B2 chickens

CEJ,J,UJ..AR

38, 350-364

IhlhlliR‘C,J.OGY

Immunity

(197s)

to Transplantable

Carcinogen

Induced

Fibrosarcomas

in B2/B2 Chickens1 II. Macrophage

Dependency

MICHAEL

Department

A.

of Adoptive

PALLADINO

3 AND

T Cell Mediated G.

JEANETTE

Tumor

Immunity*

THORBECKE

of Pathology, New York Ulziversity School of Medicine, 550 First Avenue, New York, New York 10016 Received

February

22, 1978

The nature of immunity to transplantable chemically induced fibrosarcomas in SC (BZ/BZ) chicken was examined using Winn tests performed in the wing web. Immunity in spleen cell donors was induced by pretreatment with C. parvum or BCG followed by tumor cells + bacterial adjuvant in one and tumor cells alone in the other wing web. The T cells mediating the adoptive immunity were sensitive to anti-T + C, nylon wool nonadherent, mitomycin resistant and radiation (1000 R) sensitive. The adoptive immunity could not be expressed in heavily irradiated recipients or in hosts pretreated with trypan blue or silica. The host contribution could be reconstituted by iv injection of spleen or bone marrow cells from agamma-globulinemic (AT) unimmunized donors 2 days prior to Win11 tests or by the local injection into wing webs of spleen cells or purified peripheral blood monocytes from Ay donors. It was concluded that cooperation between immune donor T cells and normal monocytes of host origin mediated the inhibition of SCFS growth in Winn tests.

The introduction of fibrosarcomas in B2/B2 histocompatible SC chickens was described previously (1). The transplantable lines, SCFS I-III, which were derived from such tumors, lacked demonstrable tumor virus products and maintained genetic markers upon transplantation (1). Tumor-line-specific immunity was induced in both normal and agammaglobulinemic ( Ay ), bursectomized chickens with the aid of bacterial adjuvants (2). Adoptive immunity to SCFS could be demonstrated in Winn tests performed in the wing web. The cellular basis of this adoptive immunity is the subject of the present study. Since spleen cells from tumor immune AY donors were able to transfer immunity, T cells were most likely involved. However, two major types of tumor-specific T-cell mediated immunities are known: (1) cytotoxic T cells, and (2) T cells responsible for delayed hypersensitivity (DH) reactions, which 1 This work was supported by Grant CA16247 and Contract NO1 CB 64043 from the National Institutes of Health. 2 In partial fulfilment of doctoral thesis requirements. 3 Fellow under U.S. Public Health Service Training Grant STOl-GM00127.

OOOS-8749/78/0382-0350$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

iI1

~IIc

111011.W

UC'

hJ\\'ll

(0

I~'h~lg

11, ;L tlif'fcwllt

'1'

cC'Il

Xdldkdi

(?I).

Ii

C\~~dtl\iC.

‘1’ cells \vel-c reslmisible, they nw~ld lx exl~tccl to kill tumor target cells~dircctl~~ in the Winn tests. If, however, DH T cells were mediating the specific immunity, they would be likely to require interaction with monocgtes for their inhibitory effect on tumor growth. The elegant series of experiments by Mackaness (4), Volkmau and Collins (5)) and Tuttle and North (6) has deuloustrated that interaction between antigen and specific inmune T cells leads to nonspecifically activated macrophages with strong bacteriocidal and tumoricidal activity. Activation of macrophages renders them cytostatic (7) or cytolytic (8, 9) to tumor cells. This can also be accomplished b!r agents mimicking the effect of T cells, such as bacterial products, which cau exert a direct effect on macrophages even in thymusless mice (10). It has been suggested that cytotoxic macrophages can distinguish between normal and tumor cells (9, 11)) although activated macrophages are also known to be cytostatic for PHA responsive T lymphocytes (12). The need for T cell-macrophage cooperation was shown in the transfer of resistance to I,. ~umcyfogems (13) and in adoptive immunity to some transplantable tumors (14, 15) in mice. It will be shown in the following that synergy between immune T cells and recipient macrophages is also required for the expression of adoptive immunity to SCFS in the chicken. hlATEKIALS

AND

RlE’I’HOL~S

ZInirmls a& i~~mu&~tions. SC (B2/B2) chick embryos were obtained from Hj line Poultry Farms (Dallas Center, Iowa). IIormonal bursectomy was performed on Day 11 of embryonation by injection ot 3.75 mg testosterone propionate (Elkins Sinn, Cherry Hill, N. J.) . After hatching, these chicks received cpclophosphamide (Cytoxan, Meade Johnson, Evansville, Idiana) on Ihys 1 (4 itlg), 2 (3 rug) ? and 3 (3 nirr) b , ip, and at 8 weeks of age their sera lvere analyzed for the presence of IgbI and IgG by double diffusion in agar (16). .!‘lnimals that failed to show precipitin lines (< 2% of normal Ig levels) Ivere designated -4~. Irradiation of either recipient chickens or of spleen cells itz vitro was 1)~ a laiCs y-irradiation source (Gammator M, Radiation Machinery Corp., Parsippanr;, N. J.) at 630 Rads/min. Chickens were immunized to SCFS tumor lines by injection of 10F SC‘FS into each wing web, combined on one side with either 10” killed C‘ol-ll)lc,l’crcfrrjlrl!i ~arz~zmz (C. @~mm, Burroughs ~$‘ellcome, Durham, N.C., now designated as Propio~~ibacfcrilllz acnes) or 2 x lo7 live Bacillus Calnlette Guerin (IKX;, TMClOll, Trudeau Institute, Saranac Lake, N.Y.). Such animals had been pretreated iv with the corresponding bacterial antigen 1 week previously. These animals regressed their tumors after several weeks. Lyrlplzoid cdl preparations. Spleens were removed and gently teased {\-it11 forceps in modified Dulbecco’s phosphate buffered saline (DI’BS, pH 7.2, 0.165 M). After filtration through gauze all cells were washed three times, counted and resuspended to the desired concentration in DPES. Nonadherent and adherent spleen cell populations were separated on nylon wool co1u111ns (Fenwal I,aboratories, Morton Grove, Ill.), according to the method of Julius ct al. (17), exceljt that cells were in RPM1 1640 (Associated Biomedic Systems, Huffalo, K.Y.) with 2% normal chicken serum. Previous experiments (unpublished observations 1

352

PALLADINO

AND

TIIORRECKE

have showli tllat ikyloii wool nonadherent chicken sljleen cells 1)rolifcratc ~II resl)unsc to pllytollenlagglutiiiiii and to cuncauavalin A, while adherent cells du nut. Bone marrow cells were obtained from the tibiae and femurs of 6- to lo-week old Ay chickens by flushing the diaphysis with DPBS. Peripheral blood monocytes were obtained by centrifugation of whole blood layered on ficoll-hypaque (specific gravity : 1.090). The mononuclear cells were washed three times with DPBS and incubated 15 X 100 mm petri dishes (2x 107/ dish) for 24 hr. Th e nonadherent cells were then removed by rinsing with warm media. The remaining cells were further incubated for 4 more days at which time the adherent monocytes were removed by treatment for 10 min with 5 m?/l EDTA in DPBS at room temperature. For some experiments, spleen cells (2 x 10r/ml) were incubated with rabbit anti-thymus serum (1: 90) and guinea pig C (1 : 18) for 45 min at 37°C. Control cells were treated with normal rabbit serum and C. The anti-thymus serum + C kills 60% of spleen cells, > 95% of thymus cells, and < 10% of bursa cells from 4-week old chickens as described previously (18). Mitomycin treatment (ICN Pharmaceuticals Inc., Life Sciences Group, Cleveland, Ohio) was performed at 100 pg and 10’ cells per ml for 45 min at 37°C. Macrophage inactivating agents. Silica particles of an average size distribution of 5 p were obtained through the courtesy of Dr. K. Robach, Steinkohlenberg Bouverein, Germany. It was injected iv in doses of 3 mg. Trypan blue, obtained from Allied Chemical and Dye Corp. (New York, N.Y.), was dissolved (10 mg/ml) in and dialyzed against saline for 24 hr at 4°C (19). It was injected iv 5 mg/dose. Poly 2 vinyl pyridine-N-oxide (PVNO, Polysciences, Inc., Warrington, Pa.) was given at a dose of 150 mg/kg subcutaneously 24 hours before silica (20,21). Cortisone acetate (Cortone, Merck Sharp and Dohme, West Point, Pa.) was injected im, three daily doses of 15 mg. Tumor cells. Minced tumor tissue was placed in 0.25% collagenase (Worthington Biochemicals,, Freehold, N.J. ) f or 15 min at 37°C with constant stirring. This was followed by incubation for 15 min with 0.5% trypsin (ICN). The resulting cell suspensions were filtered through gauze and washed in DPBS. Wirrn tests. Tumor immunity was assayed by the Winn test (22). Usually mixtures of 10Gtumor cells and 5 to 200 x 10” lymphoid cells were injected into wing webs of 2- to 4-week old recipients. Tumor growth was measured for at least two weeks using a metric vernier caliper and tumor sizes were recorded in cm in all tables. Statistical analyzes were performed using the combined Student’s t test for tumor sizes and chi square for tumor incidences. RESULTS Nature of iwltune spleen cells mediating adoptive tumor imnmnity. Spleen cells from chickens that had rejected wing web injections of lo6 SCFSI cells after pretreatment with bacterial adjuvant and tumor cells as indicated under Methods were able to inhibit growth of 10F SCSFI ceils in Winn test recipients. This was also shown previously as was the specificity of this immunity with respect to the SCFS line inducing the immunity (2). Immunity to SCFS lines II and III could not be shown with spleen cells immune to SCFSI. Neither did cells from chickens pretreated with either BCG or C. parvum alone transfer any detectable immunity to

‘I‘AI
Expt. NO.

1

Pretreatment* of donor cells

2

NRS + c Ii anti-T + C I-nfractionated N.Lt:. nonadherent N.W. adherent

3

Irnfractionated .U\V nonadherent K\i- adherent

1

Spleen to tumor cell ratio

20 20 10 10 10 0 5 20 5

20 S 20 0

Tumor

growth

Size f SE (cm )

N.1). N.Ij. 0.12 f 0.04 0.15 f 0.06” 0.63 f 0.08 0.66 f 0.02” 0.08 f 0.01 0.05 f 0.0s 0.07 f 0.07” 0.00 0.93 f 0.08 0.89 f 0.05 0.83 f O.OSh -___~

Tumors present/total test sites 1, 8h 8, 8” 4 ‘8 4, 8 IO;‘10 6,6 2, 6 1.6 1 ‘5 0, S $1 *,!-I s/s

a Expt. 1: Immune spleen cells were incubated at lo7 cellsjml with 1: 7.5 NRS or R anti-chicken T cells and 1: 18 guinea pig C. Expts. 2 and 3: Nylon wool (NW) fractionation: 45 min at 37 Y’ incubation on column. *P < .OOl.

SCFSI (2). The results in Table 1 allowed further characterization of the immune spleen cells responsible for the adoptive immunity. Pretreatment with rabbit anti-T and C abolished the immunity, and fractionation on nylon wool columns cleari! identified the immune cells as nylon wool nonadherent. In fact, nylon wool adherent spleen cells totally failed to transfer immunity. Data obtained from tumor sizes were in agreement with those from tumor incidences and statistically highly significant. The conclusion that this immunity was T cell mediated is in agreement with previous observations that spleen cells from agammaglobulinemic chickens immune to SCFS transplants also mediate this adoptive immunity (2), even nhen transferred into bursectomized recipients. Further characterization of the T cells mediating immunity was obtained through data in Table 2. Treatment with mitomycin C in &fro did not noticeably affect their activity, suggestin g that mitotic activity was not needed for these T cells to mediate their effect. On the other hand, they appeared relatively y-irradiation sensitive. Although 500 R did not completely wipe out the immunity since there was still significant tumor inhibition as compared to the control without spleen cells, the degree of immunity was significantly less than that transferred 1,~ untreated cells. In addition, higher doses of irradiation (1000-2000 R) completely abolished immunity. Sysfemic transfer of turnor i~~vzunity. The results in Table 3 show that the intravenous injection of spleen cells from SCFSI immune donors on the day Or challenge with lo6 SCFSI cells in the wing web caused a transient, but statisticall! highly significant inhibition of tumor growth. 1Vhen compared to the effect of 2 X 10’ immune spleen cells in the wing web, the systemic transfer of immmiity

354

PALLADINO

Mitulll),cill

Mitomycin

THORBECKE

Kcsistauce and Irradiation Spleen Cells as Detected

Pretreatment of spleen cells’&

Untreated

AND

spleen cells Cd

500 R r-irradiation 1000 R y-irradiation 2000 R r-irradiation

Spleen to tumor cell ratio”

Sensitivity of SCFS Immune in Winn Assay

Expt. 1 Tumors present/total test sites

20 200 20 200 20 20 20 200

Expt. Tumor cm f

sizec SE

0.24 f

O.OSc

2 Tumors present/total test sites 6/8

O/4 O/5 O/5

No spleen cells

0.61 f 0.03e 0.93 f 0.03 0.89 zk 0.04

8/8 8/8 8/8

0.86 f

8/8

0.02e

n Donors were immunized to SCFSIII with the aid of BCG in Expt. 1 and to SCFSI with Cl’ in Expt. 2. b Tumor cell dose was always 10G. c Tumor incidence was recorded 10 days (Expt. 1) or 12 days (Expt. 2) after injection. Size measurements were performed only in Expt. 2. d Cells (lO’/mI) were incubated for 45 min at 37°C with 100 pg/ml of mitomycin C. e P < .OOl.

caused only slightly less growth inhibition on Day 7, although the locally injected cells were much more inhibitory as judged by measurement on Day 14. Cooperation of donor and host in mcdiafion of adoptive tumor immunity. It seemed important to evaluate the role of the recipient in the expression of tumor immunity in Winn tests. First, the ability of y-irradiated hosts to support the expression of local tumor immunity was determined (Table 4). It was found that 2 x lo7 immune spleen cells could inhibit growth of lo6 SCFS cells much more effectively in normal than in irradiated hosts. A single exposure of the hosts to TABLE Inhibitory

3

Effect of Tumor Immune Spleen Cells Given Intravenously or in Wing WTeb Simultaneously with lo6 SCFSI on Tumor Growth in Wing Web Tumor

Immune spleen cells injected* Day 7

None 2 X lo’, in wing web 2 X 1O8, iv a Immunized by injection of lo6 SCFSI other wing web 6 weeks prior to transfer. “P < 0.001. CP = NS. d P < 0.001.

0.70 f 0.04b 0.05 f 0.03 0.19 f 0.05b

growth Day

on

14

0.81 zt 0.03C.d 0.19 It 0.05d 0.74 i 0.06c

Tumor present/totaI test sites

8/8 6,‘lO

8/8

+ C. pnrvunz in One wing web and 10G SCFSI

in the

T CELL

MACKOPHAGE

COOPERATION

IN

TUMOR

TABLE Inhibitory

Expt.

Effect

Pretreatment recipients

IMMUXITY

IN

355

4

of Y-Irradiation or Macrophage Inactivation Expression of Adoptive Immunity to SCFSI of

CHICKENS

Immune spleen celW added to 106 SCFS in LVing Cl’eb

of Host on the

Tumor Tumor

Growth

size f

SE

(Clll)

Da>. T Tumors present, tumor sitea

1

None Trypan Blue* y-Irradiation

2 x 10’ 2 x 107 2 x 107

0.22 f 0.09 0.40 f O.OY 0.36 f 0.02’

6,‘8 9,‘lO 6, ‘0

2

None None ‘I‘qyan Blue6 yIrradiationc Trypan Blue”

None 2 x 107 None 2 x 10’ 2 x 10’

0.55 * 0.02r~ O.OOh 0.48 z!z 0.03g 0.53 * 0.02 0.50 zk 0.02”

10,‘lO 0 #‘lo 10, 10 0, ‘6 10’10

3

None

None 2 x 10’ 2 x 10’ 2 x 10’ Kane

0.57 0.18 0.11 0.51 0.59

* f It * Ik

O.OJi.j 0.0-l,” 0.06k 0.03” 0.04,

10’10 8,‘12 15 18 11 12 8,‘8

0.60 0.28 0.68 0.29

f f f +

O.O,P 0.06” 0.02k 0.05”

10,:10 5 ‘0 X8 12/N

XOIlCC

Silica” yIrradiation< y-Irradiationc 4

None None Silica? I’V,1:0

+ Silica?

None 1 X lo7 N\nT Nonadherent 1 X lo7 NW Nonadherent 1 X lo7 NW Nonadherent

is I>onors made immune to SCFSI + C. parr’zlm; iV\4; = n),lon wool nonadherent. * Five milligram trypan blue iv X 2, Day - 1 in Expt. 1 ; 5 mg tr).pan blue iv I)a!. -3, and .5 nlg iv X 2 Day -1 in Expt. 2. c 760 R day -1 in Expt. 1; 2 X 630 Ii Days -2, - 1 in Espts. 2 and 3. i’ 3 X 3 mg silica iv; Days -2, 0, +2. r 2 X 3 mg silica iv; Days -1, 0, with or without 150 mg/kg I’VNO subc. on ])a). -2. j P < 0.05. ‘J I’ = N.S. * I’ << 0.001. ’ P < 0.001. i I’ = N.S. A-P < 0.001,

760 R (Expt. 1) was less inhibitory than two daily exposures to 630 R (Expts. 2 and 3). In the absence of immune spleen cells tumor growth was similar in irradiated and normal recipients (Expt. 3). Additional results (not in Table) showed that tumor growth in 12 irradiated and 10 normal hosts n-as also similar when measured on day 12: 1.03 +- 0.16 (Xl) vs. 1.23 -t 0.12 (y-irradiated). Next, the effect of several agents used to inactivate macrophage activity in recipients was examined. It was found that pretreatment of prospective hosts with trypan blue or with silica appeared to mimic the irradiation effect. The total abolition of the expression of tumor immunity in silica-pretreated hosts was particularly clear in Expt. 4 (Table 4), where nylon wool nonxtthei-ettt inmltme splren rolls were used as the donor cells. Confirmation that the silica had acted througl~ macrophages was obtained by the protective effect of PVNO (20, 21) against the silica inhibition in this experiment. The inhibitory effect of host pretreatment with

356

PALLADINO

AND

THORBECKE

TABLE Use of BCG to Reverse the Effects

of Irradiation

Pretreatment of recipients

07 Expts. 1 and 2

-

(10, 6) (12, 6)

2 X 107 BCG ivb

C--,8)

1260 R r-irradiation Day -2, -1 2 X lo7 BCG iv* + 1260 R r-irradiation Day -2, -1

(12,4)

5 on Adoptive

Immunity

in SC Chickens

Injected with106 SCFSI in wing web

Expt. 1 Tumor size f SE (cm) Day 7

Expt. 2 Tumor size f SE (cm) Day 10

-

0.57 f 0.03

0.53 f 0.06"

0.18 f

O.OOd

2 X lo7 immune spleen cells 2 X 10T immune spleen cells

(12, 6)

Tumor

0.54 f 0.03d

0.46 f 0.05c~e 0.64 f 0.0~5~

0.31 f

0.13 f

-

2 X lo7 immune spleen cells

0 Animals were made immune with CP and SCFSI. * BCG pretreatment was 2 X lo7 live bacteria iv Day

0.04d

-9

0.04

(Expt.

2) and Day

-11

0.08”

(Expt.

1).

cP = N.S. dP <<0.001. eP < 0.01. trypan blue was also very striking (Expts. 1 and 2, Table 4). This was considered as further proof that monocytes were involved in the expression of tumor immunity particularly since other experiments have shown that treatment of donors and recipients with similar doses of trypan blue does not interfere with the transfer of (1) antibody production by bursa cells or (2) agammaglobulinemia by splenic suppressor T cells (Lerman et al., manuscript in preparation). Another approach towards identification of the host contribution to the Winn test immunity was by an attempt to stimulate bone marrow production of monocytes. For this purpose, 2 X lo7 live BCG were injected iv, 9 to 11 days prior to transfer. In such pretreated recipients, the usual dose of irradiation was unable to TABLE

6

Lack of Effect of Cytoxan or Cortisone Pretreatment of Host in the Expression of Adoptive Immunity to SCFSI Expt. NO.

Pretreatment

of

recipient

Weight Total body

N0ne

134

(pm)

on Day

BUSa

0.18

0 of*

Spleen

NW Nonadh immune spleen cells, lo’* in wing web

Tumor Size

3 X 20 -3, -2. 3 X 15 -3, -2,

mg. ip, -1 mg. im, -1

Cytoxan, Days

3 x 20 mg. -8, -7, -6

93

0.10

0.06

114

0.08

0.11

1.09 + + -

n Average wicights taken from thrrr animals in rnch group killrd on Day 0. b Donors immunized by injection of 10” SCFSI f C. porvum in one wiw web and weeks prior to transfer. c P < 0.001. dP =NS.

SE

14

TllUl0r test

+ Cytoxan. Days Cortisone. Days NOW2

f

Day

present/total 0.72

0.30

growth

0.84 0.03 0.81 0.19 0.36

101 SCFSI

f O.OSc+’ 0.00 f 0.04c 0.00 * 0.06a * 0.03 * 0.03 l o.ov f O.lld

in thr

other

sites

616 016 lO/lO

O/6 lO/lO

l/6 8/8 6110 6110

wing

wrh

10

T

CELL

MACROPHAGE

COOPERATION

IN

TABLE Reconstitution Adoptive Tumor A? IIonors Pretrratment r-Irradiation (Day -3,

-2)

TUMOR

1260

1160 Ii 1260 Ii

357

(‘HICKEXS

of the Ability of Irradiated KYnn Test Recipients to Support Expression of Immunity by Intravenously Injected Spleen or Bone Marrow Cells from Normal

of recipients

Expt.

Injected with 106 SCFSI= in wing web (Day 0)

Rwonstitution (Day -2)

XOllt~ Nonv R

I?;

7

Tumr

No11r 2 x lo? xy splrrn iv 2 x 10” A”/ Imnc marrow

iv

None 10: immune spleen cells 10’ immune spleen crlls 10’ immune spleen cells 10’ immune spleen cells

i SE (cm)

1) or nylon

rnnrtl~

wool

Incirlcnrt~

Tumor ~~Size

0.66 0.15

0.x.3 * 0.06 i

0.05 0.03

x;x .i,lL

0.46

*

0.0x

12,14,

0.02

*

0.02~

1, 16~

0.01

*

0.00*

1 /‘lXC

-.-__ ” Unfractionated immune spleen cells (Exut. immunized with SCFSI + C. porsum. b Tumor size determined on days 7 and X. c I’ < 0.001 (size) ; P < 0.001 (incidence). ,’ I’ < 0.001 (sizr).

Espt.

1b

.~ Sizr

None N<,llt~

IMMVNITT

nonadll~wnt

+ SE (cm)

~rrnvtl~ 111ridtwre

0.02 0.02

(I 0 .i x

0,.5x *0.0.3’~

8 x

O.lh

* *

2”

*

10 10

0.02”

__~~_.. cells

(lil~t.

2) r,btained

from

~101101

prevent the expression of imnunity. BCG pretreatment of hosts without the local injection of tumor spleen cells did not significantly diminish tumor growth in the ?Vinn tests. Attempts to mimic the effect of irradiation by pretreatment of the prospective hosts with 3 im injections of 15 mg cortisone acetate on Days -3, -2, and -1, or with cyclophosphamide, 3 ip injections of 20 nlg each either on Days -3, -2, and - 1 or Days -8, -7, and -6, were unsuccessful (Table 5 j. This indicated that high doses of these drugs had relatively little effect on the host component of tumor immunity, even though both bursa and spleen Lveights had been drastically reduced by the pretreatment. They were 50% of normal for bursa and 20 to 307% for spleen at the initiation of the experiments. Body weights were also reduced by these high doses of drugs (Table 6). In addition, in contrast to the relative lack of effect of irradiation on tumor growth in the al,seiice of immune cells, cyclophosphamide appeared to cause a significant enhancement of tumor gro\vth. Recomtitution of tlze irhlity ilwnamity. In order to identify

of M’iarz test 1-ccipicnts

to support

adoptive

turllor

further the contribution of j$‘inn test recipients to the expression of tumor immunity, irradiated recipients were reconstituted, 2 days prior to the actual IVim test, with iv injection of bone marrmv or spleen from A7 donors. The results in Table 7 show that these reconstituted recipients had regailletl the ability to supllort the expression of tumor immunity hy inimune spleen cells, whether unfractionatecl (Expt. 1) or nylon wool nonadherent (Expt. 2) in the wing web. Reconstitution with bone marrow cells on Day -1 was less effective than on Day -2 (not in Table). Since the reconstituting cells canle from .\r donors, F? cells could he excluded as contributors. Other 0l)servations in this Inlmrntrn-y have rst;tl)lislie(l tliat A7 ccl1 (1011ors sl10bv < I :I; ~(~11s witI1 surf:lcc Jg it1 their slkens (~.erniaii ct nl., uiq~ublislietl). I,ocal reconstitutiol~ of tlw expression of tumor immunity was also obtained with norinal spleen cells fl-olll .I;, donors, injected into wing webs together with tumor and i~ii~iiuine nylon n.001

358

PALLADINO

AND

THORBECKE

TABLE Local Reconstitution to Support Pretreatment of recipients 1260 R Days -2, -1

Injected with 10” in wing web

Expt.

1

Expt.

2

Expt.

3

SCFSI

(Day NA immune Spleens

f +

a Donors b Normal c Tumor

8

of the Ability of y-Irradiated \&Ynn Test Recipients Expression of Adoptive Tumor Immunity

Tumor

0) Normal .&y Spl&-Jl*

-

-

IO’ 10’ 10’ 10’

2 x 10’ 1 to 5 x 101

Size

f

gruwtlv SE

Tumor

Incidence

Size

(cm)

0.57 0.05 0.59 0.54 0.38 0.30

f 0.10 f 0.03 * 0.04 * 0.03d * 0.07 i 0.09

f

growths SE

Tumor

Incidence

Size

(4

f SE (cm)

growtlic Incidecce

818

z/a

818 818 414 818

0.00 0.54 0.16 0.12

f 0.040 f 0.06 * 0.07e

O/4

616

0.18 f 0.53 * 0.61 f

0.05 0.02 0.03/

S/8 R/8 8/8

313 213

0.25

0.02/

616

immune to SCFSI i- C. paruum. Immune donor and BX recipients used in spleen cells from agammaglobulinemic chickens aged >8 weeks. growth was determinrd on Day 7 or 8. made

Expt.

f 3.

d P < 0.01. 6 P < o.oot. f P < 0.001.

nonadherent spleen cells, even when cell numbers as low as 3 x lo6 were used. In fact, higher doses of normal Ay spleen cells were relatively less effective, possibly because of inefficient cell-to-cell contact due to crowding. Comparison between results in Tables 7 and 8 shows that 2 x lo8 Ay spleen cells iv 2 days prior to Winn tests gave complete reconstitution, while local injection in the wing web of 1 to 5 X lo6 cells on the day of the Winn test was almost as effective. It should be noted that the inhibition of the expression of tumor immunity and its local reconstitution with unimmunized spleen could also be demonstrated when cell donors and recipients were bursectomized 4 (Expt. 3). Although bone marrow from Ay donors was likely to contribute monocytes rather than normal T cells in this reconstitution phenomenon, it was thought necessary to show that isolated monocytes from peripheral blood could also accomplish this. The results in Table IX show clearly that local injection of 0.5 to 1 X lo6 isolated monocytes (cultured for 5 days ill vitro prior to use) caused complete to highly significant, partial reconstitution of the expression of immunity by nylon wool nonadherent spleen cells in the wing web. These isolated monocytes had been derived from peripheral blood cells of Ay donors. The injection of these monocytes alone together with tumor cells into the wing web did not affect tumor growth at all (line 2, Table 9). DISCUSSION The results presented here show clearly that a collaboration between specific tumor-,immune nylon wool nonadherent spleen cells and non-immune non T, non B, monocyte-like cells is needed for the inhibition of fibrosarcoma cell growth in the chicken. The immune donor cells were identified as T cells and came from donors exhibiting DH reactivity to irradiated tumor cells. They did not need to 4 I )u;‘ lo pcrsislillg malcrnal I& at lhc age used for Wiun lest recil)irnts, tllc tlcgrcc of Ay < 90% of burscctomized SC chickens in these recipients could not be ascertained. However, are totally Ay at the age of 8 weeks.

T CELL

MACROPHAGE

COOPERATIOF

IN

TABLE

TUMOR

IMMUNITY

IN

9

Reconstitution by Purified Cultured Monocytes of the Ability of T-Irradiated Recipients to Support the Expression of Adoptive Tumor Immunity Kccipients lL60 K (DWS -2, -1)

(A-o. Expts. 1, 2)

-

(5, 10) (5, N.D.) (10. IO) (N.D., X) (10. 8) (10 7)

-

+ + +

359

CIIICI
\\%m Test

Injected in wing web with 106 SCFSI (Day 0) Ii.4 spleen MonoWllSC cytesn -10’ 10’ 10’

+ +

0.97 * 0.04 O.Y4 l 0.0.x 0.08 * 0.04

100"

0.72 f 0.07 0.11 zt 0.06

1ooi

0.65 * 0.o.i

100

0.04 0.66 0.60 0.27

SO 1OU ,OO” 9,w

100”

.iO'

.iO”

i

* i * *

0.03 0.02 0.04 o.oi

0 :1dherent peripheral blood monocytes cultured for 5 days prior to injection; ccl1 dose IW site 5 X 10” (Exist. Lj or 106 (Expt. 1). b Tumor growth determined Day 10 (Expt. 2) or Day 14 (Expt. 1). c Sylon wool nonadherent spleen crlls from donor immunized with SCFSI f C‘. ~u?‘w~~r. d I’ = NS. e I’ = NS. 1 P < 0.001. Q I’ < 0.001.

proliferate in order to inhibit tumor growth in the local TZ’inn test, since their prior treatment with mitomycin had no deleterious effect on the transfer of immunity. They were relatively sensitive to y-irradiation, which may be due to the known lymphocytic effect of y-radiation. These findings are in agreement with those of others who also found that y-irradiation rendered immune cells inactive in local Jf?nn tests (14, 23), but they are at variance with those of Howell ct al. (24) who did not find much decrease in effectiveness of immune cells in this assay even after high X-irradiation doses. Such differences might be due to the interval between immunization and irradiation, to dose rates of y-irradiation exposure of the cells in zrifro, or to the type of T cell involved in the immunity transfer. Although antibody to transplantable SCFS has recently been noted in our immutie donors (Galton, Palladino, and Thorbecke, unpublished), the adoptive immunity could be demonstrated with cells from Ay donors into Ay recipients. Thus, antibody production could be totally excluded as an important factor in these immunity transfers. Some other authors have reported that serum antibody was absent in their tumor immunity models (2.5)) but a complete exclusion of the possibility of local antibody formation is difficult except in Ay animals. In view of the need for macrophage collaboration, it seemed unlikely that cytotoxic T cells were responsible for the observed adoptive immunity. In addition, direct evidence for T cell cytotoxicity on SCFS cells in vitro has not yet been obtained upon mixing of immune spleen cells with Yr-labeled tumor cells (data not shown). It should be noted that there is relatively little evidence that birds are able to respond with cytotoxic T-cell immunity. Quails were reported to shop cytotoxic effects of their lymphoid cells on RSV-induced primary sarcoma cells ilz vitro (26). The inhibition of colony formation was sho\vn mith cells from animals regressing their tumors but not with cells from birds with progressively growing tumors. Bursectomy did not inhibit immunity, suggesting the involvenwtlt Of 'I' cells. 12’ainberg- et al. (27) 5.llowed that spleeil cells from chickens 1,itll RSV-induced sarcomas caused 61Cr release from autochthonous tumor cells as well

360

PALLADINO

AND

TABLE Review

of Studies on Cellular

Specie

Tumor

BALB/c

mice

BALB/c

mice

CBA/J

mice

CB6Fl mice

AB~FI

mice

NZB and BALB/c

mice

Syrian Golden hamsters Strain 2 guinea pigs BZ /B2 chickens

SV-40 kidney cells SV-40 kidney cells

NW nonadb. SplWl Spleen or LN

Chem-induced fibrosarcoma Cbem-induced fibrosarcoma (M&h A) Spindle cell sarcoma (SAl) My&ma Myel. leukemic thymoma Allogeneic lymphosarcoma Chem-induced hepatoma

Nonadh. PE cells LN (replicating* T cells) LN (T cells needed) PE cells, spleen TDL or LN (T cells needed) Spleen, LN. PE cells Nonadb. PE cells

Chem-induced fibrosarcoma

NW nonadh. spleen PBL

0 Systemic transfer, or glass nonadherent.

10

Mechanism

Donor lymphoid cells

THORBECKE

of Adoptive

Tumor-Specific

Recipient cell contribution T cells excluded Silica + X-ray sensitive, BM-derived cell reconstitute X-ray sensitive, macropbages reconstitute T cells excluded 8.50 R-no rffrct

Immunity Suspected mechanism

Ref. NO.

Cytotoxic T cells unlikely Activated macrophages

24

Activated macrophages or K cells Cytotoxic T cells

15 29

14

X.50 R--no

effect

Cytotoxic

T cells

30

4.50 R-no

rffect

Cytotoxic

T cells

23

850 R sensitive, BM or PE cells do not reconstitute Not examined

Cytotoxic

T cells

31

X-ray, trypan blue + silica sensitive, BM-derived macrophages reconstitute

DH reaction induced mononuclear cell infiltration DH reaction induced activated macrophages

all others were local Winn tests; NW nonadh. = nylon wool nonadherent;

3L

nonadh. = plastic

as from allogeneic tumor cells and xenogeneic RSV transformed cells, but the cytotoxic cell type involved was not characterized. Killing of viral antigen-bearing lymphoma cells, or else inactivation of the Marek’s disease virus itself, was shown with splenic T cells from virus infected chickens (28). Thus, the only available evidence for cytotoxic activity in the literature on chicken immunity related to tumor virus antigens. Clear T cell cytotoxic effects on allogeneic cells due to Blocus determined antigens have not yet been described. The need for collaboration of accessory cells with immune T cells in Winn tests was also noted by others ( 14, 15)) although in some tumor systems (23, 29, 30), T cells alone seemed to suffice for the transfer of immunity. Various systems for which host cell contributions in Winn tests were suggested or demonstrated are included in Table 10. In experiments with myeloma, leukemias and lymphomas (23), the existence of cytotoxic T cells was clearly demonstrated in the donor inocula and were assumed responsible for the tumor inhibition in z&o. Even cytotoxic T cells generated by exposure to syngeneic myeloma cells in vitro were able to inhibit tumor growth in viva (23). Since most of the transfers in these experiments were done into 450 R irradiated mice, a relatively low dose, it is not clear whether monocytes in the recipients could have contributed to the immunity, but it was shown that they were not needed in the donor population. In the experiments of North and collaborators (29, 30) even 850 R of recipients did not prevent tumor inhibition, but no attempt was made to remove adherent cells from the donor inocula for the transfers into irradiated recipients. However, in similar experiments by these authors on the transfer of nonspecific tumor immunity, a role of radiation sensitive host cells was more readily demonstrated (6). Thus, they also concluded that specific immune donor T cells were capable of inhibiting tumor growth by direct T cell cytotoxicity.

T

CELL

MACROPHAGE

COOPERATION

IN

TUMOR

IMMUNITY

IN

CHICKENS

361

Obviously, more than one T cell system can be involved in the adoptive immunity to any given tumor under study. However, the monocytic component contributed by the host in MJinn tests appears of great importance in a large variety of systems and explains, as also stressed by Zarling and Tevethia (14) and by Zbar ct (11. (32), the low ratios of non-dividing immune cells to tumor cells needed in z~iz~ as compared to the one needed for T cell cytotoxicity ill zritro. Howell et nl. (,24j have also compared Winn tests with in vitro cytotoxic T cell tests and came to the conclusion that cytotoxic T cells in their donor inocula could not explain the adoptive in viz~ immunity observed. Unfortunately, quantitative measurements on these various components of tumor immunity in viz~ and in vitro are still lacking for most tumor systems. It should be noted that even in vitro the demonstration of tumor specific cytotoxic activity which is inactivated by killing of immune T cells does not prove the presence of cytotoxic T cells unless the cytotoxic effect persists after removal of macrophages (33). T cell-macrophage collaboration in specific tumor immunity in vifro was shown by a number of authors (11, 33-35). It was clear from the present results that systemic transfer required more immune cells than local transfer, but was at least transiently effective in inhibiting local tumor growth. This was also found by others (29, 36) ; systemically transferred immtme cells lvere recently found to act even against small established tumors (36). *A host component of tumor immunity is even more evident when a study is made of nonspecific immunity. Table 11 summarizes the results of others (13, 35, 3741) and of ourselves (42), suggesting that tumor growth inhibition can be obtained 1)~ the induction in the tumor site of a specific DH reaction to an antigen unrelated to the tumor. A requirement of a radiation sensitive host component for this tyl)e TABLE

11

Review of Studies Showing Local Inhibition of Tumor Growth by Reaction to Immunologically Unrelated Antigensa Tumor

cells in

Local L1H reaction

Local adoptive DH reaction

Local host vs. graft reac.tion

Species

Tumor

DNCB BCG BCG Other hepatoma C. parcum L. monocytogenes PPD C. parvuna HGG BCG

Human Human Guinea pig

Basal cell carcinoma Melanoma Chem.-induced hepatoma

Mouse Mouse Mouse Chicken

C‘hem-induced Chem-induced Lymphoma Chem.-induced

Salmonella C. parvum C. parvum HGC

Mouse Mouse Chicken

Ehrlich ascites Chem.-induced sarcoma Chem:induced sarcoma

Rl011sr (‘hicken

(‘hellI.-intluretl (‘Ilent.-induced

Antigen

Allogeneic Allogeneic

used

cells cells

iz In some of these examples, the mechanism of action was not entirely between bacterial agents and tumor antigens could not be excluded.

Ref. NO.

sarcoma sarcoma sarcoma

sifrconx* si~rcon~i~

clear since crossreactivitl

362

PALLADINO

AND

THORBECKE

of inhibition in a local adoptive DH reaction was found in all three systems examined (6, 42,43). Thus, in these examples of specifically induced nonspecifically mediated inhibitions of tumor growth, activated macrophages are almost certainly responsible as well as in concomitant immunity to unrelated tumors (30) and in local host vs. graft reactions (4’2, 44). It should also be stressed that tumor infiltration with monocytes has been observed by numerous authors to be a prominent feature of tumor rejection (45-48). Pyran and glucan have recently been used as agents that cause (possibly direct) activation of macrophages. In systems where these agents enhanced tumor immunity in viva accumulations of macrophages around tumor sites were noted in man (49) as well as in animals (50, 51). It has also been reported that BCG and MER can cause direct activation of macrophages in thymusless mice and that tumor immunity in such mice is abrogated by silica (10). Preliminary data in this laboratory suggest that injection of “activated” macrophages together with SCFS tumor cells can cause inhibition of tumor growth (Palladino and Thorbecke, unpublished observations). Further studies on this aspect are in progress. Although in the present study no long term follow up was made of tumor immunity development in Winn test recipients, previous studies with these tumors indicated that local immune reactions to unrelated antigens in SCFS tumor sites not only retarded their growth but frequently induced specific immunity to the tumor line used (2). It is quite important to differentiate between transient tumor inhibition due to a local immune reaction to antigen (tumor or unrelated) and induction of active immunity in the recipient (41). In studies in man, it has been noted that local BCG therapy acts effectively only on melanoma metastases in the same lymphatic drainage region (38). This might be explained by a need for interaction between local antigen, immune T cells and macrophages without the presence of actual specific tumor immunity. The irradiation sensitivity of the monocytic component of tumor immunity may depend solely on the rate of peripheral blood monocyte turnover, or else may also be a function of the reported sensitivity of certain macrophage functions to irradiation (52). The fact that BCG pretreatment rendered recipient chickens more resistant to irradiation is in line with observations on the stimulation by such bacterial agents of monocyte production in bone marrow (53, 54). It is also of interest that high doses of cyclophosphamide and of cortisone were much less effective than irradiation in interfering with the expression of adoptive tumor immunity, particularly since these agents are such favorites in clinical tumor chemotherapy. The inhibitory activity of recipient pretreatment with trypan blue and with silica are also noteworthy. It will be important to determine how long the effect of these agents may last and whether repeated treatment with macrophage inactivating agents can affect the rate of primary tumor induction. It should be noted that chronic parasitic infection has been reported to result in a lower frequency of spontaneous tumors (55). ACKNOWLEDGMENT The exc~llrnt tcclmical assistance of Pedro Sanchez and Pamela Pulver is greatfully acknowledged. The help of Dr. Michael Rabinovitch (Dept. Cell Biology, this Medical Center) and Dr.

Marc Grebenau (this department) in developing tnethods for obtaining purified peripheral blood monocytes is greatly appreciated.

‘1‘ CIII,L

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

1\1:\CROI’TI~\(;I

(‘OOI’ER:\TION

IX

TUMOR

Thlhl 1’KlTY

1N

C’III(‘l
30.1

Let-man, S. I’., Palladino, M. A., and Thorbecke, G. J., J. A’& CUUU Inst. 57, 2% 1976. Palladino, M. A., and Thorbecke, G. J., Ccllltlav I?w~~mlOgY 38, 336, 1978. Cantor, H., and Boyse, E. A., J. Exp. Med. 141, 1376, 1975. Mackaness, G. B., J. Exp. Med. 129, 973, 1969. Volkman, A., and Collins, F. M., Cclldnr I~mmrz~log~ 2, 552, 1971. Tuttle, R. L., and North, R. J., J. Natl. C07rccr Inst. 55, 1403, 1975. Krahenbuhl, J. L., and Remington J. S., J. Z~nw~tcnol. 112, 507, 1974. den Otter, W., Evans, R., and Alexander, P., Tva~pla?ztntio~t 18, 421, 1974. Hibbs, J. H., J. Reticztloc~~~otl~cliu~ Sot. 20, 223, 1976. Hopper, D. G., Pimm, M. V., and Baldwin, R. W., Cnlrcc,r lw~!t/~ol. Inlmzmoflrey. 1. 113. 1976. Piessens, V. F., Churchill, W. H., and David, J. R., J. Z+rc~tt~1to1.114, 293, 1975. Nelson, D. S., Aiatzwc 246, 306, 1973. Youdim, S., Cancer Rescarclz 37, 991, 1977. Zarling, J. M., and Tevethia, S. S., J. hratl. Ca~ccr Iwf. 50, 149, 1973. Simes, R. J., Kearney, R., and Nelson, D. S., lat~twtolo~q~~ 29, 343, 1975. Palladino, M. A., Lertnan, S. P., and Thorbeckc, G. L., .I. 1wwt~toI. 116, 1673, 1976. Julius, M. H., Simpson, E., and Herzcttberg, J. A., E~IY. 1. I*ttwttlriol. 3, 645, 1973. McArthur, W. P., Chapman, J., and Thorbecke, G. J., J. E.rp. dfcti. 134, 1036, 1971. Hibbs, J. B., Tyarzsplmfntiou 19, 77, 1975. Rios, A., and Simmons, R. L., 7‘yousplanfc~tion 13, 343, 1972. Lotzova, E., and Cudkowicz, G., J. Iw~~torol. 113, 798, 1974. Winn, H. J,, J. Zwttwzol. 86, 228, 1961. Rouse, B. T., Rollinghoff, M., and Warner, N. L., Eny. J. I~nmuuol. 3, 218, 1973. Howell, S. B., Dean, J. H., Esber, E. C., and Law, 1~. W., Iufcynnt. J. Cmccr- 14, 662, 1974. Tuttle, R. I,., and North, R. J., J. Rctic~~lorlzdotl~clinl Sot. 20, 197, 1976. Hayami, X., Hellstrom, I., Hellstrom, K. E., and Tamanouchi, K., Iltfrrrltrt. J. (‘n,tr~ 10, 507, 1972. Wainbet-g, M. A., Yu, M., Sch\vartz-I*uft, E., and Jst-ael, E., Ilrtcrlrttf. J. Currccy 19, 6x0, 1977. Ross, L. J. N., IVatwc 268, 644, 1977. Tuttle, R. L., and North, R. J., J. Rcfic~~loc~~dofl~clinl SOC. 20, ZO’), 1976. North, R. J., and Kirstein, D. P., J. Exp. Med. 145, 275, 1977. Nomoto, K., Gershon, R. K., and Waksman, B. H., J. Natl. C~IKCY Ittst. 44, 739, 1970. Zbar, B., Wwk H. T., Rapp, H. J., Stewart, L. C., and Borsos, T., J. Natl. C,ltlcrr Imf. 44, 701, 1970. Kearney, R., Basten, A., and Nelson, D. S., Znfrr-rzat. J. Corrc~y 15, 438, 197j. Youdim, S., and Sharman, M., J. I~uvw~oZ. 117, 1860, 1976. Alexander, I’., Natl. Cmrccr Inst. Mouog~nplz 39, 127, 1973.

36. Smith, H. (i., Harmcl, R. P., Hanna, M. G., Zwilling, B. S., Zbat-, B., and Raljp, H. J., J. Nuti. Cancer Zmt. 58, 1315, 1977. 37. Klein, E., Holtermann, O., Milgrom, H., Case, R. W., Klein, D., Rosner, D., and Djer:lssi, I., Med. c‘hics of Nortl~ Awericn 60, 389, 1976. 38. Mastrangelo, M. J., Sulit, H. L., Prehn, L., Bornstein, R. S., Yarbro, J. W., and l’t-chn, R. T., C‘n~ccr 37, 684, 1976. 39. Zbar, B., Bernstein, I. D., and Rapp, H. J., J. Nntl. I‘mcry Inst. 46, 831, 1971. 40. Zbar, B., Wegsic, H. T., Borsos, T., and Rapp, H. J., J. Xatl. Cnuccr I,zst. 44, 473, 1970, 41. Scott, M. T., J. Katl. Caeccv Inst. 56, 675, 1976. 42. Palladino, M. A., and Thorbecke, G. J., Ew. J. 1~w+tzwo1., in press. I., and Hardy, D., I~n~r~tr~rology 32, 1, 1977. 43. Ashley, M. P., Kotlarski, 44. Prehn, R. T., I?r “Immunological Parameters of Host-Tumor Relationshil,es” (1). W, ~~~~~~~ Ed.), Vol. 2. Academic Press Inc., New York, 1972. 45. Haskill, J. S., Rador, L. A., Yamamura, Y., Partltenais, E., Korn, J. H., and Ritter, 17, I~,, J. Rrtic~~loc~~dothclial Sot. 20, 233, 1976.

364

I’ALLADIiXO

AND

TIIORBECKE

46. l’ross, 11. I;., :ud I\cihcl, It. S., J. Nutl. ~‘~II~~~~~Y Iusf. 57, 1157, 1976. 47. Hamla, M. (;., Zhar, B., and Kapp, H. J., J. Nnfl. (.‘trrrccr Irrsf. 48, 1441, 1972. 48. Berd, D., and Prehn, R. T., J. Nut/. Cmccr Zmt. 58, 1729, 1977. 49. Mansell, P. W. A., Ichinose, H., Reed, R. J., Krementz, E. T., McNamee, R., and DiLuzio, N. R., .I. Natl. Cancer Imt. 54, 571, 1975. SO. Harmel, R. P., and Zbar, B., J. Natl. Cancer Inst. 54, 989, 1975. J. D., Leutzeler, J., Ruiz, P., and Chirigos, M. A., 51. Schultz, R. M., Papamatheaksis, Cancer RES. 37, 358, 1977. 52. Gallily, R., and Zylberlicht, D., Il)zlrzzl,zoclzelltistry 12, 611, 1975. 53. Wolmark, N., and Fisher, B., Cancer Research 34, 2869, 1974. 54. Fisher, B., Taylor, S., Levine, M., Saffer, E., and Fisher, E. F., Cancer Research 3.4, 1665,

1974. 55. Hibbs, J. B., Lambert,

1972.

L. H., and Remington,

J. S., Proc.

Sot. Exp. Biol.

Med.

139, 1053,