled tumor cells. In the assay, spontaneous (background) release of “Cr from YAC was 25 to 30% of total cpm. For each sample tested, a straight line was obtained for the percentage specific lysis versus effectortarget ratio. The percentage specific lysis at one or more effector-target ratios was used in calculating a significant reduction (P < 0.0 1, Student’s t test) by the antiserum and C compared to C alone control according to our previously defined criterion (4, 7). Results herein were represented as percentage specific lysis or as positive (+) or negative (-) depending on whether the P value was < or >O.O 1, respectively. Preparation of antisera. The antisera used herein were all produced by hyperimmunization of mice as previously described (4, 5, 7). Sera from individual mice and/ or pools of sera from groups of mice were tested for complement (C) dependent depletion of splenic NK activity against “Cr-labeled YAC targets in vitro before the final pools of sera were made for use in these experiments. Testing of antisera. The effects of antisera and C on NK cells were usually tested in a two-stage procedure. Antiserum was added to 1O7 spleen cells in 1O-ml centrifuge tubes and the mixtures were incubated at 37°C in 5% CO2 in air for 30 min. The tubes were then centrifuged at 600g and the cells were washed once with 10 ml of TCM and resuspended in 1 ml of dilute rabbit C (Pel Freez Biologicals, Inc., Rogers, AK). They were incubated again for 30 min at 37°C in 5% CO2 in air, washed twice with TCM, and resuspended to the original (pretreatment) volume for use in NK analyses. In the analysis of the back-cross mice, a microtray was used in place of the conical tubes and the procedure was carried out as follows. Quadruplicate samples of 1O6and 5 X 1O5spleen cells in 100 ~1 of culture medium were placed in the tray wells
GENETIC
STUDIES ON MURINE
NATURAL
KILLER
447
CELLS
with 100 ~1 of antiserum; the trays were incubated at 37°C in 5% CO* in air for 30 min and then centrifuged at 150 g for 3 min. The cells were washed once, resuspended in 200 ~1 diluted C, and again incubated for 30 min, after that they were again washed and resuspended. FCS was omitted from the TCM during the time of treatment with antiserum and C, but was introduced immediately after the C had been removed and the cells were being prepared for analysis of NK activity. To avoid enriching the surviving cells and potentially introducing artifacts, the antiserum and C or C alone treated spleen cells were resuspended to the starting volume (200 ~1) in all assays. RESULTS
AND
DISCUSSION
In our past genetic analysis of CE anti-CBA alloantisera we used as indication of a positive result, a reduction of 50% or more of in vitro lysis of YAC by splenic leukocytes treated with alloantiserum and C as compared to the C control (4). In our analysis of C3H anti-ST alloantisera we applied the same criterion (7). These reductions in lysis were always highly statistically significant (P < 0.0 1 or 0.00 1, Student’s t test). In the analysis of (C3H X BALB/c)F, anti-CE and NZB anti-BALB/c alloantisera, Pollack and Emmons (5) used the criterion of a 30% reduction with the first antiserum and a 25% reduction with the second, this being significant for the genetic analysis of those antisera. We have produced these alloantisera but have found that their anti-NK titers were usually lower than those of CE anti-CBA and C3H anti-ST alloantisera. We have therefore applied the one criterion (P < 0.01, Student’s t test) to the genetic analyses and strain typing of all four alloantisera. This is illustrated in Table 1 which shows the results of typing (CE X CBA)F2 mice for simultaneous expression of NK- 1.1, NK2.1, and NK-3.1 with three anti-NK alloantisera (C3H X BALB/c)F, anti-CE, CE anti-CBA, and C3H anti-ST. Table 2 sets out the strain distributions reported for the mm-me NK cell alloantigens, NK- 1.1, NK-2.1, and NK-3.1 in 26 strains of mice (2,4-9). In this report spleen cells from 11 strains were treated with (C3H X BALB/c)F, anti-CE (anti-NK- 1. l), CE antiCBA and NZB anti-BALB/c (anti-NK-2. l), and C3H anti-ST (anti-NK-3.1) antisera TABLE 1 Expression of NK Cell Alloantigens Recognized by Three Anti-NK Alloantisera in Seven (CE X CBA) F2 Mice Mouse No.
C control
NK-1.1 (C3H X BALB/c)F, anti-CE
I 2 3 4 5 6 7
46f la 64-c 1 54 -c 2 43 + 2 58 f 3 39 f 3 28 f 2
48 + I (-)* 21 * 1 (+) 2 IL 2 (+) 38 + 2 (-) 53 + 2 (-) 8 + 2 (+) 6 zk 1 (+)
NK-2.1 CE anti-CBA 17 + 44 * 52 -t 37 f 41 It 25 + 2+
2 (+)” 3 (+) 2 (-) 3 (-) 3 (+) 2 (+) 2 (+)
NK-3. I C3H anti-ST 46 50 37 44 54 26 22
+ 3 (-) f 2 (+) f 2 (+) z!z0 (-) Ii 4 (-) +- 1 (+) -t 4 (-)
a Mean specific lysis t SEM of four replicates at the highest effector-target ratio. ’ Mouse scored negative because P > 0.01 for antiserum + C versus C control (Student’s t test). ’ Mouse scored positive because P < 0.01 for antiserum + C versus C control (Student’s f test).
448
BURTON
ET AL.
TABLE 2 Strain Distribution of Murine NK Cell Alloantigens Recognized by Alloantisera: (C3H X BALB/c)F, Anti-CE (anti-NK-1. 1), CE Anti-CBA a NZB Anti-BALB/c (Anti-NK-2. l), and C3H Anti-ST (Anti-NK3.1) NK-2.1 Group I”
Strain C5lBL/6
NK-1.1
CE anti-CBA
NZB anti-BALB/c
NK-3.1
+
+
+
+ + + NT
IIb
NZB Ma/My SJL CE CBA C3H BALB/c A
+ + + + -
+ + + +
IIF
C58 BIO.A 129 C57Br
+ + + +
IV*
DBA/ 1 DBA/Z RF AKR
+ + NT -
+ + + + + -
+ + + +
+ + NT NT
+ + + +
V*
LP ST SM C57L C57BL/lO BlO.Br BlO.DZ AL BLB
+ + + NT NT NT NT NT
+ + NT NT + + + + -
+ NT NT NT NT NT NT
NT + NT NT + + + NT NT
+ + + + -
* Strains expressing all alloantigens. b NK- 1.i and NK-3.1 are clearly different from NK-2.1. ’ NK-2.1 defined by CE anti-CBA is different from that defined by NZB anti-BALB/c. *These strains type differently for NK- 1.1 and NK-3.1. e Other strains.
made in our laboratory, and the results of 10 of these strains-C57BL/6, NZB, CE, C58, BlO.A, DBA/2, 129, CBA, C3H, and BALB/c-confirmed the published results. For strain DBA/ 1 the result with (C3H X BALB/c)Fr anti-CE (anti-NK- 1.1) was in accord with the results published by Koo and Peppard for their anti-NK- 1.1 monoclonaf antibody (6), and this was negative. This, however, conflicts with the data of Pollack and Emmons who reported that DBA/l NK cells were NK-1.1 positive by sorting studies (5) although by C depletion with an anti-NK-1.1 antiserum was negative. We would argue that the NZB anti-BALB/c antiserum they produced could possibly contain non-C fixing alloantibodies to Qa-5, Ly-1 1, Ly-7.3, and Ly-24.1 (8, 10) whose
GENETIC
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CELLS
449
expression by NK cells is unknown. These antibodies could have produced the positive results obtained by the cell sorter. It is important to stress that the genetic characterizations of the NK specific alloantigens NK- 1.1, NK-2.1, and NK-3.1 have been performed using alloantisera and Cmediated removal of NK activity in back-cross and F2 mice (4, 5, 7). In order to be certain that the same antibodies were being detected by flow cytometry, similar genetic analyses, or, at the very least a strain distribution analysis, would have to be performed by flow cytometry. Thus, we believe that DBA/l should be designated as NK- 1.1 negative. We had previously reported that CE anti-CBA sera and C did not significantly decrease the NK activity of DBA/l spleen cells (4). The pool of CE anti-CBA serum used in the genetic analyses described herein was used to type strain DBA/I positive in 3/3 experiments. Therefore, we have revised the typing of strain DBA/l from negative to positive with CE anti-CBA sera. Of the 26 strains listed in Table 2, 20 have been typed with anti-NK- 1. I and antiNK-3.1,24 with CE anti-CBA antisera, and 16 with both CE anti-CBA and NZB antiBALB/c antisera. This Table has been arranged into five groups based on whether the alloantigens were similarly expressed in the strains and whether alloantisera, CE antiCBA and NZB anti-BALB/c typed similarly for NK-2.1. As can be seen, only strain C57BL/6 was positive for all four antisera (Group I). Eight strains typed the same (Group II), and five (Group IV and strain 129) typed differently for NK- 1.1 and NK3.1. NK- 1.1 and NK-3.1 were easily distinguished from NK-2.1 by the very different strain typing (Group II). CE anti-CBA and NZB anti-BALB/c have both been proposed as anti-NK-2.1 alloantisera (5, 8). As can be seen in Group III, four strains typed differently with these two antisera for C-dependent removal of NK activity as was previously reported (8). To confirm that these antisera detect different NK alloantigens we have performed the relevant F2 genetic analysis as proposed by Pollack and Emmons (5). Table 3 summarizes the results of the genetic analyses in the (CE X CBA)F2 mice tested with the three antisera. A total of 35 mice were tested with the same pools of anti-NK- 1.1, anti-NK-2.1, and anti-NK-3.1 alloantisera. An additional 2 1 mice were tested with a second pool of CE anti-CBA (anti-NK-2.1) antiserum. As can be seen, the results indicate that NK-1.1 and NK-2.1 are determined by independently segregating loci as are NK-2.1 and NK-3.1. This, however, is not the case for NK-1.1 and NK-3.1 since of the 35 mice tested 33 typed either NK-1.1 and NK-3.1 positive (29 mice) or NK-1.1 negative and NK-3.1 negative (4 mice). The remaining 2 mice were clearly NK- 1.1 positive and NK-3.1 negative. These results are not compatible with independently segregating loci and, taken together with the strain distribution result for NK-1.1 and NK-3.1 (Table 2) indicate that the most likely relationship between these two loci is that they are distinct but linked on the same chromosome. Using BXD recombinant inbred mice and anti-NK- 1.1 MoAb, it has been shown that the gene which codes for NK- 1.1 is located on chromosome 6 (9). These results indicate that there are at least three murine NK specific alloantigens, NK-1, NK-2, and NK-3, determined by independent loci, but that two of these, NK1.1 and NK-3.1, are linked on the same chromosome. The strain distribution in Table 2 suggests, however, that there may be a fourth alloantigen because of the discrepant typing of four strains with two putative anti-NK-2.1 alloantisera CE anti-CBA and
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ET AL.
TABLE 3 Inheritance of NK Phenotypes in (CE X CBA)Fz Mice NK- 1.1 and NK-2.1
F2 Observed F2 Expected”
NK-l+ NK-2+
NK- I + NK-2-
NK- l- NK-2’
40 (24) 31.5 (20)
10 (17) 10.5 (6.5) P=NSh
3 (2) 10.5 (6.5)
NK-1
NK-2-
3 (2) 3.5 (2)
NK-2. I and NK-3.1
Fz Observed F, Expected
NK-2+ NK-3+
NK-2+ NK-3-
NK-2- NK-3+
NK-2- NK-3.
23 20
5 6.5 P=NS
6 6.5
I 2
NK-1.1 and NK-3.1
F, Observed F, Expected
NK-l+ NK-3+
NK-l+ NK-3-
NK-I- NK-3’
NK-lm NK-3-
29 20
2 6.5 P
