lymphocyte Mitogen Reactivity and Enumeration of Circulating B- and T-Cells During Feline leukemia Virus Infection in the Cat 1, 2, 3

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1 lymphocyte Mitogen Reactivity and Enmeration of Circlating B- and T-Cells Dring Feline lekemia Virs Infection in the Cat 1, 2, 3 G. L. Cockerell, E. A. Hoover, S. Krakowka, R. G. Olsen, and D. S. Yohn 4 ABSTRACT-Mitogen-indced blast transformation of peripheral blood lymphocytes and qantitative changes in circlating T- and B-cells were stdied serially in cats inoclated with feline lekemia virs (FeLV). Concanavalin A-indced blast transformation sharply declined beginning at 5 weeks post inoclation (PI) in FeLV-infected cats when compared to age-matched ninfected control cats. Similar bt less consistent changes were seen in responses to pokeweed mitogen-indced stimlation. In most infected kittens this defect persisted ntil they died from thymic lymphosarcoma, weeks PI. An early lymphopenia, de primarily to a decrease in circlating B-cells, occrred in infected cats 5-8 weeks PI. Following a retrn of total and B-Iymphocytes to control vales, infected cats developed increased nmbers of T-cells at 16 or more weeks PI, which correlated with circlating lymphoblastic lymphocytes bearing T-cell markers. These reslts correlated neoplasia arising in a thyms-derived 'lymphocyte poplation with mitogenic hyporeactivity in the preneoplastic period and sggested that FeLV-indced immne alterations may be a necessary antecedent of lekemogenesis in the cat.-j Natl Cancer Inst57: 5-, 176. The observation by Old et al. (1) in 160 that infection of mice with Friend lekemia virs interfered with the prodction of antibodies against sheep red blood cells sparked a series ofinvestigations aimed at the evalation of the inflence of oncogenic virses on immne mechanisms [reviewed in (2)]. Nmeros investigators have now demonstrated that immnodepression in both birds and mammals is a conseqence of infection with oncogenic virses. Striking analoges of impaired immnologic competence exist in spontaneos hman neoplastic diseases, althogh a viral etiology remains nproved (3-5). It has been proposed that the impairment of normal immne fnction is necessary for the srvival and proliferation of neoplastic cells (2, 6-8). Feline lekemia (lymphosarcoma), a horizontally transmitted, virs-indced lymphoproliferative disorder, is the most common neoplastic disease ofthe cat ( 11). The disease occrs spontaneosly or can be prodced by inoclation with FcLV. Thymic atrophy (12, 13), paracorticallymphoid depletion (12), a rnting syndrome (12, 13), increased ssceptibility to intercrrent infections (12), and prolonged ctaneos allograft rejection (14) have been observed in prelekemic Fe LVinfected cats, which sggests that altered immne fnction is an important component of lekemogenesis in the cat. Recently developed procedres for measrement of LBT as adapted to feline PBL (15), and identification of thyms-dependent (T) and -independent (B) lymphocytes in the cat (16), now permit frther evalation of the immnologic abnormalities that occr dring lekemogenesis in the cat. We report here reslts of serial stdies of mitogen-indced blast transformation of PBL and qantitative changes in circlating T - and B cells in kittens inoclated with FeLV. MATERIALS AND METHODS Cats and lymphosarcoma indction.-we sed 68 specific-pathogen-free cats from a closed breeding colony of gnotobiotic ancestry (17). Thirty-for newborn kittens were inoclated ip with 5 focs-forming nits of FeLV-R (). This inoclm prodces a greater than 5% incidence of lymphosarcoma with a latent period of weeks (13). Infected kittens were tested for in vitro mitogen reactivity at intervals from 1 to 24 weeks PI and compared with 34 ninfected control kittens of different ages. Ten infected cats and 8 age-matched ninfected controls were tested serially for total, B-, and T -lymphocytes. Lymphocyte preparation.-pbl were isolated by centrifgation of defibrinated blood on a Ficoll-Hypaqe gradient as previosly described (15). The final concentration was adjsted to 1x 6 cells/rnl in complete medim, which consisted of Eagle's minimm essential medim for sspension cltres spplemented with 20% heatinactivated fetal calf serm, 2% of 1 M NaHC0 3, and 1% penicillin (2x 6 U/ml) and streptomycin (1 g/loo ml). LBT assay.-this assay was detailed in (15). We incbated 0.1 ml of the PBL sspension (1 x 6 cells/ml) together with an eqal volme of complete medim containing either {-kg Con A (Sigma Chemical Co., St. Lois, Mo.) or 4 {-kg PWM (Grand Island Biological Co., Grand Island, N.Y.) in MicroTest plates (Falcon Plastics, Oxnard, Calif.) for 72 hors. Control cells were incbated only with complete medim. Dring the final 18 hors of incbation, 0.5 {-kci of[ 3H]thymidine (6.7 Ci/ mmole; New England Nclear Corp., Boston, Mass.) was added. We terminated the cltres by harvesting cells onto glass fiber paper with a semiatomatic mltiple sample processor (Otto Hiller Co., Madison, Wis.) and conted them in a liqid scintillation spectrometer. Net cpm of qadrplicate wells were averaged to obtain the mean cpm. We calclated SR by dividing mean cpm of mitogen-stimlated cells by mean cpm of control cells. ABBREVIATIONS USED: FeLV=feline lekemia virs; LBT=lymphocyte blast transformation; PBL=peripheral blood lymphocytes; Fe LV R=Rickard strain of FeLV; PI=post inoclation; Con A=concanavalin A; PWM=pokeweed mitogen; cpm=conts per minte; SR= stimlation ratios; PHA=phytohemaggltinin; LPS=lipopolysaccharide; MLV-G=Gross mrine lekemia virs; FOCMA=feline oncornavirs-associated cell membrane antigen. j Received December 1, 175; accepted April, Spported by Pblic Health Service (PHS) contract NOI CP53571 from the Division of Cancer Case and Prevention, National Cancer Institte (NCI), and by PHS grant CA from the NCI. 3 A preliminary report of this work was presented at the Seventh International Symposim on Comparative Research on Lekemia and Related Diseases, Copenhagen, Denmark, October 13-17, Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, 125 Coffey Rd., Colmbs, Ohio 432, VOL. 57, NO.5, NOVEMBER J NATL CANCER INST

2 6 COCKERELL ET AL. Identification oft- and B-cells.-Techniqes for identification of feline T- and B-cells were described in (16). T-cells were identified by their formation of spontaneos rosettes with ginea pig erythrocytes (E-rosettes). B-cells were identified by the presence of a complement receptor, which was detected by formation of rosettes with sheep erythrocytes coated with canine IgM antibody against sheep erythrocytes and complement (EACrosettes). Total and differential lekocyte conts were made on whole venos blood. RESULTS '"' I : '"' LBT The response of PBL from FeLV-inoclated kittens to Con A is shown in text-figre 1. Beginning at 5 weeks PI, a precipitos decline in Con A-indced blast transformation occrred in infected kittens as compared with age-matched ninfected controls. In most kittens this defect persisted ntil they died from thymic lymphosarcoma between 15 and 24 weeks of age. Text-figre 2 depicts the response of these same cats after stimlation with PWM. A similar depression in PWM reactivity was noted beginning at 5 weeks PI, bt it was not as marked or persistent as was the response to Con A. Morphologically distingishable circlating lymphoblastic cells were present in most cats sampled after 7 weeks PI (see below). In 7% of the samples, circlating lymphoblasts were associated with increased basal ptake of [3H]thymidine in PBL incbated in medim withot mitogen (text-figs. 1, 2). In some instances mitogen stimlation, measred by cpm, was eqal to or greater than stimlation in ninfected control kittens (table 1). However, the increased basal ptake of [3H]thymidine reslted in SR far below those seen in control kittens. Regardless of whether blast cells cased increased basal [3H]thymidine ptake, they were consistently associated with (althogh not a prereqisite for) decreased mitogen reactivity I6 o TEXT-FIGURE WEEKS OF AGE (WEEKS PJ) I.-Con A-indced blast transformation of PBL from FeLV-infected cats (e--e) and age-matched ninfected control cats (e-----e). For 72 hr, I X]05 cells were incbated with ]0 /Lg Con A; dring the last 18 hr of incbation, 0.5 /LCi [3H]thymidine was added. Each point v-x x s«. Hatched area represents ±l SE of control cats. Nmbers adjacent to points=nmber of cats tested. 04 o To determine if PBL from Fe LV-infected cats might respond better to different concentrations of mitogens, dose-response experiments were performed dring the period of mitogenic hyporeactivity. Neither one-forth nor for times the optimal concentration of Con A (2.5 f.lg and 40 f.lg, respectively) or PWM (1 f.lg and 16 g, respectively) improved blast transformation. Frthermore, we fond no difference between control and infected cats in the percent of viable PBL (as determined by trypan ble dye exclsion) after 72 hors' incbation with optimal concentrations of Con A and PWM; this indicated that PBL from infected cats were not ndly ssceptible to toxic effects of the mitogens. Circlating Total, B-, and T-Lymphocytes Text-figre 3 illstrates reslts obtained when PBL from FeLV-infected cats were assayed seqentially for absolte nmbers of total, E-, and EAC-forming lymphocytes from 5 to 24 weeks PI. An early lymphopenia seen in infected cats 5-8 weeks PI was de mainly to a decrease in circlating B-cells. Following a retrn of total and B-Iymphocytes to control vales, slight bt consistently elevated nmbers of T-cells were fond in infected cats from 16 to 24 weeks PI; this correlated with the presence of circlating lymphoblastic E-rosetteforming cells which were particlarly nmeros in some cats dring this period. Blast forms cold be fond in small percentages «5%) as early as 7 weeks PI and appeared in 60% of the FeLV-inoclated cats sometime dring the infection. DISCUSSION WE EKS OF AGE (WEEKS PI) TEXT-FIGURE 2.-PWM-indced blast transformation of PBL from FeLV-infected cats (e--e) and age-matched ninfected control cats (e-----e). For 72 hr, I X]05 cells were incbated with 4 /Lg PWM; dring the last 18 hr of incbation, 0.5 /LCi [3H]thymidine was added. Each point=x±se. Hatched area represents ± I SE of control cats. Nmbers adjacent to points=nmber of cats tested. A widely sed measre of celllar immnocompetence is the degree to which mitogens case lymphocytes to ndergo immnologically nonspecific, polyclonal blast transformation (3, 18, 1). Moreover, mitogens have been shown to stimlate particlar cell types. For J NATL CANCER INST VOL. 57, NO.5, NOVEMBER 176

3 IMMUNODEPRESSION DURING LEUKEMOGENESIS IN THE CAT 7 TABLE I.-Effect ofcirclating lymphoblasts on basal and mitogen-stimlated reactivity ofpbl from FeLV-infected kittens Percent circlat- Kittens tested Wk, PI ing lymphoblasts" Mean cpm and SR after incbation of 1x 5 cells for 72 hr b with Medim alone cpm Medim + /Lg Con A Medim + 4 /Lg PWM cpm SRc cpm SRc Control ninfected" , FeLV-infected: # , # , , # , #3R , a Morphologically distingishable lymphoblastic lymphocytes bearing T-cell markers. b Dring final 18 hr of incbation, 0.5 /LCi tritiated thymidine was added. cpm of mitogen-stimlated cells c SR=---=-,-----::-:---'=--.,, :---- cpm of cells incbated with medim only d We tested 6 ninfected kittens. example, investigators sing hman or mrine lymphoid cells have reported that PHA and Con A selectively stimlate T-cells (20-23) and LPS stimlates solely B cells (21,24,25), while PWM is mitogenic for B- as well as T-lymphocytes (20, 22). However, definitive stdies have not been performed to determine the tropism of Con A and PWM for feline lymphocytes. LPS was not sed in the present stdy becase it is not mitogenic for feline PBL (Cockerell GL: Unpblished observations). However, extrapolations from the reported behavior of other species pls or mitogen data sggest to s that FeLV-R affects T-cell fnction more than it does B-cell fnction. This is consistent with previos observations of delayed ctaneos allograft rejection with concomitant retention of nearly normal hemaggltinating (lgm) antibody response to sheep red blood cells in FeLV-Rinfected kittens (14). Moreover, the characterization of the FeLV-R-transformed cell as a T-cell (16) sggests that this cell type might even be the target for the virs. Therefore, as Perryman et al. (14) sggested, FeLV-R infection in cats may be analogos to MLV-G infection in mice. MLV-G-infected mice have decreased ability to reject homografts (2, 26) and decreased response of splenic lymphocytes to PHA (2). Neonatal thymectomy in sch mice redces the incidence of lekemia (27). In this system, the thyms is the target organ for neoplastic transformation by the virs. Evidence of the effect of FeLV on B-cell fnction is the early depletion in circlating B-cells, which is the main contribtor to the lymphopenia seen in infected cats between 5 and 8 weeks PI. A comparable lymphopenia was reported in healthy cats infected with FeLV that showed an increased risk for disease development (28). This early B-cell depletion may be associated with decreased prodction of antibody directed toward FOCMA, the absence of which was shown to correlate with increased disease incidence or severity (2-31). Alternatively, the B-cell depletion may be coincidental and the reported decrease in FOCMA antibody titer de to defective helper T-cell fnction. The mechanism of depressed mitogen-indced LBT remains nresolved. This change was probably not de to a redistribtion of peripheral lymphoid cells, since it occrred in the presence of normal nmbers of circlat ' <l: ("l 4 $2 x 3 X / v» -- >- 0J: 2 c, ::E ~... 0 ij 0 -.t._ l--- r - _-1 z 5 4 <l: 3 2, _.-. i i i i I I i I I I I I I i I i I i WEEKS OF AGE (WEEKS PI) TEXT-FIGURE 3.-Total, E-, and EAC-forming lymphocytes of FeLVinfected cats (e--e) and age-matched ninfected control cats (e-----e). Each point=x±se of 8 control cats or infected cats. ing total, B-, and T-Iymphocytes. Similarly, the immnodepression cold not be de to the presence of nonmitogen-responsive lekemic lymphoblasts. Althogh we detected circlating lymphoblasts bearing T-cell markers in some kittens as early as 7 weeks PI, they did not increase the T-cell poplation ntil late in the disease. In some instances, the presence of blast forms was associated with increased basal ptake of [3H]thymidine; VOL. 57, NO.5, NOVEMBER 176 J NATL CANCER INST

4 8 COCKERELL ET AL. invariably these cells were associated with decreased mitogen reactivity. However, other kittens tested at the same time had no lekemic Iymphoblasts and had normal basal [3H]thymidine ptake; yet they were hyporeactive to mitogen stimlation. The onset of mitogen hyporeactivity correlated well with the time of initial detection of FeLV grop-specific antigen in circlating lekocytes (2). Hence the observed immnodepression is most likely de to a direct viral effect on lymphocyte fnction, which makes the lymphocytes nonresponsive to mitogens whether or not they are yet transformed. Similar reasoning has been postlated to explain the depression in lymphocyte response to mitogens in owl monkeys infected with Herpesvirs saimiri (32). Other mechanisms that might explain mitogen hyporeactivity inclde circlating immnosppressive factors or the activation of sbpoplations of cells with sppressor fnctions. Serm-blocking factors that can selectively inhibit the cytotoxicity of immne lymphocytes in vitro have been noted in tmor-bearing animals (33). Glasgow et al. (34) and Nimberg et al. (35) demonstrated that cancer patients may have abnormally high serm levels of an immnoreglatory peptide fraction capable of inhibiting PHA-indced activation of normal lymphocytes. Investigators who sed the Moloney sarcoma virs system have fond sppressor cells in spleens of tmor-bearing mice that cold inhibit PHAindced blast transformation of normal syngeneic spleen cells (36, 37) as well as DNA synthesis of cltred mrine lymphoma cells (38). Recently, both hmoral and celllar sppressive activities have been demonstrated in rats bearing progressively growing MLV-Gindced tmors (3). Similar stdies in Fe LV-infected cats indicate that sera or spleen cells from tmor-bearing cats can inhibit Con A-indced blast transformation of normal feline PBL (Cockerell GL, Hoover EA: In preparation; Cerney J: In preparation). The significance of these findings is the sbject of crrent investigation. In this stdy we have demonstrated another effect of FeLV on feline immne fnction by correlating the neoplasia of a thyms-derived lymphocyte poplation with mitogenic hyporeactivity in the preneoplastic period. Frther work is necessary to establish the precise mechanism involved and its relationship to lekemogenesis. Or reslts sggest that virs-indced depression of host immne protective mechanisms may be a necessary antecedent for the expression of the oncogenic potential of FeLV. REFERENCES (1) OLD LG, CLARKE DA, BENACERRAF B, et al: The reticloendothelial system and the neoplastic process. Ann NY Acad Sci 88: , 160 (2) DENT PB: 1mmnodepression by oncogenic virses. 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