Immunogenetics 1:33 - 4 4 , 1974 �9 by Springer-Verlag New York Inc. 1974

"Weak" Histocompatibility Antigens Generate Functionally "Strong" Humoral Immunity

William M. Baldwin III and Nicholas Cohen

Division of Immunology of the Department of Microbiology University o f Rochester School o f Medicine and Dentistry

Rochester, New York 14642

Abst rac t

The kinetics and complexity of the immune responses generated by weak histocornpatibility antigens on transplants of normal tissues were investigated by analyzing (1) the survival times of test skin grafts on mice that had been initially immunized with skin, liver, or kidney implants, (2) the survival times of test skin grafts on mice that had received putatively immune sera or lymphocytes harvested from an allografted host, and (3) the cytotoxic potential of these cells and sera in vitro. The data indicate that despite chronic rejection, weak antigens rapidly immunize hosts, and that the immune response includes a major humoral component that can either accelerate graft rejection or prolong graft survival.

Introduction

Histocompatibility antigens that elicit chronic rejection of a transplant are classified as "weak" antigens, whereas those that generate an acute rejection reaction are said to be "strong." Whether chronic rejection vis ~ vis acute rejection primarily results from a slower recognition phase, the production of a quantitatively less intense immune response, or the evolution of a qualitatively different immune reaction has not been thoroughly analyzed. Data from second-set transplantation (Barnes et al. 1968, Henry and Dammin 1962), from cell and serum transfer studies (McKhann and Berrian 1961, Warren and Gowland 1970), and from in vitro cytotoxicity assays (Canty and Wunderlich 1971, Wilson 1963) indicate that strong antigens elicit readily detectable host immunity 5 to 9 days after transplantation. We have exploited these same assay procedures to define immunity generated during weak histoincompatibility interactions involving alloantigenic products of the H-Y and the H-3 + H-13 loci.

Materials and Methods

Mice and Their Immunization. Two- to 4-month-old C57BL/10ScSn and B10.LP mice (abbreviated B10 and LP, respectively) were obtained from the Jackson

33

34 William M. Baldwin III and Nicholas Cohen

Laboratory, Bar Harbor, Me., and were subsequently maintained in our vivarium for at least 10 days prior to the following surgical procedures. Small pieces of tail skin (20 mma) or small fragments of liver or kidney (15 to 25 mg)were grafted either on the kidney cortex (suprarenally (sr.)) or subcutaneously (sc.), or to the body surface ("orthotopically" (ot.)), according to previously published procedures (Baldwin et al. 1973, Wheeler et al. 1966).

Assessment of Transplantation Immunity. The immune status of mice sensitized by various routes with an initial small graft was determined by three different assays. In one experimental series (double transplant assay), first-set grafted mice received a single, ot., large (100 mm2) test graft of tail skin at various times after first-set or., sc., or sr. transplantation. The survival time of this test graft relative to control first-set ot. large skin grafts was then evaluated. In the second experimental series (transfer assay), lymphoid cells or sera from first-set grafted mice were injected I.P. into isologous re- cipients. One day after transfer, these new hosts were challenged with an ot., large, first-set test skin allograft from the original donor strain. Cell suspensions were pre- pared by mincing the spleens and lymph nodes and then sieving the fragments through a 60-mesh stainless steel screen (Billingham and Silvers 1961). One spleen equivalent or 1 to 4 lymph node equivalents of viable cells were suspended in 0.75 ml aliquots of phosphate buffered Ringer's solution and injected I.P. Serum from 2 to 4 mice was pooled and then transferred in single 0.15 to 0.25-ml atiquots by I.P. injection.

The in vitro cytotoxic potential of leukocytes from first-set immunized animals was evaluated in the final experimental series (51Cr-release assay). Spleen and lymph node cells from appropriately grafted mice were prepared as before and incubated in vitro with s 1Cr.labeled target cells from the donor strain mice (Canty and Wunderlich 1971, Denham et al. 1970). Radiation-induced B10 lymphomas (kindly provided by Dr. Gustavo Cudkowicz, State University of New York Medical Center at Buffalo, New York) were first screened as a potential source of target cells. However, within 3 months after these tumors had been introduced into our mouse colony, lymphocytes from 60 to 70% of our apparently healthy and nonimmunized mice caused marked s 1Cr-release from the labeled tumor cells in vitro. This observation is not unique, since lymphocytes from healthy relatives of a patient with leukemia or a lymphoma can also effect in vitro lysis of tumor cells from that patient (Bias et al. 1973, Rosenberg et al. 1972). Therefore, in the present experiments, the thymus provided a far superior source of target cells. Thymocytes were labeled by incubating 30-50 • 106 cells in 1.5-ml medium containing 150 to 200 tac sodium chromate (51Cr; specific activity 200 to 250/ac/#g; Amersham Searle, England) for 1 hour at 37~ Following incuba- tion, these cells were washed three times (centrifuged at 80 g for 5 min and resuspended in 6 ml of fresh medium). The concentration of target thymocytes was determined with a hemocytometer and their viability was evaluated by trypan blue dye exclusion. They were then diluted to a final concentration of 5 or 10 • 104 viable cells/0.2 ml. Such aliquots contained about 2000 cpm with only 1.3 + 0.8% of the label present in the supernatant. Labeled thymocytes were mixed with 100 times as many potential killer leukocytes or with 0.1 ml of putatively immune serum. The cultures were brought to a final volume of 0.4 ml and incubated at 37~ in corked 10 • 75 mm sterile disposable culture tubes. All cells were prepared and cultured in HEPES buffered Eagle's basic medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 15% heat-inactivated fetal calf serum. No exogenous complement was added.

Destructive and Protective Immunity to Skin Grafts 35

Incubation was stopped after 7 hours and the tubes were gently vortexed and centri- fuged. The radioactivity in 0.2 ml of the supernate, as well as in the cell pellet plus the remaining 0.2 ml of supernate, was measured in a Nuclear-Chicago solid-well scintillation counter. The rate of spontaneous s 1Cr.release was measured in control cultures that contained labeled thymocytes mixed with either 0.2 ml medium, or with lymphoid cells, or sera from nonimmune mice. Maximum slCr.release (75 to 80%) was determined by subjecting labeled thymocytes to three freeze-thaw cycles. Gener- ally, release measured from duplicate or triplicate experimental or control tubes differed by < 3%. Immune-mediated lysis was calculated by the standard formula:

(% s 1Cr-release in experimental tube) - (% spontaneous s 1Cr-release)

(% maximum s 1Cr-release) - (% spontaneous s 1Cr-release)

Results

Rejection o f ControlSkin Grafts. Median survival times (MST) for ot. control skin grafts were calculated nomographically (Litchfield 1949) and are listed in Table 1. In each donor-host combination tested, 100 ram2 control first-set skin grafts and similarly sized second-set grafts transplanted 3 to 5 weeks after first-set rejection exhibited a complex chronic rejection reaction. Although within 4 weeks post- transplantation all grafts underwent initial rejection crises in which 10 to 70% of each graft was destroyed, some grafts recovered from these episodes. When recovery occurred, the viable tissue that remained was either destroyed chronically but com- pletely 3 to 7 weeks later or retained its full viability for > 200 days. For this reason, the plot of the cumulative percent of skin graft zero viability end points versus time on logarithmic-probability coordinates according to Litchfield's method (1949) failed to produce the simple straight line that is compatible with a single mechanism under- lying rejection (Fig. 1).

Double Transplant Assays. In each donor-host combination tested, exposure to a small (20 ram2) ot. skin graft caused an obvious and progressive sensitization of

Table 1. Median Survival Times (MST) for 100 mm2 and 20 mm 2 Control First-Set Skin Grafts

95% Donor Host Histo- Graft confidence strain strain compatibility size MST limits

and sex and sex barrier (ram 2) (days) (days)

Fraction surviving

indefinitely (200 days)

100 35.5 27.5-45.8 dBlO 9BlO H-Y

20 22.5 19.6-25.9

9BlO 9LP H-3 +H-13 100 39.7 27.5-58.0

100 78.5 15.7-392.5 dB 10 dLP H-3 + 14-13

20 20.5 14.6-28.7

0/22

0/14

6/20

6/12

O/4

36 William M. Baldwin III and Nicholas Cohen

the host that was manifested by the markedly accelerated rejection of test skin grafts placed 5 to 28 days after initial transplantation (Figs. 1 and 2). The vigorous rejection of test grafts did not noticeably affect the destruction of the original sensitizing graft, which was still rejected at times typical of control first-sets. In some instances, this was weeks after rejection of the test graft.

95- ~ [ ]= Slope

"~ [1.2 2 C o n t r o l 2 ndse t 2 'F / ~ . . . . . . . . . 7 5 - Io~ , Contro l lSt set

- / [2.5] , - ~ ,.1 . . . . . . 1

,~= so-/,,~'" +,,1,,, ~- [2.3] ,1'"

,I S 9 Day implanted 25 - ~" j , , ~ , ,,

2 1, , , , " - [1.2]; { / "

5-A ~ , , , , , / / , 12 20 30 40 50 6'0 70 ) 2 0 0

Days

Fig. 1. Cumulative percent of skin graft rejection times plotted on logarithmic- probability coordinates according to Litchfield's method (1949). Control female LP mice received a second-set female B 10 skin graft after they have completely rejected their first graft. Experimental mice were pretreated 9 days before receiving their test skin graft with either a suprarenal (sr.) liver or kidney implant (pooled data) (A), or an orthotopic (ot.) skin graft (e).

Whereas the MST of test grafts on ot.-immunized mice was always curtailed regardless of the interval between the two transplants (5 to 28 days), the survival time of test grafts on recipients of sr. implants was variable and depended on the tissue type implanted, the histocompatibility barrier, the sex of the donor and host, and the inter- val between implantation and test skin grafting (Fig. 2). In certain protocols, the survival of test grafts placed 5 days after st. implantation was neither curtailed nor prolonged relative to control first-sets; in other protocols, the same interval resulted in clear-cut accelerated rejection.

It is most noteworthy that an implant-to-test graft interval of 9 days resulted in prolonged test graft survival (immunosuppression) when kidney or liver fragments from female B10 mice were sr.-implanted in female LP recipients. In this strain com- bination, the immunosuppressive influence of the implant disappeared and accelerated test graft rejection became manifest when the interval was changed from 9 to 15 days (Fig. 2). Increasing this interval in this strain combination even further (to 21, 35, or 70 days) always resulted in the curtailed survival of test grafts. Hence, once immunity as measured by accelerated rejection was established across this weak H-barrier, it remained long-lived.

The following observations indicate that the immunosuppression generated by the sr. implantation of female LP mice with kidney or liver fragments from female BIO

Destructive and Protective Immunity to Skin Grafts 37

donors and measured by the survival of a test skin allograft placed 9 days later, is not complete. First, all of the initial test grafts underwent rejection crises before they recovered and survived indefinitely. Second, hosts carrying viable test skin grafts for 200 to 600 days were capable of rejecting a second test skin graft. Finally, when long- surviving initial test grafts were removed and immediately retransplanted to a new site on the same host, they were rapidly destroyed. That implant-induced immuno- suppression is not the result of nonspecific trauma (stress) is revealed by our observa- tions that sham st. implantation (i.e., st. kidney implants from female LP donors to female LP hosts) failed to prolong the survival of the female B10 test grafts trans- planted either 5 or 9 days later. Antigen specificity of this immunosuppression was demonstrated by the fact that although implants from female B10 donors on female LP hosts significantly extended the survival of female test grafts transplanted 9 days postimplantation, they failed to alter the rejection rate of male B10 test grafts on female LP hosts from that associated with controls.

100

~ 80

~ 60

~- 40 i - -

~_ 20

o

-Ot. Sc. Sr. dB10~-gB10 v �9 Slain o ? Kidney/liver < >No. hosts

I I I I I 3 5 9 15 21

_•!i••ol 0~9 LP

l I I l I 3 5 9 15 21

Days between pretreatment and test skin grafting

dB10~dLP

I l I 5 9 15

Fig. 2. Median survival times (MSTs) of test skin grafts transplanted across three histocompatibility barriers to mice that had been pretreated with either suprarenal (st.) kidney, liver or skin implants, subcutaneous (sc.) kidney, liver or skin implants, or orthotopic (ot.) skin grafts at various intervals before test skin grafting. Each point represents data from 4 to 20 hosts.

Transfer Studies. Ot. skin grafts and st. implants of kidney or skin rapidly initiated a destructive and protective immunity that could be adoptively transferred with either lymphoid cells or serum. The immune compartment (e.g., serum, draining or distant lymph nodes, spleen) that was capable of transferring either curtailed or prolonged test graft survival at a given postimmunization interval was dependent on the site of grafting and the origin of the tissue used for sensitization. Immunity to ot. female B10 skin grafts could be transferred to female LP hosts with serum or with cells from the distant (mesenteric) and the draining (axillary and brachial)nodes harvested 5 days after sensitization and with splenocytes harvested at 12 days (Fig. 3). Ot. male B10 skin grafts also elicited both humoral and cellular immunity within 5 days in male LP hosts, but the cellular immune response was present in the spleen rather than in the regional draining axillary and brachial node compartments. Specifically, 0.15 ml of serum or 1 spleen equivalent (110 X 106 leukocytes)from either female B10 or male LP hosts harvested 5 days after immunization with ot. male B10 skin grafts could transfer an immune status to syngeneic hosts, whereas 1 to 4 axillary and brachial node equivalents (10 to 50 X 106 leukocytes) could not. The

38 William M. Baldwin III and Nicholas Cohen

immunity transferred by splenocytes was always expressed by curtailed test graft survival. The immune state transferred by sera raised in response to ot. skin grafts was referable to the strain combination tested. In both male B10 ~ male LPs and female B10 ~ female LPs (1t-3 + H-13 incompatible), sera harvested at day 5 transferred accelerated rejection. On the other hand, sera harvested from female B10 recipients of male B10 grafts (H-Y incompatible) always enhanced the survival of test grafts.

When skin from male B10 donors was sr.-implanted in either females of the same strain or in male LPs, the ability to reject test grafts in the accelerated fashion was not transferable with cells or with sera. Sera or cells from the distant axillary and brachial nodes from these male LP hosts, however, did transfer a protective immune status to the isologous recipient (Fig. 3). Again this pattern of immunization differs from that engendered in female LP recipients where sr. skin implants elicited immunity in the regional draining node (mesenteric) by day 5 and in the spleen at day 12.

In contrast to st. implants of skin, st. implants of kidney fragments in male LP recipients resulted in the ability of all lymphoid tissues tested to transfer accelerated rejections with 5 days after implantation. The ability to transfer this immune state with regional lymphoid organs (spleens and mesenteric nodes) subsided by day 12 but was

Ot. skin Sr. skin ,oo[/\ I / x / ; o - - : - - -

oL ","" :

-o_ 100i ~o~~ i~' 5 0 ~ - - - - v -

0 J ~Vo" ~Vm

5

I

5 12 5 12

Sr. kidney

m

o

Serum Spleen

L M e s e n t e r i c Axillary _~

Bra.chia L o

z~

o

g_ o 5 12

Time (days) after sensitization that cells & sera were harvested

Fig. 3. Percent test skin grafts that survived longer than 40 days (the time at which about 50% of the control grafts were rejected) on hosts that received cells or serum harvested at specific times from mice sensitized with orthotopic skin allografts, or with suprarenal skin or kidney alloimplants. Cells and sera were injected 1 day before test skin grafting. Each point represents an average of 5 mice for sera and spleen transfers and 4 mice for lymph node transfers.

Destructive and Protective Immunity to Skin Grafts 39

still transferable at this time with sera or with cells from the distant axillary and brachial nodes (Fig. 3). St. kidney implant also elicited destructive cellular and humoral immunity in female LP hosts, but these responses did not maximize until after day 5 (Fig. 3).

In Vitro CytotoxicityAssay. To date, only leukocytes and sera from LP recipients of B10 ot. and sr. grafts have been surveyed for their cytotoxicity by the in vitro s lCr-release assay (Table 2). The most striking results can be summarized as follows. First, cells as well as sera harvested at various intervals from female B10 mice were cytotoxic, whereas only the sera from males proved capable of regularly killing target cells. Indeed, the frequency with which sera from males immunized by the ot. and sr. routes proved cytotoxic at each postimmunization interval tested was greater than it was for comparable serum samples collected from females. Second, in vitro measurements of serum cytotoxicity coincided with the effectiveness of sera in passively transferring destructive immunity to syngeneic hosts. For example, sera harvested from male LP mice 5 or 12 days after they had been implanted with a kidney fragment showed marked slCr-release (10 to 25%), whereas sera from LP females were cytotoxic when harvested at day 12, but not earlier. Similar kinetics were revealed by the transfer assay (Fig. 3).

Cytotoxic activity of cell preparations from various lymphoid organs was con- stant in duplicate or quadruplicate cultures of the same cell preparations, but showed marked variability between mice immunized by the same protocol and sacrificed at the same time. Such variability has also been reported in the cellular responses of individual mice and rats to allogeneic tumor transplants (DeLandazuri and Herberman 1972, Denham et al. 1970).

Discussion

The immune responses generated by grafting normal tissues across the weak H-3 + H-13 and the H-Y barriers without vascular surgery appear different from those elicited by tissues transplanted across strong H-barriers with respect to the types of immune responses elicited, the rapidity of their appearance, their relative intensities, and the ease of their detection in vitro. The following major distinctions were found. First, about 40 to 60% of the first-set, and up to 20% of the second-set, control skin grafts transplanted across the weak H-barriers in question recovered from their initial rejection crises and survived for prolonged periods. Although similar recovery from such rejection episodes has been reported for rat skin allografted across the Ag-B barrier, its frequency was low (Heslop 1971). No such recovery has been reported for 11-2 incompatible mouse skin grafts whose rejection is invariably acute. Second, sensitization with grafts of skin, kidney, or liver prolonged the survival of some sub- sequently transplanted test skin grafts. With the exception of sensitization with tumor grafts, virtually all other instances of such transplant-induced immunosuppression have been reported in systems that involved only weak histoincompatibility inter- actions (see Baldwin and Cohen 1970, for discussion). Third, serum harvested 5 or 12 days after transplantation across the weak histoincompatibilities tested and trans- ferred to isologous hosts could prolong or curtail the survival of test skin grafts on the adoptively immunized recipients. Such protective immunity may well explain why

Tab

le 2

. In

Vit

ro C

yto

tox

icit

y o

f C

ells

and

Ser

a H

arve

sted

Fro

m L

P H

osts

Tha

t W

ere

Sen

siti

zed

Wit

h S

upra

rena

l S

kin

or

Kid

ney

All

oim

plan

ts o

r O

rtho

topi

c S

kin

All

ogra

fts

Fro

m B

10 D

onor

s

Don

or ~

ho

st

% 5

1C

r-re

leas

eda

from

tar

get

B 1

0 th

ym

ocy

tes

whe

n ce

lls

or s

era

wer

e fr

om m

ice

sens

itiz

ed w

ith:

9BIO

~

9LP

dBlO

~

dLP

Cel

ls o

r o

rth

oto

pic

ski

n fo

r:

supr

aren

al s

kin

for:

su

prar

enal

kid

ney

for:

se

ra t

este

d 5

days

12

day

s 5

days

12

day

s 5

days

12

day

s

Ser

um

0, 2

5 0,

0

Spl

enoc

ytes

0,

0

0, 0

Mes

ente

ric

node

10

, 0

0, 0

Ser

um

0, 2

5 10

, 0

Spl

een

0, 0

0,

0

Mes

ente

ric

Nod

e 0

0, 0

16 d

ays

0 0,

0

10,

0, 0

0,

20,

23

,0,

5 0,

0,

5

5 5,

25

7, 3

0, 0

0,

0,

0, 3

0, 5

0,

15,

10

0 40

, 15

2

5,5

,0

0,0

,0,2

7,0

0 17

12

, 12

, 10

, 10

15

, 15

, 25

, 0

0 10

0,

0,

0, 0

0,

0,

0

0 10

0

,0

0,0

90,

O, 0

g~

Z

O

Izr

t~

aEac

h nu

mbe

r re

pres

ents

the

aver

age

rele

ase

reco

rded

in 2

-4 r

epli

cate

cul

ture

s.

Destructive and Protective Immunity to Skin Grafts 41

weakly incompatible first- or second-set control skin allografts often recovered from frank episodes of rejection and why some test grafts on previously grafted animals did not exhibit curtailed survival times. Most attempts to use serum to transfer immunity to strong transplantation antigens on normal tissues have failed (Stetson 1963). The sera used in these experiments have generally been harvested from hyperimmune donors. Whether sera harvested early after immunization with a single strongly in- compatible graft would be more effective in transferring immunity remains unknown. However, the recent observation that skin grafted across weak H-barriers elicits formation of more germinal centers in mice than do comparable grafts across strong H-barriers (Mariani et al. 1973) suggests that strong antigens may, in fact, elicit the less intense humoral response (i.e., one that is not transferable). Fourth, the cellular immune response elicited by the weak antigens we tested could be passively trans- ferred earlier after grafting (on day 5) than could the cellular response generated in an H-2 incompatible (A -+ CBA) combination (on day 8) (Warren and Gowland 1970). Fifth, whereas our weakly incompatible transplants frequently sensitized the distant lymphoid tissues of the host before the draining lymphoid organs, the reverse usually appears to be the case when stronger incompatibilities are involved (Warren and Gowland 1970). Sixth, frequently those cells that could transfer immunity to secon- dary hosts in vivo were not demonstrably cytotoxic in vitro. On the other hand, sera harvested at the same time as these cells lysed target cells in vitro as well as transferred immunity in vivo. Our in vitro demonstration of an early humoral response to weak transplantation antigens is similar to that made for mice sensitized with H-2 incom- patible tumor grafts, but contrasts with the delayed humoral immunity revealed by the same s 1Cr.release assay in mice sensitized with H-2 incompatible skin (Canty and Wunderlich 1971).

There is a noteworthy lack of correlation between some of the data obtained from the double transplant and the transfer assays. No protective immunity was de- tected in a protocol that led to overt immunosuppression (female BIO sr. liver or kidney implants to female LP hosts), whereas there was a transient transferable pro- tective immunity in a protocol that led to accelerated test graft rejection (or. male B10 skin to female B10 hosts). Positive correlations, in addition to those mentioned already that involved curtailed survival, were suggested by the fact that passive serum transfers did reveal protective humoral immunity in at least one of the two double transplant protocols, which resulted in test graft survival that was neither accelerated nor prolonged (male B10 st. skin implants to male LP hosts). In view of our (Baldwin and Cohen 1973) and others' (Jooste et al. 1973) observations that the ultimate fate of a test graft on the recipient of an aliquot of serum is critically dependent on the timing of the transfer relative to test grafting (i.e., on the state of vascularization of the test graft and on the serum recipient's own immune responses to the test graft), discrepancies between data generated from these two disparate assay systems were not unexpected.

The in vivo transfer assay and the in vitro s l Cr-release assay also gave some con- flicting data that may well reflect basic and immunologically critical differences between the types of cellular and humoral immune reactions detected by each pro- cedure. For example, the s 1Cr-release assay appears to primarily measure T-cell mediated killing as opposed to B-cell or macrophage cytotoxicity (Miller et al. 1971). The rejection of weakly incompatible test grafts by adoptively immunized hosts may

42 William M. Baldwin III and Nicholas Cohen

be mediated in large part by B-cells or macrophages that are recruited and/or activated by the injected cells themselves or by antibodies they elaborate in the new recipient. By labeling lymphocytes from immune mice with s ICr (Bainbridge et al. 1966), we have determinedthat at least 2 to 5% of the transferred leukocytes (5 to 10 • 106 cells) were still present in the host's lymphoid tissues at the time the test grafts became re- vascularized (3 to 4 days posttransfer). Such cells could expand the immune activity of the transferred inoculum by dividing or by recruiting host cells. In the in vitro test system, however, the number of cells participating in an immune reaction is artifically limited. In contrast to the transfer method, the s 1Cr-release assay allows hours rather than days for the leukocytes or serum factors to kill target cells. Others have demon- strated that those cells that do not express their cytotoxic potential after incubation with target cells for a few hours can express their immune reactivity when their ex- posure to target cell antigens is lengthened, as it is in the in vivo passive transfer system (Biesecker 1973, Denham 1970, Rosenberg et al. 1973).

Regardless of these apparent discrepancies, data from the kinetics of first- and second-set skin graft rejection, from transfer studies, and from in vitro cytotoxicity assays indicate that weak transplantation antigens on normal tissues (like weak or strong tumor associated antigens) can rapidly generate cellular and humoral immune responses, each of which exhibits a protective as well as a destructive nature. The ultimate fate of transplants grafted across such weak H-barriers is determined by the sequence of appearance, intensity, and duration of these two antagonistic immune responses. In an intact host, some of these complex immune reactions may synergize to effect accelerated rejection or prolonged survival of grafts, or they may "equally" oppose each other to result in no overt effect on graft survival. Whether chronic re- jection represents qualitative differences in the reactivities elicited by weak and strong H-antigens or whether it reflects quantitative shifts in the functional participation of qualitatively comparable cellular and humoral immunity to strong and weak H-antigens still remains to be determined.

A c k n o w l e d g m e n t s

We thank Ms. Barbara B. Hrapchak for her excellent technical assistance and expertise. Tiffs work was supported by United States Public Health Service (National Institute of Allergy and Infectious Diseases) Grant AI-08784 and by a research grant from the Monroe County Cancer and Leukemia Association. W. M. Baldwin is a clinical investigator trainee supported by United States Public Health Service Training Grant TI-AM-1004-07.

Re fe rences

Bainbridge, D. R., Brent, L., and Gowland, G.: Distribution of allogenic s 1Cr-labeled lymph node cells in mice. Transplantation 4:138, 1966.

Baldwin, W. M., III., and Cohen, N.: Liver-induced immunosuppression of allograft immunity in urodele amphibians. Transplantation 10:530-537, 1970.

Baldwin, W. M., III., Cohen, N., and Hrapchak, B. B.: Prolonged survival of murine skin grafted across a weak histocompatibility barrier as a function of skin-grafting technique. Transplantation 15:4 l 9-422, 1973.

Destructive and Protective Immuni ty to Skin Grafts 43

Baldwin, W. M., III., and Cohen, N.: Immune serum implements either accelerated rejection or prolonged survival of murine skin grafts. Transplantation 15:633- 637, 1973.

Barnes, A. D., Burr, W. A., and Staniforth, P.: A backcross assay of the number of transplant antigens controlling the successful transplantation of skin, ovary, and kidney in mice. In: J. Dausset, J. Hamburger, and G. Mathe (eds.) Advances in Transplantation; pp. 343-347. Baltimore, Williams and Wilkins Co., 1968.

Bias, W. B., Santos, G. W., and Burke, P. J.: Cytotoxic ant ibody in normal human sera reactive with acute lymphocyt ic leukemia. Transpl. Proe. 5:949-952, 1973.

Biesecker, J. L., Scollard, D., Rowley, D., Stuart, F., and Fitch, F.: Assessment of cell- mediated immunity and serum blocking factors in renal allografted rats. Fed. Proc. 32:1005, 1973.

Billingham, R. E., and Silvers, W. K.: Transplantation o f Tissues and Cells. Philadelphia: Wistar Press, 1961.

Canty, T. G., and Wunderlich, J. R.: Quantitative assessment of cellular and humoral responses to skin allografts. Transplantation 11:111-116, 1971.

deLandazuri, M. O., and Herberman, R. B.: Immune response to Gross virus-induced lymphoma. III. Characteristics of the cellular immune response. J. Nat. Cancer Inst. 49:147-154, 1972.

Denham, S., Grant, C. K., Hall, J. F., and Alexander, P.: The occurrence of two types of cytotoxic lymphoid cells in mice immunized with allogeneic tumor cells. Transplantation 9:366-383, 1970.

Henry, L., and Dammin, G. L.: Time and dose relationships in the development of immunity to skin homografts. J. Immunol. 89:841-848, 1962.

Heslop, B. E.: Spontaneous deceleration of skin allograft rejection in the rat. Transplantation 11:497-499, 1971.

Jooste, S. V., Winn, H. J., and Russell, P. S.: Destruction of rat skin grafts by humoral antibody. Transpl. Proc. 5:713-716, 1973.

Litchfield, Jr., J. R.: A method for rapid graphic solution of t ime-percent effect curves. J. Pharmacol. Exp. Ther. 98:399-408, 1949.

Mariani, T., Demhof, J., and Good, R. A.: Germinal center formation in response to skin grafted across weak and strong histocompatibi l i ty barriers. Adv. Exper. Med. Biol. 29:597-602, 1973.

McKhann, C. E., and Berrian, J. H.: Immunologic properties of weak his tocompatibi l i ty genes. J. Immunol. 86:170-176, 1961.

Miller, J. F. A. P., Brunner, K. I., Sprent, J., Russell, P. J., and Mitchell, G. F.: Thymus- derived cells as kiUer cells in cell-mediated immunity. Transpl. Proc. 3:915-917, 1971.

Rosenberg, E. B., Herberman, R. B., Levine, P. H., Halterman, R. H., McCoy, J. L., and Wunderlich, J. R.: Lymphocyte cytotoxic i ty reactions to leukemia-associated antigens in identical twins. Int. J. Cancer 9:648-659, 1972.

Rosenberg, E. B., Hill, J., Ferrari, A., Herberman, R. B., Ting, C. C., Mann, D. L., and Fahey, J.: Prolonged survival of tumor allografts in mice pretreated with soluble transplantation antigens. J. Nat. Cancer Inst. 50 : 1453-1458, 1973.

Stetson, C. A.: The role of humoral ant ibody in the homograft reaction. Adv. Immunol. 3:97-130, 1963.

Warren, J., and Gowland, G.: Cell transfer studies on the persistence of homograft sensitivity in the mouse. J. Path. 100:39-51, 1970.

44 William M. Baldwin III and Nicholas Cohen

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