Leukemia prevention and long-term survival of AKR mice transplanted with MHC-matched or...

CELLULAR IMMUNOLOGY 101,476-492 (1986)

Leukemia Prevention and Long-Term Survival of AKR Mice Transplanted with MHC-Matched or

MHC-Mismatched Bone Marrow’

RossE. L~NGLEY*ANDROBERTA.GOOD~

Department of Biological Sciences. University of Central Florida, Orlando, 32816 and Department of Pediatrics, All Childrens’ HospitaI, St. Petersburg, Florida 33701

Received September 13, 1985; accepted April 4, I986

The current studies were designed to evaluate the effectiveness of marrow transplantation within and outside the major h&compatibility complex (MHC) on the long-term survival and occurrence of spontaneous leukemia in AKR mice. AKR mice, which were lethally irradiated and received MHC-matched marrow from CBA/J mice (CBA - AKR), never developed leukemia and were alive and remained healthy for up to 280 days post-transplant. These long-term surviving chimeras possessed substantial immune vigor when both cell-mediated and humoral responses were tested. Lethally irradiated AKR mice, which had received MHC-mismatched marrow (anti-Thy-l.2 treated or nontreated) from C57BL/6J mice (B6 - AKR), never developed leukemia and survived up to 170 days post-transplant. However, both groups of these chimeras began dying 180 to 270 days post-transplant due to a disease process which could not be readily identified. Histological analysis of B6 - AKR chimeras revealed severe lymphoid cell depletion in thymus and spleen; however, none of these chimeras exhibited classical features of acute graft versus host disease. Concanavalin A mitogenesis, primary antibody responses to sheep red blood cells and the production of interleukin 2 (IL-2) were suppressed in B6 - AKR chimeras. IL-2 treatment of B6 - AKR chimeras was shown to partially correct these deticien- ties without stimulating mixed lymphocyte responsiveness to donor or host lymphocytes. These studies indicate that the use of MHC-mismatched marrow for the prevention of spontaneous AKR leukemia may rely on augmentative IL-2 therapy for complete immune reconstitution of leukemia-free chimeras. Q 1986 Academic PXSS, ITIC.

INTRODUCTION

One of the major problems associated with bone marrow transplantation for treatment of various leukemias and immune disorders has been the occurrence of graf% vs host disease (GVHD),2 when marrow from donor is not matched to the recipient’s major histocompatibility complex (MHC) (l-3). Previous work from our laboratory

’ This work supported in part by the Department of Sponsored Research, University of Central Florida; Florida Division, American Cancer Society Lucretia B. Troyer Research Grant (R.E.L.); and National Institutes of Health Grants AI-22360-02 and AG-05628-O 1 (R.A.G.).

* Abbreviations used GVHD, graft versus host disease; MHC, major histocompatibility complex; B6, C57BL/6J; Con A, concanavalin A; PI-IA, phytohemmaglutinin; SRBC, sheep red blood cells; K-2, interleukin 2; PMA, 4Bphorbo1, 12&myristate, 13a-acetate; MLC, mixed lymphocyte culture; SI, stimulation index.

476

0008-8749/86 $3.00 Copyright 0 1986 by Academic Press, Inc. AU rights of reproduction in any form reserved.

LEUKEMIA PREVENTION AND MARROW TRANSPLANTATION 477

has shown that bone marrow transplantation in mice across the MHC barrier can be successful if, at first, the bone marrow is purged of immunocompetent T lymphocytes by treatment with heterologous anti-Thy antiserum, with or without complement, and then infused into lethally irradiated recipients (4-6). These fully allogeneic chimeras subsequently develop an immune system which has considerable vigor and which is fully reactive to third-party antigens but tolerant to both donor and host- type antigens, as measured by the mixed lymphocyte reaction. Studies by Onoe et al. (7) demonstrated that lethally irradiated AKR mice, which had been infused with anti-Thy-treated marrow from C57BL/6J (B6 - AKR) mice, demonstrated no GVHD, and most immune functions remained intact, with the exception of failure of these allogeneic chimeras to respond to primary immunization with sheep red blood cells (SRBC). In subsequent studies, Onoe et al. showed these animals also to be deficient in autologous mixed leukocyte reactivity (8). Upon secondary stimulation, however, antibody responses returned. These immune responses were measured ap proximately 100 days post-transplant.

The following study was undertaken to determine if allogeneic (CBA - AKR) and fully allogeneic (B6 - AKR) chimeras would survive longer than the previously reported loo-day post-transplant period and if these chimeras resisted the develop ment of spontaneous AKR leukemia. Our data demonstrated that although both chimeric combinations resisted the development of leukemia, fully allogeneic B6 - AKR chimeras died 180-270 days post-transplant and these deaths were attributed to a strange secondary disease or wasting syndrome. Immune analysis of these chimeras revealed severe immune deficiencies which were partially corrected both in vitro and in vivo with interleukin 2 (IL-2). These data establish the importance of long-term immunological and histological monitoring of chimeras in the evaluation of the effectiveness of bone marrow transplantation across major histocompatibility barriers.

MATERIALS AND METHODS

Mice. Female AKR/J, C57BL/6J, and CBA/H mice were obtained from Jackson Laboratories (Bar Harbor, Maine). Recipient AKR/J mice were housed in laminar air flow units (PCS-80; Hazelton Laboratories, Aberdeen, Md.), five animals per cage, following bone marrow transplantation and received mouse laboratory chow and acidified water ad libitum.

Antisera and typing. The antiserum used for elimination of post-thymic T cells in donor marrow (anti-Thy- 1.2) was obtained from Accurate Chemical and Scientific Corporation (Westbury, N.Y.). This antiserum was prepared by immunizing AKR/ J female mice with C3H thymocytes. The cytotoxic titer against Thy- 1.2-positive strains was approximately 15000 while the titer against Thy- 1.1 strains was less than 1:40. Sera for typing of splenocytes (anti-H-2k, anti-H-2b, anti-Thy- 1.1, and anti-Thy- 1.2) was obtained from Dr. John G. Ray, Jr., National Institutes of Health (Bethesda, Md.). Typing of lymphoid cells, using the specific antisera described above, was ac- complished with the microcytotoxicity assay described by Terasaki and McClelland (9). Nylon wool-purified splenocytes were used for T-subset analysis of chimeras.

Preparation of radiation bone marrow chimeras. Female 8-week-old AKR mice were used as irradiated recipients of bone marrow. Bone marrow from lo- to 12- week-old donor mice, matched or mismatched at the major histocompatibility locus

478 LONGLEY AND GOOD

(H-2), was infused into AKR recipients that had been lethally irradiated. Recipient mice were placed in a plastic chamber and given 1000 R from a cobalt source (72 cm, dose rate 85 rad/min). Bone marrow from donor mice was obtained by gently aspirating marrow from excised femurs and tibia with cold RPM1 1640 medium supplemented with heat-inactivated fetal calf serum, 100 pg/ml penicillin, 100 pg/ml steptomycin, and glutamine (Kansas City Biologicals, Lenexa, Kans.) (complete medium). Cells were washed one time with medium and the concentration was adjusted to 1 X 1 O8 cells/ml. To prevent subsequent development of graft vs host disease when H-2-mismatched donors were used, marrow was treated in vitro with anti-Thy- 1.2 alloantiserum (Accurate Chemical and Scientific) as per the method of Onoe et al. (7) to remove post-thyrnic T lymphocytes. An equal volume of anti-Thy- 1.2 antiserum, diluted 1:5, was added to 1 X 10’ bone marrow cells per milliliter and the suspension incubated on ice for 30 min with occasional mixing. When the cells have been treated in this way, clumping characteristically occurs, and these clumps have been observed to contain T lymphocytes. The cells were washed three times with medium and the concentration was adjusted to 25 X lo7 cells/ml. No complement was used in the antiserum treatment. One milliliter of the suspension was injected via tail vein into irradiated AKR recipients.

Animals were removed to laminar air-flow cages and provided with acidified water and mouse chow ad libitum. Chimeras were transferred to conventional facilities at 120 days and were maintained for up to 280 days post-transplant. Tissues from mice which had succumbed to GVHD, leukemia, wasting disease, or of normal mice were fixed in Formalin, sectioned, and stained with hematoxylin and eosin for histological study.

Mitogen stimulation. Measurement of spleen cell responses of chimeras to phytohemmaglutinin (PHA) and conconavalin A (Con A) was used to monitor T-cell activity. Briefly, 2 X lo5 spleen cells were cultured with 2.0 and 1.0 pg of PHA (Grand Island Biologicals Co., Grand Island, N.Y.) and 2.0 and 1 .O pg of Con A (Pharrnacia, Piscataway, N.J.) per milliliter for 96 hr. Sixteen hours before harvest, 0.5 PCi of methyl-[3H]thymidine (NET-027; New England Nuclear Corp., Boston, Mass.) was added to each well. The plates were harvested on glass fiber filter paper and the discs placed in scintillation fluid and counted.

In vivo PFCresponses to SRBC. The ability of chimeras to respond to in vivo sensitization to SRBC was evaluated. Chimeras were injected with 1 .O ml of a 1% SRBC suspension. Five days later, spleens were removed and PFC responses were analyzed using the Jerne plaque assay according to the method of Cunningham and Szenberg ( 10).

In vitro immunization and response to SRBC. Measurement of in vitro antibody responses to SRBC was used to evaluate T-cell and B-cell cooperation. Spleen cells were cultured according to the method of Mishell and Dutton (11) except that culture medium was supplemented with 5 X lop5 M 2-mercaptoethanol. Cultures were im- munized with SRBC by adding 50 ~1 of a 0.5% SRBC suspension to each tissue culture dish. Five days later, antibody response was measured using the Jerne hemolytic plaque assay as modified by Cunningham and Szenberg ( 10).

Assays for spleen cell production of interleukin 2. Spleen cells from chimeras were cultured in RPM1 1640 medium at a density of 5 X lo6 cells/ml with Con A at 2 pg/ ml to test for production of IL-2. Supernatants were collected and filter sterilized through 0.45-pm filters. Aliquots were added to thyrnocytes from C57BL/6J mice, together with Con A, to assay for IL-2 activity. Thymocytes (2 X 105/0.1 ml/well)


were added to triplicate wells of microtiter test plates. A volume of 0.1 ml Con A and 0.050 ml of test supematant was added, so that the final concentration was 2 pg/ ml. In some wells medium was added in place of IL-2 test supematants. Other wells contained only Con A or medium as controls. The plates were incubated at 37°C for 4 days. Sixteen hours before harvest, t3H]thymidine was added, the plates were harvested, and the radioactive incorporation was determined. Thymocytes with Con A alone responded very poorly without the addition of IL2. Therefore, a high level of incorporation by thymocytes cultured with test supematants indicated IL-2 activity in the supematant.

An additional assay for IL-2 was used which is based on the measurement of growth of an IL-2-dependent cell line in a standard microassay (12). Briefly, HT-2 cells, an IL-2-dependent, murine helper-T-cell line (kindly provided by Dr. James Watson) was cultured in 200~~1 volumes in wells of microtiter test plates in RPM1 complete medium. Each well contained 4 X lo3 HT-2 cells with a log2 dilution of test supematant. After 24 hr of incubation, the plates were pulsed with 0.5 PCi of [3H]thymidine and incubated an additional 4 hr. Cultures were harvested onto glass fiber filter strips and [3H]thymidine incorporation was determined by liquid scintillation counting. Results were quantified by probit analysis. The activity was expressed in units defined as the reciprocal of the dilution of test supematant that gives 50% of the maximum incorporation of [3H]thymidine by HT-2 cells.

IL-2. Purified rat IL-2 used for in vitro addition to splenocyte cultures was obtained from Collaborative Research (Lexington, Md.). This IL-2 preparation was assayed using HT-2 cells and then added at a concentration of 50 units/ 1 ml SRBC, lymphocyte culture.

Partially purified murine IL-2, used for iv injection, was obtained from the IL-2- producing EL-4 subline designated EL-4.IL2 (American Type Culture Collection, Rockville, Md.). EL-4.IL2 cells (1 X 106/ml) were cultured in RPM1 1640 medium supplemented with 1% fetal calf serum and antibiotics plus 10, 50, or 100 pg/ml of 4&phorbol, 12 /I-myristate, 13 a-acetate (PMA). Supematants from these three cultures were filtered through a 0.45-pm filter, pooled, and assayed for IL-2 activity using the IL-2-dependent HT-2-cell line. The average IL-2 activity for this pool was 109 units. The pooled IL-2 supematant (117 ml) was concentrated and dialyzed against 1800 ml of serum-free RPM1 1640 using vacuum dialysis (MW cutoff = 10,000) (Pro Di Con Mem; Biomolecular Dynamics, Beaver-ton, Oreg.). The concentrated supematant was then dialyzed against 1 liter of 0.02 M phosphate buffer, pH 7.15, and then applied to an Affi-Gel Blue column (Biorad, Richmond, Calif.) equilibrated in the sample buffer. The supematant was collected along with two column washes, dialyzed against RPM1 1640, and concentrated. Mouse serum ( 10% by volume) was added to the concentrate and IL-2 activity determined using HT-2 cells. The activity of the concentrated sample was 1024 units. Chimeras received 1 .O ml of appropriately diluted partially purified IL-2 iv, 48 hr prior to immune analysis.

MLC assay. Mixed lymphocyte responses of long-lived B6 - AKR chimeras and normal B6 mice were evaluated using whole splenocyte preparations. Spleens were excised and single cell suspensions were made in complete medium. The cell concen- trations were adjusted to 1 X 106/ml. Stimulator splenocytes were prepared by irradi- ating splenocyte suspension with 2000 R. A volume of 0.1 ml of responder and 0.1 ml of stimulator splenocytes were cultured alone as controls. Cells were cultured for 5 days with the addition of 1 .O &i of [3H]thymidine on Day 4. Cells were harvested


---------- -___ --~~-(-~UWFJJIA- _

i ONTROL $io MARROW) I I

i.. . . I I , I

1 i I 11111111111111111111l1111

10 30 50 70 90 110 130 150 170 190 210 230 250 270 280 DAYS POST-TRIW’LAN~

FIG. 1. Survival of lethally irradiated AKR mice transplanted with marrow: ---, anti-Thy-l .2-treated, allogeneic H-2-matched (CBA - AKR); -, syngeneic (AKR - AKR); . . . , allogeneic H-2-mismatched (B6 - AKR); and -. -. -. , anti-Thy- 1.2-treated, allogeneic H-2-mismatched (B6 - AU). Positive and/or negative confirmation of leukemia was verified by gross and histological analysis of thymus, spleen, lymph nodes, and livers of chimeras at time of death.

and the [3H]thymidine incorporation was determined. The stimulation index (SI) was calculated as follows:

sI = (cpm of stimulators + irradiated responders) - (cpm of stimulators alone) (cpm of stimulators + irradiated stimulators)

RESULTS

Survival and Incidence of Leukemia in Bone Marrow-Transplanted AKR Mice

Figure 1 shows the survival of lethally irradiated AKR mice transplanted with syngeneic (AKR - AKR), H-Zcompatible allogeneic (CBA - AKR), H-Zmismatched allogeneic (B6 - AKR), and anti-Thy- 1.2 antiserum-treated (B6 - AKR) bone marrow. All controls died of the effects of lethal body radiation by Day 14. Greater than 90% of syngeneic AKR - AKR chimeras survived to 170 days post-transplant at which time chimeras began dying of lymphoblastic leukemia, with 100% mortality and 100% leukemia-lymphoma by Day 280. Ninety-eight percent of allogeneic chimeras (CBA - AKR) were alive 280 days post-transplant. Histological analysis of thymus, spleen, and lymph nodes revealed no evidence of leukemia in any of these mice.

Forty percent of the AKR mice transplanted with allogeneic, untreated B6 marrow cells (B6 - AKR), died due to graft vs host disease between Days 30 and 100 post- transplant. By contrast, greater than 90% of the AKR mice transplanted with anti- Thy- 1.2 antiserum-treated B6 allogeneic marrow were still living 170 days post-transplant with no evidence of GVHD. Both groups of B6 - AKR chimeras began dying 170 days post-transplant due to a disease process of as yet undetermined etiology. However, in neither group was there histological evidence of either classical GVHD lesions or leukemia-lymphoma.


These results indicate that H-Zcompatible allogeneic (CBA - AKR) and H-2 mismatched allogeneic (B6 - AKR) bone marrow transplantations rendered AKR recipients completely resistant to the spontaneous development of leukemia. However, mice receiving untreated or anti-Thy- 1.2-treated B6 marrow had a low incidence of survival and died between 170 to 280 days post-transplant. These late deaths could not be attributable to either GVHD or leukemia, nor could an etiological agent be identified by standard bacteriologic or fungal cultures, parasitic, and histological ex- aminations. The most probable cause of death in these chimeras was a wasting syndrome or secondary disease in the context recently described by Rayfield and Brent ( 13).

Characteristics and Mitrogen Responsiveness of Radiation Bone Marrow Chimeras 170-280 Days Post-transplant

Table 1 shows that the average body weight and spleen weight of the three chimeric groups and normal B6 mice, 170-280 days post-transplant, were similar. Thymus weight, however, was higher in AKR - AKR chimeras since some of the mice in the latter group were already showing evidence of a thymoma. Total spleen cell numbers of B6 - AKR chimeras (anti-Thy-treated marrow) tended to be lower as compared to the other groups. Con A mitogenesis was 50% lower in the B6 - AKR chimeras compared to the other groups. The results indicated that spleen cell numbers and their mitogenic response to Con A tended to be depressed in long-lived B6 - AKR chimeras.

Histological Analysis of Tissues from AKR - AKR and B6 - AKR Chimeras

Histological analysis of thymus (Fig. 2), spleen (Fig. 3), and liver (Fig. 4) of AKR - AKR chimeras showed classical lymphoblastic leukemia. Gross enlargement of the thymus (thymoma) and massive invasion with lymphoblastic cells in the thymus, spleen, and liver were apparent. Histological analysis of similar tissues (Figs. S- 7) of long-lived B6 - AKR chimeras revealed extreme involution and depletion of cortical and medullary thymocytes in the thymus (Fig. 5). Splenic tissue showed loss

TABLE 1

Characteristics and Mitogen Responsiveness of Radiation Bone Marrow Chimeras”

AKR- AKR 27.2 + 0.75 0.122 + 0.032 0.088 f 0.02 90.2 + 28.2 560&e 1505 36621 8509 + 13899 4312 + 11718 3864 k

(3) B6 - AKR’ 26.0+ 1.12 0.056kO.008 0.083+0.001 67.4k24.0 21492 +8602 19917f9741 1244Of4724 863323260

(3) CBA-AKR’ 26.4+ 1.16 0.056+0.014 0.077+0.006 78.0f 7.0 67780 e9861 52177%7450 17968f2614 16275f2983

(3) B6 (3) 26.7 2 1.05 0.049 f 0.009 0.079 ~0.008 82.0 + 6.0 55720 f 8216 49281 f 2161 12893 3612 + 8921 + 2103

’ Avemgs values of three mice f SE of the mean. b Results expressed as cpm. cAnti-Tby-l.2-tmated marrow.


FIGS. 2-4. Thymus (2), spleen (3), and liver (4) of syngeneic, AKR -+ AKR chimeras, 170-280 days post-transplant. Gross enlargement of thymus and massive invasion of lymphoblastic cells in these organs represent the classical features of AKR lymphoblastic leukemia. (hematoxylin and eosin; (2) and (3), 400X; (4), loooX).

of germinal centers and depletion of red and white pulp areas (Fig. 6). The liver appeared normal with no evidence of GVHD (Fig. 7). None of the tissues of any B6 - AKR chimeras analyzed showed evidence of leukemia. These results indicated that B6 - AKR chimeras resist the development of leukemia, but long-term survivors ( 170-280 days post-transplant) developed a histological picture resembling a wasting or secondary disease in the environment in which the animals were maintained.


1

FIGURES

Interleukin 2 Production in Long-Lived (180-270 Days Post-transplant) Chimeras

Interleukin 2 production by Con A-stimulated splenocytes of syngeneic (AKR - AKR), H-2-compatible allogeneic (CBA - AKR), and H-2-mismatched (B6 - AKR) allogeneic chimeras 170 to 180 days post-transplant was analyzed. Splenocytes from chimeras and normal mice were stimulated with 2 pg/ml of Con A for 24 hr. Supernatants were collected, filtered, and assayed for IL-2 production on mouse thymocytes. The results in Table 2 indicated that (B6 - AKR) chimeras had responses in production of IL-2 two- to threefold lower than were seen in either of the other chimer-k combinations studied or normal B6 mice. Thus, fully allogeneic bone marrow transplantation resulted in long-lived chimeras which after 180 days were deficient in production of IL-2.

T-Lymphocyte Subset Analysis of Nylon Wool-Separated Chimeric Splenocytes

To determine whether defective IL-2 production and subnormal Con A mitogenesis in B6 - AKR chimeras results from loss of specific T-cell subsets, typing of nylon wool-separated splenocytes using specific antisera and cytotoxicity assays was done. The results shown in Table 3 indicated that B6 - AKR chimeras had normal per- centages of Lyt- 1.2-, -2.2-, and -3.2-positive splenocytes compared to normal B6 controls. Typing with Thy- 1.2 and H-2b also indicated that splenocyte elements were still predominantly donor origin as late as 280 days after marrow transplantation.

PFC Response to in Vivo Sensitization to SRBC with Long-Lived (170-280 Days Post-transplant) Chimeras

Three groups of chimeras were injected ip with 1 ml of a 1% SRBC suspension. Five days later, chimeras were analyzed for antibody responses of their splenocytes


FIGS. 5-7. Thymus (5), spleen (6), and liver (7) of H-2-mismached, ailogeneic B6 - AKR chimeras, 170-280 days post-transplant. Extreme involution and depletion of cortical and medullary thymocytes is evident in thymus. Splenic tissue shows loss of germinal centers depletion of red and white pulp. Liver appears normal with no evidence of inflammatory responses usually associated with GVHD. (hematoxylin and eosin; (5) and (6), 400X; (7), 1000X).

to SRBC using the Jerne plaque assay. Our results in Table 4 demonstrate that long- lived B6 - AKR chimeras that cross the MHC did not respond to in vivo stimulation with SRBC, whereas syngeneic AKR - AKR and splenocytes from allogeneic CBA - AKR (that did not cross the MHC) are capable of in vivo SRBC sensitization. While one AISR - AKR chimera had a low PFC response, upon examination of


splenic and thymic tissue it was discovered that this animal already had lymphoblastic leukemia and a thymoma. Spleen cells had been almost totally replaced with leu- kemic blasts and this pathologic change surely accounted for the low response. Our results in long-lived chimeras agree with the earlier findings of Onoe et al. (7) in which younger B6 - AKR chimeras (100 days post-transplant) were analyzed.

PFC Responses to in Vitro Sensitization to SRBC in Long-Lived (I 70-280 Days Post- transplant) Chimeras

Splenocytes from three groups of chimeras cultured for 5 days with SRBC and PFC responses were determined using the Cunningham slide technique (10). Our results

TABLE 2

Production of Interleukin 2 by Chimeric Splenocytes”

lG2(+) Con Ab ILZ(-) Con A

AKR-AKR 12539d+4122 11351+3945 7993 + 2485 5530 2 1080 1130f392 993+448 584+271 211i69

(3) B6-AKR’ 5896 ~2101 5205 k 1483 3718k 725 2965 + 269 499 * 232 238t 88 124+ 43 128 * 58

(3) CBA - AKR’ 15774 * 258 15451+ 751 10083+ 951 6514+ 1067 1489+136 1166k 61 514+ 69 193 k45

(3) B6 (3) 13250 + 1020 11110* 954 10190f 621 94OOr 589 1212f 121 806+116 954f;212 712+58

Nofe. Con A + thymocytes, 3 133; media + thymocytes, 66. a Average values of three mice f SE of the mean. ‘Con A at 2 ,@Jml. ’ IL2 supematants diluted with medium and added as 30% volume to tat wells. *Results expressed as cpm. = Anti-Thy-l .2-treated marrow.


TABLE 3

T-lymphocyte Subset Analysis of Nylon Wool-Separated Splenocytes

Antiserum treatment

Mice Thy- 1.2” H-2b Lyt-1.2 Lyt-2.2 Lyt-3.2

B6 - AKR” 76’ B6-AKR 14 B6-AKR 73 B6 78 B6 81 B6 79

B Anti-Thy- 1.2-treated marrow. b Percentage cytotoxicity.

98 53 21 10 98 64 27 22 98 44 24 11 98 55 30 10 98 57 28 7 98 49 21 9

in Table 5 demonstrated that long-lived B6 - AKR chimeras responded poorly in vitro to SRBC sensitization as compared to the other chime& groups. Typing of splenocyte preparations from all chimeric groups revealed that splenocytes had remained predominantly of donor type over the 280-day post-transplantation period.

Efect ofAddition of IL-2 on the in Vitro SRBC Response of Normal and Long-Lived (170-280 Days Post-transplant) Chimeric Mice

Splenocytes from normal B6 mice and long-lived B6 - AKR chimeras were cultured with SRBC for 5 days, with or without the addition of purified rat K-2, at the time of initiation of the cultures. PFC responses were measured using the Cunning- ham slide method (10). The results in Table 6 show that the addition of rat IL-2 to B6 - AKR chimeric splenocyte cultures restored the PFC responses of B6 - AKR

TABLE 4

Plaque-Forming Cell Responses to SRBC by Chimeric Splenocytes (in Viva Sensitization)

Chimera 1 x 106” 5x lo5

CBA+ARR-1” 36w NT CBA - ARR-2 230 NT CBA - AKR-3 250 NT ARR-AKR-ld 65 25 AKR - AKR-2 375 245 AKR - AKR-3 765 265 B6-+AKR-lb 0 0 B6 - AKR-2 0 0 B6 4 AKR-3 0 0 B6 -+ AKR-4 0 0

a Number of spleen ceils cultured. b Anti-Thy- 1 .Ztreated marrow. ’ Number of plaques per culture. d Animal with thymoma.


TABLE 5

Typing Analysis and in Vifro SRBC Response of Long-Lived ( 170-280 Days Post-transplant) Chimeras

Chimera H-2b H-2’ Thy-l.1 Thy- 1.2 Plaques/ 1 06” CBA - AKR’ 5a.b 98 5 49 329 CBA - AKR 4 98 7 62 354 CBA + AKR 5 98 6 49 335 AKR-AKR 5 97 58 3 304 AKR-AKR 3 98 64 7 278 AKR-AKR 6 98 47 6 361 B6 + AKR’ 98 5 9 47 6 B6-AKR 98 7 5 45 0 B6-AKR 98 8 6 40 12

a SRBC response of whole splenocytes. b Percentage cytotoxicity. ’ Anti-Thy- I .2-treated marrow.

chimeras to SRBC. No plaques were formed in cultures of B6 - AKR chimeras with IL-2 but no SRBC added (data not shown). Our results show that the addition of IL-2 to chimeric splenocyte cultures corrected deficiencies of SRBC immunization usually present in the B6 - AKR chimeras.

In Vivo Efects of IL-2 Administration on the in Vitro Response to SRBC and the Production of IL-2 by Splenocytes of Normal and Long-Lived (170-280 Days Post- transplant) Chimeric Mice

B6 --t AKR chimeras were injected via tail vein with various doses of partially purified murine IL-2 48 hr prior to analysis. Splenocytes were then cultured with SRBC and the PFC response was measured on Day 5. IL-2 production was determined by analysis of IL-2 activity of supematants obtained from 24-hr Con A-stimulated splenocyte cultures assayed on HT-2 cells. Since the supply of IL-2 for injection was limited, responses of individual mice were evaluated. Our results of two experi-

TABLE 6

Effect of the Addition of IL-2 on the in Vitro SRBC Response of Normal and Long-Lived ( 170-280 Days Post-transplant) Chimeric Mice

Plaques/ 1 O6

Mice (-)IL-2 (+)IL-2”

B6 -+ AKRb 0 285 B6-AKR 0 321 B6-+AKR 0 336 B6 373 303 B6 398 345 B6 392 316

’ 50 units of IL2 added at beginning of culture. b Anti-Thy-l .2-treated marrow.


TABLE I

In Vivo Effects of IL-2 on the in Vitro Response to SRBC and the Production of IL-2 by Splenocytes of Normal and Long-Lived ( 170-280 Days Post-transplant) Chimeric Mice

Mice IL-2

(Units) Plaques/ 1 O6 IL-2 production

(Units)

Experiment 1 B6 - AKR“ B6-AKR B6-AKR B6-AKR B6 B6

Experiment 2 B6 + AKR” B6-AKR B6-+AKR B6 B6

1024’ 153 10.1’ 512 0 12.1 256 0 5.7

0 0 8.6 1024 399 19.1

0 496 20.0

1024 63 9.7 1024 67 13.0

0 0 7.6 1024 407 21.6

0 411 23.0

’ Anti-Thy-l .Ztreated B-6 marrow (B6 - AKR). b IL2 obtained from PMA-stimulated EL4.IG2 subline. Mice were injected iv 48 hr prior to sacrifice

and initiation of cultures. ‘Normal and chimeric splenocytes were stimulated with Con A and the 24-hr supematant was assayed

for IL-2 activity using the IGZdependent HT-2-tell line.

ments shown in Table 7 indicate that IL2 treatment of B6 - AKR chimeras resulted in a measurable but small restoration of their splenocyte SRBC responsiveness. IL-2 production tended to be higher in IL-Ztreated B6 - AKR chimeras; however, the level never reached that of normal B6 mice. IL-2 treatment of normal B6 mice had no demonstrable effect on the response of their splenocytes or their ability to produce IG2. These results suggest that in vivo IL-2 treatment oflong-lived B6 - AKR chimeras restored to a slight degree the deficiency in SRBC production.

In Vivo Eflects of IL-2 on the MLC Response of Splenocytes from Long-Lived (170- 280 Days Post-transplant) Chimeric Mice

B6 - AKR chimeras were injected via tail vein with various dosages of partially purified murine IL-2 48 hr prior to sacrifice. Responder chimer-k and normal splenocytes were cultured with irradiated stimulator splenocytes for 5 days. Results are expressed as stimulation indices. Table 8 shows that long-lived B6 - AKR chimeras retained ability to respond to cells of third party origin but not to host or donor-type lymphocyte antigens in MLC reactions. In vivo treatment of chimeras with IL-2 did not trigger or facilitate MLC responsiveness of the cells of the chimeras to donor or host lymphocytes; however, the responsiveness to third party (BALB/C) lymphocytes in the chimeras injected with a large dose of murine IL-2 appeared to be increased. These findings suggested that IL-2 given in vivo was effective in the enhancement of cellular elements that participate in MLC responses.


DISCUSSION

The present study confirms previous preliminary work from our laboratory which shows that leukemia in AKR mice can be prevented by allogeneic bone marrow transplantation when donor and recipient are H-2 compatible at the major histocompatibility complex and the donor is a strain resistant to the development of leukemia (CBA - AKR) (6). These chimeras appeared initially to be in excellent health with progressive weight gain and remained healthy upon being returned from a protected to a conventional environment. These long-lived chimeras were shown to possess normal splenic mitogenic activity to Con A or PHA as well as a normal capacity to produce interleukin 2. In addition, these allogeneic chimeras responded well with PFC responses to SRBC after either in vitro or in vivo stimulation.

Our experience with H-2-mismatched allogeneic chimeras was quite different. The lack of responsiveness of spleen cells in our long-lived chimeras to SRBC and normal proliferative responses to phytomitogens and in mixed leukocyte reactions has been previously reported by Onoe et al. (7) with similarly constructed chimeras. The latter investigations also demonstrated that splenocytes from B6 - AKR chimeras prolifer- ated well when stimulated with Con A, PHA, or lipopolysacharide and were completely normal in natural killer and antibody-dependent cell-mediated cytotoxicity. However, those observations were made using chimeras which had lived for only approximately 100 days post-transplant.

The studies reported herein utilized allogeneic B6 - AKR chimeras which were observed for up to 280 days post-transplant. While we (4-7) and others ( 14- 16) have reported good survival of fully allogeneic radiation chimeras, the observation period in the former studies was too short. Our studies and those of Rayfield and Brent (13) point to the importance of time in the evaluation of long-term survival and the possibility that a form of secondary disease regularly develops in fully allogeneic chimeras kept for longer periods. Our results show that although leukemia is prevented by fully allogeneic bone marrow transplantation, the clinical usefulness of

TABLE 8

In Vivo Effects of IL-2 on the MLC Response of Splenocytes from Long-Lived (170-280 Days Post-transplant) Chimeric Mice

Responder IL-2’

(Units) B6

Stimulator

AKR BALB/C

B6 - AKRb 1024 1.7b.c 2.0 19.1 B6-AKR 512 1.8 2.5 10.5 B6-+AKR 256 2.0 2.1 13.9 B6-AKR 0 2.5 2.6 12.8 B6 0 1.0 4.6 4.4 AKR 0 11.6 1.0 6.1 BALB/C 0 17.5 10.5 1.0

’ IL2 obtained from PMA-stimulated EG4.IL-2 subline. Mice were injected iv 48 hr prior to initiation of MLC cultures.

b Anti-Thy-l .2-treated B6 marrow (B6 - AKR). ’ Stimulation index.


this approach in the treatment of leukemia may be negated by this strange wasting syndrome, immunoincompetence, and by immunodeficiency in primary antibody responses. Although we could not demonstrate overt signs of bacterial, fungal, proto- zoal, or viral infection in the wasting B6 - AKR chimeras, the possibility exists that these animals died of infectious complications of a more subtle nature. Zinkemagel ( 17) postulated that the failure of fully allogeneic chimeras to resist bacterial or viral infections is related to the inability of immunological cells to cooperate with each other in the bodily defense. Our findings at the present time do not speak directly to this issue.

Our studies demonstrate that the addition of partially purified interleukin 2 to splenocyte cultures of B6 - AKR chimeras resulted in impressive restoration of the in vitro PFC response to SRBC. They suggest that the necessary cellular elements may be present, but the cooperative signals such as those provided by interleukin 2 are deficient. Because the IL-2 preparations were only partially purified, we cannot determine from the experiments carried out whether these factors are replacing T- cell help (TRF) or whether they are augmenting T-helper functions by the cells of the chimeras. However, TRF generally must be added later in the culture period for positive effects rather than what was done here. Previous studies of Coico et al. (5) demonstrated that in a similar mismatched chimeric combination (B6 - CBA), failure to produce antibody to SRBC was not the result of impaired B-cell, T-cell, or macro- phage function, but rather ineffective cellular interaction between these cells or between some these cells. They reported that stimulation of chimeric macrophages with LPS or chimera spleen cells with Con A resulted in the release of soluble helper factors that restored in vitro responses of isolated B lymphocytes. Furthermore, studies by Onoe et al. (7) demonstrated that adherent cells of recipient MHC type in which matches involved IA and IE compatability served to correct these functional defects (18, 19).

In the present study, we did not use isolated B lymphocytes of the spleen to measure response to SRBC but still restored chimeric splenocyte response to SRBC. In addition, we report that B6 - AKR chimeras had normal proportions of T-cell subpopu- lations defined by Lyt antisera, but the cells produced subnormal amounts of IL-2 as compared to normal B6 splenocytes. These results suggest that the failure of long- lived B6 - AKR chimeras to respond in vivo and in vitro to SRBC may be correlated to inefficient IL-2 production which in turn might be consequent to deficient interactions of T lymphocytes with adherent cells.

The rationale for the use of IL-2 in vivo in efforts to restore antibody responsiveness of B6 - AKR chimeras to SRBC is based on our in vitro studies described above as well as prior work by Onoe et al. (7). These investigators found that B6 - AKR chimeras respond to a secondary challenge with SRBC in vivo.

Our preliminary experiments indicated that IL-2 given in vivo could partially restore the in vitro response to SRBC in B6 - AKR chimeras. Other experiments using IL-2 in vivo followed by in vivo challenge of SRBC were not successful. An interesting observation in IL-Ztreated chimeras was the increase in responsiveness of their splenocytes in MLR to third party but not to host or donor-type lymphocytes. Jadus and Peck (20) reported that 10 units of IL-2 given to (parental - F,) bone marrow chimeras on Days 1, 3, and 5 post-transplantation resulted in an increase in the incidence and severity of GVHD. Their results coupled with our observations emphasize that timing of the administration of IL-2 to immunodeficient chimeras may be crucial.


Such a manipulation might ultimately allow reconstitution of primary antibody responses but without triggering GVHD during the initial 60 days following bone marrow transplantation.

In summary, our studies establish that leukemia in AISR mice can regularly be prevented by allogeneic H-Zcompatible marrow transplantation. Long-lived ( 170- 280 days post-transplant) H-2-mismatched, allogeneic B6 - AKR chimeras were also histologically free of leukemia and overt evidence of acute GVHD but died of a still incompletely unanalyzed secondary disease which indeed might represent an unusual manifestation of very low grade GVHD. This possibility is supported by more recent studies from our laboratory which showed that in some strain combinations where donor and recipient differ across the MHC, pretolerizing the prospective donor to prospective recipient strain, eliminate this late wasting syndrome (21). These chimeras demonstrated low responsiveness of spleen cells to Con A and low IL-2 production. The defective antibody of B6 - AKR chimeras to SRBC was corrected in vitro and to some extent in vivo by administration of partially purified IL-2.

Our study has important implications for the use of allogeneic bone marrow transplantation in the treatment of disease. If fully allogeneic bone marrow transplantation is to be successful, these findings suggest that cellular interactions must take place via interleukins to permit full immunologic reconstitution. A recent report by Azogui et al. (22) showed that in patients that had undergone HLA-matched bone marrow transplantation, IL-2 production was either absent or low in 32 of 34 patients analyzed for at least 2 years following bone marrow transplantation. The latter study and an additional report by Warren et al. (23), which indicated that there was long-lasting defective IL-2 production even in HLA-matched bone marrow transplanted patients, strongly suggest that IL-2 may be useful in this situation, and our results suggest it might play an important role as adjunctive therapy in HLA-mismatched marrow transplantation. However, until the problem of GVHD in human bone marrow transplantations is completely resolved, the use of IL-2 in bone marrow transplantation will have to be approached with caution. Further studies from our laboratories will be designed to determine at what time interval following bone marrow transplantation in MHC chimeras the deficiency in IL-2 production occurs and when following bone marrow transplantation IL-2 must be administered in vivo to restore primary antibody responses to SRBC, but without inducing GVHD.

REFERENCES

1. Thomas, E. D., Buckner, C. D., Cliff, R. A., Fefer, A., Johnson, F. L., Neiman, P. E., Sale, G. E., Sanders, J. E., Singer, J. W., Shulman, H., Storb, R., and Weiden, P. L., N. Eng. JMed. 301,591, 1979.

2. Hansen, J. A.., Woodruff, J. M., and Good, R. A., In “Immunodermatology” (B. Safai, and R. A. Good, Eds.), Vol. 7, p. 229. Plenum, New York, 198 1.

3. Shulman, H. M., Sullivan, K. M., Weiden, P. L., McDonald, G. B., Striker, G. E., Sale, G. E., Hack- man, R., Tsoi, M., Storb, R., and Thomas, E. D., Amer. J. Med. 69,204, 1980.

4. Krown, S. E., Coico, R., Scheid, M. P., Femandes, G., and Good, R. A., Clin. Exp. Immunol. Immuno- pathol. 19,268, 1981.

5. Coico, R. F., Krown, S. E., Good, R. A., and Hoffman, M. K., J. Zmmunol. 128, 1590, 1982. 6. Tanaka, T., Obata, Y., Fernandes, G., Onoe, K., Stockert, E., and Good, R. A., Proc. Amer. Assoc.

Cancer Res. 20,115, 1979. 7. Onoe, K., Femandes, G., and Good, R. A., J. Exp. Med. 151,115, 1980. 8. Iwabuchi, K., Onoe, K., Ogasawara, M., Yasumizu, R., and Morikawa, K., J. Hokkaido Med. Sci. 58,

232,1983.


9. Terasaki, P. I., and McClelland, J. D., Nature (London) 204,998, 1964. 10. Cunningham, A. J., and &e&erg, A., Immunology 17,599,1968. 11. Mishell, R. I., and D&ton, R. W., .Z. Exp. Med. 126,423, 1967. 12. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A., .Z Zmmunol. 120,2027, 1978. 13. Rayheld, L. S., and Brent, L., Transplantation 3621, 183, 1973. 14. Pollard, M., and Truitt, R. L., Proc. Sot. Exp. Biol. Med. 144,659, 1973. 15. Truitt, R. L., In “The Handbook of Cancer Immunology” (H. Waters, Ed.), Vol. 5, p. 432. Garland

STPM Press, New York, 1982. 16. Vallera, D. A., Soderling, C. C. B., Carlson, G. J., and Kersey, J. H., Transplantation 31,2 18, 198 1. 17. Zinkemagel, R. M., Zmmunol. Rev. 42,224, 1978. 18. Onoe, K., Yasumizu, R., Geng, L., Iwabuchi, K., Ogasawara, M., Kakinuma, M., Okuyoma, H.,

Good, R. A., and Morikawa, K., Zmmunobiology 169,7 1,1985. 19. Onoe, K., Yasumizu, R., Noguchi, M., Iwabuchi, K., Ogasawara, M., Kakinuma, M., Okuyoma, H.,

Good, R. A., and Morikawa, K., Zmmunobiology 169,70,1985. 20. Jadus, M. R., and Peck, A. B., Transplantation 36(3), 281, 1983. 21. Himeno, K., and Good, R. A., Fed. Proc. 44(4), 958, 1985. 22. Azogui, O., Gluckman, E., and Fradelizi, D. F., J. Zmmunol. 131, 1205, 1983. 23. Warren, H. S., Atkinson, K., Pembrey, R. F., and B&s, J. C., Immunology 131(4), 1771, 1983.

Date post:	18-Nov-2023
Category:	Documents
Upload:	independent
View:	0 times
Download:	0 times

Leukemia prevention and long-term survival of AKR mice transplanted with MHC-matched or...

Documents